dear dr/prof. guido barbujani, • for · guido barbujani guido barbujani dipartimento di biologia...
TRANSCRIPT
Dear DrProf Guido BarbujaniHere are the proofs of your article
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6amp7 5th Street Radhakrishnan Salai Chennai Tamil Nadu India ndash 600004Re Human Genetics DOI101007s00439-007-0403-6
Genetic variation in prehistoric SardiniaAuthors David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina
Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot RobertTykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot GuidoBarbujani
I Permission to publishDear Springer Correction TeamI have checked the proofs of my article andq I have no corrections The article is ready to be published without changes
q I have a few corrections I am enclosing the following pagesq I have made many corrections Enclosed is the complete article
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Date Authorrsquos signature ______________________________________________________________________
Journal Human Genetics101007s00439-007-0403-6
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DateSignature DateSignature
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Guido Barbujani Guido BarbujaniDipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
Dipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
q q
Metadata of the article that will be visualized in OnlineFirst
ArticleTitle Genetic variation in prehistoric SardiniaArticle Sub-Title
Journal Name Human Genetics
Corresponding Author Family Name BarbujaniParticle
Given Name GuidoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Email gbarbujaniunifeit
Author Family Name CaramelliParticle
Given Name DavidSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name VernesiParticle
Given Name CristianoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Author Family Name SannaParticle
Given Name SimonaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name SampietroParticle
Given Name LourdesSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name LariParticle
Given Name MartinaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name CastrigraveParticle
Given Name LoredanaSuffix
Division Dipartimento di Biologia Evoluzionistica e Sperimentale
Organization Universitagrave di Bologna
Address Bologna Italy
Author Family Name VonaParticle
Given Name GiuseppeSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FlorisParticle
Given Name RosalbaSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FrancalacciParticle
Given Name PaoloSuffix
Division Dipartimento di Zoologia e Antropologia Biologica
Organization Universitagrave di Sassari
Address Sassari Italy
Author Family Name TykotParticle
Given Name Robert
Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
Query Details required Authorrsquos response 1 Please check and confirm the city in all
the affiliations
2 Please check and confirm the occurrence of ldquoAmicon Beverly Mardquo under the subsection ldquoAmplification of associated animal remainsrdquo
3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
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DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
123
439 403 xxxxJournal Article MS Code
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Hum Genet
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83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
123
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149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
123
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Hum Genet
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
123
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300
301302303304305306307308309310311
312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
123
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425426427428429430431432433434435436437438439440
UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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Fax to +44 870 622 1325 (UK) or +44 870 762 8807 (UK)From Springer Correction Team
6amp7 5th Street Radhakrishnan Salai Chennai Tamil Nadu India ndash 600004Re Human Genetics DOI101007s00439-007-0403-6
Genetic variation in prehistoric SardiniaAuthors David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina
Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot RobertTykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot GuidoBarbujani
I Permission to publishDear Springer Correction TeamI have checked the proofs of my article andq I have no corrections The article is ready to be published without changes
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Journal Human Genetics101007s00439-007-0403-6
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Guido Barbujani Guido BarbujaniDipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
Dipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
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ArticleTitle Genetic variation in prehistoric SardiniaArticle Sub-Title
Journal Name Human Genetics
Corresponding Author Family Name BarbujaniParticle
Given Name GuidoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Email gbarbujaniunifeit
Author Family Name CaramelliParticle
Given Name DavidSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name VernesiParticle
Given Name CristianoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Author Family Name SannaParticle
Given Name SimonaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name SampietroParticle
Given Name LourdesSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name LariParticle
Given Name MartinaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name CastrigraveParticle
Given Name LoredanaSuffix
Division Dipartimento di Biologia Evoluzionistica e Sperimentale
Organization Universitagrave di Bologna
Address Bologna Italy
Author Family Name VonaParticle
Given Name GiuseppeSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FlorisParticle
Given Name RosalbaSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FrancalacciParticle
Given Name PaoloSuffix
Division Dipartimento di Zoologia e Antropologia Biologica
Organization Universitagrave di Sassari
Address Sassari Italy
Author Family Name TykotParticle
Given Name Robert
Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
Query Details required Authorrsquos response 1 Please check and confirm the city in all
the affiliations
2 Please check and confirm the occurrence of ldquoAmicon Beverly Mardquo under the subsection ldquoAmplification of associated animal remainsrdquo
3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
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8910111213141516171819
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DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
123
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83848586
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888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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116
117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148
149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
123
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
123
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301302303304305306307308309310311
312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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425426427428429430431432433434435436437438439440
UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
123
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466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
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Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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Journal Human Genetics101007s00439-007-0403-6
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Guido Barbujani Guido BarbujaniDipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
Dipartimento di Biologia ed Evoluzionevia Borsari 46Ferrara 44100 Italy
q q
Metadata of the article that will be visualized in OnlineFirst
ArticleTitle Genetic variation in prehistoric SardiniaArticle Sub-Title
Journal Name Human Genetics
Corresponding Author Family Name BarbujaniParticle
Given Name GuidoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Email gbarbujaniunifeit
Author Family Name CaramelliParticle
Given Name DavidSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name VernesiParticle
Given Name CristianoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Author Family Name SannaParticle
Given Name SimonaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name SampietroParticle
Given Name LourdesSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name LariParticle
Given Name MartinaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name CastrigraveParticle
Given Name LoredanaSuffix
Division Dipartimento di Biologia Evoluzionistica e Sperimentale
Organization Universitagrave di Bologna
Address Bologna Italy
Author Family Name VonaParticle
Given Name GiuseppeSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FlorisParticle
Given Name RosalbaSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FrancalacciParticle
Given Name PaoloSuffix
Division Dipartimento di Zoologia e Antropologia Biologica
Organization Universitagrave di Sassari
Address Sassari Italy
Author Family Name TykotParticle
Given Name Robert
Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
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the affiliations
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3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
A1A2A3
A4A5A6
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A11A12A13A14
A15A16A17
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8910111213141516171819
2021222324252627282930
DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
123
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83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
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608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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Metadata of the article that will be visualized in OnlineFirst
ArticleTitle Genetic variation in prehistoric SardiniaArticle Sub-Title
Journal Name Human Genetics
Corresponding Author Family Name BarbujaniParticle
Given Name GuidoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Email gbarbujaniunifeit
Author Family Name CaramelliParticle
Given Name DavidSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name VernesiParticle
Given Name CristianoSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Author Family Name SannaParticle
Given Name SimonaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name SampietroParticle
Given Name LourdesSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name LariParticle
Given Name MartinaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name CastrigraveParticle
Given Name LoredanaSuffix
Division Dipartimento di Biologia Evoluzionistica e Sperimentale
Organization Universitagrave di Bologna
Address Bologna Italy
Author Family Name VonaParticle
Given Name GiuseppeSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FlorisParticle
Given Name RosalbaSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FrancalacciParticle
Given Name PaoloSuffix
Division Dipartimento di Zoologia e Antropologia Biologica
Organization Universitagrave di Sassari
Address Sassari Italy
Author Family Name TykotParticle
Given Name Robert
Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
Query Details required Authorrsquos response 1 Please check and confirm the city in all
the affiliations
2 Please check and confirm the occurrence of ldquoAmicon Beverly Mardquo under the subsection ldquoAmplification of associated animal remainsrdquo
3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
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A11A12A13A14
A15A16A17
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1
2
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8910111213141516171819
2021222324252627282930
DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
123
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31
323334353637383940414243444546474849505152535455565758596061626364656667686970717273747576777879808182
83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
123
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111112113114115
116
117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148
149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
123
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183184185186187188
189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
439 403 xxxxJournal Article MS Code
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Address Barcelona Spain
Author Family Name LariParticle
Given Name MartinaSuffix
Division Dipartimento di Biologia Animale e Genetica Laboratorio di Antropologia
Organization Universitagrave di Firenze
Address Firenze Italy
Author Family Name CastrigraveParticle
Given Name LoredanaSuffix
Division Dipartimento di Biologia Evoluzionistica e Sperimentale
Organization Universitagrave di Bologna
Address Bologna Italy
Author Family Name VonaParticle
Given Name GiuseppeSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FlorisParticle
Given Name RosalbaSuffix
Division Dipartimento di Biologia Sperimentale
Organization Universitagrave di Cagliari
Address Cagliari Italy
Author Family Name FrancalacciParticle
Given Name PaoloSuffix
Division Dipartimento di Zoologia e Antropologia Biologica
Organization Universitagrave di Sassari
Address Sassari Italy
Author Family Name TykotParticle
Given Name Robert
Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
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Hum Genet
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DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
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UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
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Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
439 403 xxxxJournal Article MS Code
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Suffix
Division Department of Anthroopology
Organization University of South Florida
Address Tampa FL USA
Author Family Name CasoliParticle
Given Name AntonellaSuffix
Division Dipartimento di Chimica generale ed Inorganica Chimica Analitica e ChimicaFisica
Organization Universitagrave di Parma
Address Parma Italy
Author Family Name BertranpetitParticle
Given Name JaumeSuffix
Division Dept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva
Organization Universitat Pompeu Fabra
Address Barcelona Spain
Author Family Name Lalueza-FoxParticle
Given Name CarlesSuffix
Division Unitat drsquoAntropologia Dept Biologia Animal
Organization Universitat de Barcelona
Address Barcelona Spain
Author Family Name BertorelleParticle
Given Name GiorgioSuffix
Division
Organization Dipartimento di Biologia ed Evoluzione
Address via Borsari 46 44100 Ferrara Italy
Schedule
Received 4 June 2007
Revised
Accepted 29 June 2007
Abstract We sampled teeth from 53 ancient Sardinian (Nuragic) individuals who lived in the Late Bronze Age andIron Age between 3430 and 2700 years ago After eliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducible results we obtained 23 sequences of themitochondrial DNA control region which were associated to haplogroups by comparison with a dataset ofmodern sequences The Nuragic samples show a remarkably low genetic diversity comparable to thatobserved in ancient Iberians but much lower than among the Etruscans Most of these sequences have exact
matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
Query Details required Authorrsquos response 1 Please check and confirm the city in all
the affiliations
2 Please check and confirm the occurrence of ldquoAmicon Beverly Mardquo under the subsection ldquoAmplification of associated animal remainsrdquo
3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
A1A2A3
A4A5A6
A7A8A9A10
A11A12A13A14
A15A16A17
A18A19A20A21
A22A23A24A25
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A30A31A32
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2
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8910111213141516171819
2021222324252627282930
DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
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83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148
149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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matches in two modern Sardinian populations supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populations appear to be part of a large and geographicallyunstructured cluster of modern European populations thus making it difficult to infer their evolutionaryrelationships However the low levels of genetic diversity both within and among ancient samples asopposed to the sharp differences among modern Sardinian samples support the hypothesis of the expansionof a small group of maternally related individuals and of comparatively recent differentiation of the Sardiniangene pools
Footnote Information Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6)contains supplementary material which is available to authorized users
Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
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Hum Genet
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DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
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UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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300
301302303304305306307308309310311
312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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425426427428429430431432433434435436437438439440
UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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441442443444445446447448449450451452453454455456457458459460461462463464465
466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
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491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543
544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
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608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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Journal 439 Article 403 Dear Author During the process of typesetting your article the following queries have arisen Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proofonline grid or in the lsquoAuthorrsquos responsersquo area provided below
Author Query Form
Query Details required Authorrsquos response 1 Please check and confirm the city in all
the affiliations
2 Please check and confirm the occurrence of ldquoAmicon Beverly Mardquo under the subsection ldquoAmplification of associated animal remainsrdquo
3 Please check and provide initial for the last author in the reference Excoffier et al (2005)
4 Please check and confirm the ESM
Hum Genet
A1A2A3
A4A5A6
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A15A16A17
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A30A31A32
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1
2
345
67
8910111213141516171819
2021222324252627282930
DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
123
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83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
123
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117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148
149150151152153154155156
157
158159160161162163164165
166
167168169170171172173174175
176
177178179180181182
UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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8910111213141516171819
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DOI 101007s00439-007-0403-6
UNCORRECTED PROOF
ORIGINAL INVESTIGATION
Genetic variation in prehistoric Sardinia
David Caramelli middot Cristiano Vernesi middot Simona Sanna middot Lourdes Sampietro middot Martina Lari middot Loredana Castrigrave middot Giuseppe Vona middot Rosalba Floris middot Paolo Francalacci middot Robert Tykot middot Antonella Casoli middot Jaume Bertranpetit middot Carles Lalueza-Fox middot Giorgio Bertorelle middot Guido Barbujani
Received 4 June 2007 Accepted 29 June 2007copy Springer-Verlag 2007
Abstract We sampled teeth from 53 ancient Sardinian(Nuragic) individuals who lived in the Late Bronze Ageand Iron Age between 3430 and 2700 years ago Aftereliminating the samples that in preliminary biochemicaltests did not show a high probability to yield reproducibleresults we obtained 23 sequences of the mitochondrialDNA control region which were associated to haplogroupsby comparison with a dataset of modern sequences TheNuragic samples show a remarkably low genetic diversitycomparable to that observed in ancient Iberians but muchlower than among the Etruscans Most of these sequenceshave exact matches in two modern Sardinian populations
supporting a clear genealogical continuity from the LateBronze Age up to current times The Nuragic populationsappear to be part of a large and geographically unstructuredcluster of modern European populations thus making itdiYcult to infer their evolutionary relationships Howeverthe low levels of genetic diversity both within and amongancient samples as opposed to the sharp diVerences amongmodern Sardinian samples support the hypothesis of theexpansion of a small group of maternally related individu-als and of comparatively recent diVerentiation of the Sar-dinian gene pools
Electronic supplementary material The online version of this article (doi101007s00439-007-0403-6) contains supplementary material which is available to authorized users
D Caramelli middot S Sanna middot M LariDipartimento di Biologia Animale e Genetica Laboratorio di Antropologia Universitagrave di Firenze Firenze Italy
C Vernesi middot G Bertorelle middot G Barbujani (amp)Dipartimento di Biologia ed Evoluzione via Borsari 46 44100 Ferrara Italye-mail gbarbujaniunifeit
L Sampietro middot J BertranpetitDept Ciegravencies de la Salut i de la Vida Unitat de Biologia Evolutiva Universitat Pompeu Fabra Barcelona Spain
L CastrigraveDipartimento di Biologia Evoluzionistica e Sperimentale Universitagrave di Bologna Bologna Italy
G Vona middot R FlorisDipartimento di Biologia Sperimentale Universitagrave di Cagliari Cagliari Italy
P FrancalacciDipartimento di Zoologia e Antropologia Biologica Universitagrave di Sassari Sassari Italy
R TykotDepartment of Anthroopology University of South Florida Tampa FL USA
A CasoliDipartimento di Chimica generale ed Inorganica Chimica Analitica e Chimica Fisica Universitagrave di Parma Parma Italy
C Lalueza-FoxUnitat drsquoAntropologia Dept Biologia Animal Universitat de Barcelona Barcelona Spain
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Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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544
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UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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83848586
87
888990919293949596979899100101102103104105106107108109110
UNCORRECTED PROOF
Introduction
The population of Sardinia is one of the main Europeangenetic outliers (Cavalli-Sforza and Piazza 1993) Whencompared with populations from all over the worldSardinians are clearly part of a European genetic cluster(Rosenberg et al 2002) However they diVer sharply fromtheir European (Barbujani and Sokal 1990) and Italian(Barbujani and Sokal 1991 Barbujani et al 1995) neigh-bours so much so that they are often excluded frommultivariate analyses lest all other samples appear identicalin comparison (Piazza et al 1988 Semino et al 2000)Mitochondrial (Morelli et al 2000) and Y-chromosome(Semino et al 2000 Quintana-Murci et al 2003) haplo-types that are rare elsewhere in Europe occur at higherfrequencies in Sardinia and an extensive linkage disequi-librium has been described for autosomal markers (Tenesaet al 2004) In addition unusually strong genetic diVer-ences are observed among Sardinian communities both forallele-frequency (Barbujani and Sokal 1991) and DNA(Fraumene et al 2003) polymorphisms
These peculiar features are the likely evolutionary prod-uct of the interaction between reproductive isolation andsmall population sizes the former historically documentedand the latter a consequence of the fragmented habitatStarting perhaps 10000 years ago people of diVerent prov-enance reached the island (Webster 1996) but strong isolat-ing factors such as the Mediterranean Sea and mountainranges as well as cultural barriers (Barbujani and Sokal1991) have probably enhanced the evolutionary role ofgenetic drift both within the island and between it and therest of Europe However the details of these processes arenot well understood and it is well known that ages of par-ticular genealogical lineages present in a geographicalregion do not contain information on the arrival of the pop-ulation in that region (Barbujani and Goldstein 2004)Therefore to gain insight into the biological features ofpast populations and into the relationships between modernpopulations and their ancient counterparts the direct studyof the DNA of ancient individuals is a crucial researchpriority
Here we present the Wrst analysis of the hypervariableregion I (HVR-I) of mitochondrial DNA (mtDNA) fromsamples of Bronze and Iron Age inhabitants of Sardiniawho are called Nuragic people from the towers (nuraghi)that they built Using a strict set of methodological criteriathat give a high probability of reproducible results (whichforced us to exclude more than half of the initial samplesfrom the analysis) we obtained sequences that were com-pared with those of modern European populations and withthe sequences of the two pre-classical European popula-tions whose DNA has been described so far the Etruscans(Vernesi et al 2004) and the Iberians (Sampietro et al
2005) In particular prior to sequencing the samples weretreated with Uracil-N-glicosidase so as to avoid as far aspossible artefacts due to post-mortem damage of DNA(Hofreiter et al 2001 Gilbert et al 2003)
Materials and methods
DNA molecules are often scarce and damaged in ancientsamples As a consequence experimental artefacts are pos-sible or even likely depending on the state of preservationof the material during the analysis of DNA from fossilremains The risk to mistake modern contaminating DNAfor endogenous genetic material is higher when dealingwith relatively recent human samples whose DNAsequence may be similar to that of the archaeologistsmuseum personnel and geneticists who manipulated themFor all these reasons in order to obtain reproducible resultsthe most stringent available standards were followed in thisstudy based on criteria listed by Cooper and Poinar (2000)and Hofreiter et al (2001)
We collected 106 teeth 2 from each of 53 diVerent indi-viduals coming from excavations at six archaeologicalsites of Sardinia (Fig 1) and dated between 4300 and3000 years before the present (Table 1) The selected teethhad not been washed which meant they had not beentouched by anthropologists for morphological analysis Byanalysing dental pulp from inside the tooth we reduced therisk of contamination from the archaeologistsrsquo DNA andcare was taken to choose integer teeth without fractures As arule to avoid the risk of sampling twice the same individual
Fig 1 A map of Sardinia showing the six Nuragic sampling sites Thetwo-letter codes are used to label the sequences in the text and in Fig 2
Carbonia (CA)
Fluminimaggiore (FL)
Perdasdefogu (PE)
Seulo (SE)
Alghero (AL)
Santa Teresa di
Gallura (ST)
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166
167168169170171172173174175
176
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UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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189
190191192193194195196
197
198199200201202203204205206207208209210211212
213
214215216217218219220221222223224225226227228229
230231232233
234
235236237238239240241242243244245246247248249250251252253254
255
256257258259260261262
263
264265266267268269270271272273274275276
UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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UNCORRECTED PROOF
whenever the teeth had not been found in place in the skullor mandible we took only left lower canines and left lowermolars Because of the limited amount of ampliWable DNApresent in most specimens we decided to concentrate oureVorts on the typing of the non-coding HVRI region
Criteria of authenticity an overview
Tooth specimens were handled (using mask gloves andlaboratory coats) in a space where no modern DNA hadever been extracted or analysed We used only disposablesterile tubes Wltered tips sterile reagents and solutionsexclusively dedicated to ancient DNA studies DiVerentsets of pipettes were used for DNA extraction PCR ampli-Wcation and analysis of the PCR products DNA wasextracted and PCR-ampliWed in separate rooms and underhoods constantly irradiating with UV rays (254 nm) PCRproducts from ancient DNA templates were analysed in athird room In each set of extractions or puriWcations weincluded a negative control represented by all the reagentsexcept the bone powder and these negative controlstogether with blanks (all ampliWcation reagents minusDNA) were regularly analysed in every PCR experiment tocontrol for presence of exogenous DNA
All phases of the analysis were replicated at least twiceFour samples for which relatively large amounts of toothpowder were available were sent to the Barcelona labora-tory where the whole analysis was independently repli-cated To test for preservation of other macromolecules asan indirect evidence for DNA survival (Poinar et al 1996Poinar and Stankiewicz 1999) we estimated the degree ofaminoacid racemisation in each sample using approxi-mately 5 mg of tooth powder (Poinar et al 1996) We alsoquantiWed the amount of target DNA by competitive PCRPCR products were cloned an average 25 clones weresequenced for each individual and the sequences thusobtained were aligned and compared across clones Theconsensus sequences were Wnally compared with a databaseof European mitochondrial sequences to test whether theancient sequences obtained make phylogenetic sense
DNA was also extracted from cattle (Bos taurus)remains retrieved in one burial (PE25) and we tried toamplify it using both human and cattle primers The pres-ence of human DNA sequences in extracts from non-humanbones would suggest contamination in other bones in theburial and possibly in other burials as well All personswho worked in this study were mtDNA genotyped andnone of their sequences matched those obtained
DNA extraction
After brushing and irradiating each tooth surface (1 h underUV light) the root tip of each tooth was removed and thecrown was repositioned into its mandibular alveolus Theroot was powdered and DNA was extracted by means of asilica-based protocol (modiWed from Caramelli et al 2003)For each individual we obtained two independent extractsfrom diVerent teeth A negative control was included ineach extraction
Competitive PCR
A competitor was used containing a 94 bp deletion (fromposition 16106 to 16189 nt position according to Caram-elli et al 2003) in the mitochondrial HVR-I PCR compo-nents were the same as described below for the sequencingof mitochondrial HVR-1 and the primers were those usedfor the second fragment ampliWcation Thermal cycler con-ditions consisted of an initial 10-min incubation at 95degCfollowed by 45 cycles of 50 s at 94degC 50 s at 48degC and50 s at 72degC with a Wnal extension step at 72degC for 5 min
Real-time PCR
Real-time PCR ampliWcation was performed to conWrm theresults of competitive PCR We used Brilliantreg SYBRreg
Green QPCR Master Mix (Stratagene) in MX3000P (Strat-agene) using 05 M of appropriate primers (forwardprimer at H 16107 and reverse primer at L 16261) Ther-mal cycling conditions were 95degC for 10 min 40 cycles at
Table 1 Samples analysed
N1 is the number of specimens initially available N2 the number of specimens whose mitochondrial sequences could be determined
Site Series Time 14C Dating Burial N1 N2
Alghero Lu Maccioni Iron Age 700 sect 70 BC Natural Cave 6 1
Carbonia Su cungiareddu lsquoe SeraWni Middle Bronze Age 1430 sect 70 BC Natural Cave 7 2
Fluminimaggiore Capo Pecora Late Bronze Age 1265 BC Natural Cave 3 1
Perdasdefogu Perdasdefogu Late Bronze Age 930 sect 60 BC Natural Cave 10 5
Seulo Stampu Erdi e Cannisoni e Gastea Middle Bronze Age 1300 sect 60 BC Natural Cave 10 6
STeresa di Gallura STeresa di Gallura Late Bronze Age 1200ndash1300 BC Tomb of Giants 17 8
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230231232233
234
235236237238239240241242243244245246247248249250251252253254
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UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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312313314315316317318319320321322323324325326327328329330331332333334335336337
UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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UNCORRECTED PROOF
95degC for 30 s 53degC for 1 min and 72degC for 30 s followedby SYBRreg Green dissociation curve steep Tenfold serialdilutions of the puriWed and quantiWed standard wereincluded in the experiment to create the standard curvethus estimating the number of initial DNA molecules in thesamples
UNG treatment
Uracil-N-Glycosylase (UNG) reduces sequence artefactscaused by post-mortem damage of DNA resulting in appar-ent C to T and G to A mutations and subsequent errors inthe sequence (Hofreiter et al 2001) For each specimenconsidered 10 l of DNA extract were treated with 1 U ofUNG for 30 min at 37degC to excise uracil bases caused bythe hydrolytic deamination of cytosines
AmpliWcation
Two microliters of DNA extracted from the bone andtreated with UNG was ampliWed with this proWle 94degC for10 min (Taq polymerase activation) followed by 50 cyclesof PCR (denaturation 94degC for 45 s annealing 53degC for1 min and extension 72degC for 1 min) and Wnal step at 72degCfor 10 min The 50-l reaction mix contained 2 U of Amp-liTaq Gold (Applied Biosystems) 200 M of each dNTPand 1 M of each primer The 360-bp long HVR-I was sub-divided in three overlapping fragments using the followingprimer pairs L15995H16132 L16107H16261L16247H16402 (Vernesi et al 2004) Each extract wasampliWed at least twice Since overlapping primers wereused throughout the PCR ampliWcations it is highlyunlikely that we ampliWed a nuclear insertion rather thanthe organellar mtDNA
Cloning and sequencing
PCR products were cloned using TOPO TA Cloning Kit(Invitrogen) according to the manufacturerrsquos instructionsWhite recombinant colonies were screened by PCR trans-ferring the colonies into a 30-l reaction mix [67 mM TrisndashHCl (pH 88) 2 mM MgCl2 1 M of each primer0125 mM of each dNTP 075 U of Taq Polymerase] con-taining M13 forward and reverse universal primers After5 min at 92degC 30 cycles of PCR (30 s at 90degC 1 min at50degC 1 min at 72degC) were carried out and clones withinsert of the expected size were identiWed by agarose gelelectrophoresis After puriWcation of these PCR productswith Microcon PCR devices (Amicon) a volume of 15 lwas cycle-sequenced following the BigDye Terminator kit(Applied Biosystems) supplierrsquos instructions The sequencewas determined using an Applied BioSystems 3100 DNAsequencer Finally to test for possible contamination within
the laboratory four teeth were subjected to DNA extrac-tion ampliWcation cloning and sequencing in Barcelona Inthis lab the following primer pairs were used L16022H16095 L16055H16218 L16209H16401
AmpliWcation of associated animal remains
We tried to amplify the DNA extracted from cattle bonesusing both human-speciWc and bovine-speciWc primersnamely primers for a 152-bp fragment of the Bos taurusmtDNA D-loop and for a fragment of the human D-Loop(respectively L16030H16137 and L16107H16261)The PCRs were performed with 2 l of DNA 1 M of eachprimer 200 M of each dNTP 1pound reaction buVer (AppliedBiosystem) 15 mM MgCl2 and 2 U of AmpliTaq Gold(Applied Biosystem) in a total volume of 50 l The cattleD-Loop was ampliWed using the following thermal cycleinitial denaturation at 94degC for 10 min followed by 40cycles of 94degC for 1 min 57degC for 1 min 72degC for 1 minand Wnal extension at 72degC for 5 min The conditionsdescribed above for human mitochondrial DNA ampliWca-tion were used in the ampliWcation of bovine DNA withhuman-speciWc primer After a run on a 15 agarose gelbands of the appropriate size were excised from the gel andpuriWed with Ultra Free DNA (Amicon Beverly Ma)Cloning and sequencing of the PCR products were asdescribed above
Detection of long ampliWcates
Appropriate molecular behaviour was also tested by ampli-Wcation of longer mtDNA fragments (443 and 724 bp)which have been reported to be very unusual for ancientDNA PCR conditions were those described for mtDNAanalysis above the primers used were L15995 andH16401 (for the 443 bp fragment) and L16247 andH00360 (for the 724 bp fragment)
Data analysis
We estimated three measures of intrapopulation diversity inthe Nuragic sample Nucleotide diversity () is the averagenumber of nucleotide diVerences per site (Nei and Kumar2000) over the 360 sites screened [from position 16024 to16383 of the Cambridge reference sequence (CRS)] Thehaplotype diversity (h often referred to as heterozygosity)is deWned as h = 1 iexcl q i
2 where qi is the frequency ofeach haplotype and summation is over all haplotypesobserved (Nei and Kumar 2000) The average pair wisesequence diVerence or average mismatch (k) is the averagediVerence between sequences in the sample
Phylogenetic trees ie reduced median networks(Bandelt et al 1995) summarising mtDNA variation were
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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UNCORRECTED PROOF
estimated by means of Network 41 using a reductionthreshold = 2 and the same weights for all loci To putNuragic DNA diversity in the proper evolutionary contextwe used a database of HVR-I sequences from 60 popula-tions namely 57 modern populations from Europe CentralAsia and the Southern Mediterranean shores plus the twopre-classic European populations typed so far at the DNAlevel the Etruscans (Vernesi et al 2004) and Iberians(Sampietro et al 2005) and a medieval sample from Spain(Casas et al 2006) The modern database includes a samplegenerically labelled as Sardinia (Di Rienzo and Wilson1991) and two samples from two regions of Sardinianamely Ogliastra in the Southeast (Fraumene et al 2003)and Gallura in the Northeast (Morelli et al 2000) Com-plete references are available at this URL httpwebunifeitprogettigeneticapdatahtm
Genetic distances between populations Fst distanceswere calculated by the Arlequin ver 301 software(ExcoYer et al 2005) based on Kimurarsquos two-parametermodel and using = 026 A two-dimensional representa-tion of population relationships was obtained by multi-dimensional scaling (MDS) using the software Statistica55 (Statsoft Inc)
Results
For 10 of the 53 samples the D enantiomer of Asp repre-sented more than 10 of the total amino acid (Table 2)implying that there was little hope to retrieve well-pre-served DNA molecules from those samples (Poinar et al1996 Serre et al 2004) Therefore we extracted the DNAfrom the remaining 43 samples Sporadic contamination isconsidered unlikely when the number of molecules thatPCR will use as template (target DNA) is greater than 1000(Poinar et al 1996) in 7 samples quantitative PCR showedthat the DNA available was less than that whereas 36 sam-ples yielded suYcient DNA for ampliWcation cloning
(about 30 clones for each sample) and sequencing RealTime PCR conWrmed that these 36 samples were suitablefor further analysis In comparing sequences across clonesnucleotide substitutions observed in one clone only wereconsidered due to Taq-polymerase errors Conversely insix samples the repeated occurrence of the same substitu-tion in several (but not all) clones made it impossible toinfer a consensus sequence without ambiguity (Handt et al1996)
Globally a low rate of Taq misincorporation was esti-mated (021 substitutions every 1000 bp within the HVRI)with at least 95 of the clones showing the consensusnucleotide at each position This result suggests that theDNA templates were not damaged On the other handseven samples yielded multiple sequences and wereexcluded from the analysis at this stage thus bringing downthe number of nuragic HVR-I sequences to 23 Additionalteeth from four of these 23 individuals (PE23 PE20 SE14SE2) were available and were sent to the Barcelona labora-tory the sequences determined there were identical to thoseobtained in the Florence laboratory AmpliWcation of largeDNA fragments unusual for ancient DNA was notobserved and the Wnal consensus sequences (Table 3)make phylogenetic sense ie do not appear to be combina-tions of diVerent known sequences Finally in the only casein which cattle remains were available PCR ampliWcation
Table 2 Elimination of the specimens that did not comply with qual-ity standards
a This test was carried out for four specimens onlyb This test was carried out for one specimen only
Initial sample size(individuals)
Test Eliminated Kept
53 AA racemisation 10 43
43 Quantitative PCR 7 36
36 Cloning sequencing 6 30
30 Sequence comparisons 7 23
23 Replication in a second lab 0a 23
23 AmpliWcation of non-human associated remains
0b 23
Table 3 DNA sequences of 23 Nuragic specimens
CRS Cambridge reference sequence
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
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UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
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544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
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608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
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368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397
UNCORRECTED PROOF
was successful only using primers speciWc for Bos taurusand not human primers
Ten sites appeared polymorphic in the Nuragic sampleswith nine transitions and one transversion leading to iden-tiWcation of ten distinct haplotypes Nucleotide diversitywas 00041 and haplotype diversity a low 083 close to thevalue observed in Ogliastra 078 By contrast haplotypediversity values of 096 (Di Rienzo and Wilson 1991) and097 (Morelli et al 2000) were observed in the other mod-ern Sardinian samples and cedil093 in all other Europeansamples with the exception of Saami The low haplotypediversity among the Nuragic people is due to the occur-rence at relatively high frequencies of Wve haplotypesincluding the CRS (9 individuals 39 of the total) Theother Wve haplotypes AL07 PE15 SE02 SE60 and ST16are observed once in the Nuragic sample and so despitethe small sample sizes diVerent Nuragic populations shareone or more haplotypes (Table 4) The average diVerencebetween pairs of sequences or average mismatch was alow 143 sect 090 compared to the Etruscans (390 Vernesiet al 2004) and the modern samples (average 468) butclose to the Iberiansrsquo 212 (Sampietro et al 2005) and to themodern Ogliastra 263 (Fraumene et al 2003)
The phylogenetic relationships of the Nuragic sequenceswere summarised in a network comparing them with themodern sample from Ogliastra (Fraumene et al 2003) forwhich the RFLPs data necessary for haplogroup identiWca-tion were available (Fig 2) Eight Nuragic sequences rep-resenting 21 individuals appear to fall into the haplogroupH cluster whereas the remaining two appear to belong to
haplogroups V and J However in the FL04-ST15-ST54sequence a C is observed in position 129 a substitutioncommonly observed in subhaplogroup U2 (Achilli et al2005) Therefore in the absence of RFLP data it is impos-sible to attribute with conWdence this sequence to eitherhaplogroup H or U
The comparison with the database including sequencesfrom 57 modern populations ancient Etruscans and Iberi-ans and a medieval sample from Spain is clearly aVectedby the diVerent sample sizes However 8 of the sequencesrepresenting 19 individuals (83) found an exact match inmodern European samples to the exclusion of PE15 (withsubstitutions at 16129 and 16298) and FL04-ST15-ST54(with a substitution at 16129) These two sequences Wnd nomatch in comparisons with 92 African samples either (datanot given) Six haplotypes are shared between modern andancient Sardinians representing 61 of the ancient individ-uals Four and two haplotypes representing respectively 52and 48 of the total Nuragic sequences is the level of shar-ing between the Nuragic people and both Etruscans andancient Iberians The minimum allele sharing was one forthe Sardinian sample from Gallura (and for a few EuropeanCaucasian Near Eastern and North African populationsincluding Tuscany and the Ladin linguistic isolate of North-ern Italy) Two modern samples from Sardinia (Di Rienzoand Wilson 1991) and Ogliastra (Fraumene et al 2003)share four haplotypes each with the Nuragic sample and inparticular the Ogliastra sample shares three haplotypeswith the ancient samples from the same area (Seulo andPerdasdefogu) The CRS which represents 391 of the
Table 4 Number of haplotypes shared between the population of Nuragic Sardinia and other regions of Eurasia (relative frequencies betweenparentheses)
The three modern Sardinian samples are from Di Rienzo and Wilson (1991) (Sardinia SR in the legend to Fig 3) Fraumene et al (2003) (Oglia-stra SO) and Morelli et al (2000) (Gallura GR)
Haplotype Frequency North Africa
Near East
Caucasus Central Asia
Europe Ancient Iberians
Etruscans Sardinia Ogliastra Gallura
Sample size 456 261 397 205 3379 17 27 73 175 27
CA02 PE11PE20 PE23SE81 SE84ST10 ST38 ST47
391 44 (96) 18 (69) 56 (141) 11 (54) 451 (133) 3 (176) 2 (74) 15 (205) 78 (445) 5 (185)
ST16 44 11 (24) 6 (22) 4 (10) 2 (10) 89 (26) 1 (37) 3 (41) 12 (71)
AL07 44 2 (04) 3 (08) 2 (10) 46 (14) 1 (14)
SE02 44 1 (04) 1 (03) 5 (01) 2 (12)
SE60 44 1 (04) 11 (03)
PE25 ST08 87 1 (04) 1 (03) 1 (00) 1 (37)
SE01 ST30 87 1 (04) 2 (03) 22 (06) 2 (118) 2 (74) 3 (18)
CA14 SE13 87 7 (15) 1 (03) 1 (00) 1 (14)
PE15 44
FL04 ST15 ST54 130
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UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
123
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466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543
544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
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LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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398399400401402403404405406407408409410411412413414415416417418419420
421422423
424
425426427428429430431432433434435436437438439440
UNCORRECTED PROOF
Nuragic sequences and only 133 all over Europe reachessimilarly high frequencies only in the modern Sardinians(globally 355) and especially in Ogliastra
An analysis of molecular variance (ExcoYer et al 2005)showed no diVerence among the Perdasdefogu (n = 5)Seulo (n = 6) and Santa Teresa di Gallura (n = 8) sampleswith a negative estimate of the among-population variancecomponent (iexcl060) Among the Etruscans the diVer-ences among populations albeit insigniWcant accountedfor 111 of the overall variance
The multidimensional scaling graph (Fig 3) summarisesthe genetic relationships inferred from Fst distancesbetween populations Saami were eliminated from thisgraph because their high level of diVerentiation drasticallyreduces the apparent diVerences among all other popula-tions A mild stress value was observed in the analysis019 meaning that the projection of the data on a plane dis-torted population relationships only to a limited extent Inthis global representation Nuragic Sardinia falls in a largeEuropean cluster where little or no geographical structureis apparent All outliers are either populations separated bylarge geographic distances from the other Europeans(mainly North Africans and Central Asians) or well-known
genetic outliers such as the modern population of Ogliastra(Fraumene et al 2003) and the ancient non-Indo-Europeanspeaking Etruscans
Discussion
The Wrst archaeological evidence for the Nuragic civilisa-tion dates back to the Middle Bronze Age 3800ndash3300 years ago Populations previously scattered in smallunits integrated in larger communities still having onlysporadic contacts with non-Sardinian people (Webster1996) In the Late Bronze Age pre-existing nuraghi wereenlarged and metallurgy intensiWed leading to increasedtrade across the Mediterranean Sea Population growth andwarfare can be inferred from the archaeological record ofthat period the former being a possible cause of the latter
The Late Bronze Age Sardinians whose mtDNA wecould type show a remarkably low level of genetic diVeren-tiation Despite sampling covering an area of approximately24000 km2 and a 700-year time interval (up to the earlyIron Age 2700 years BP) the same sequences wererepeatedly found at diVerent locations and all belong to
Fig 2 Reduced median networks of Nuragic (white) and modernOgliastra (grey) samples The size of the circles represents the haplo-typersquos absolute frequency Positions of HVR-I mutations are indicatedminus 16000 and transversions by the nucleotide change RFLP sites
are indicated as follows iexcl7025AluI = 7025 iexcl14766MseI = 14766+12308HinfI = 12308 +4216NlaIII = 4216 Capital letters refer tothe haplogroups inferred from sequence variation and RFLP status
V
H
T
J
U
123
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441442443444445446447448449450451452453454455456457458459460461462463464465
466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543
544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
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Hum Genet
441442443444445446447448449450451452453454455456457458459460461462463464465
466467468469470471472473474475476477478479480481482483484485486487488489490
UNCORRECTED PROOF
only 3 or 4 of the 20 haplogroups documented in WesternMediterranean populations (Sampietro et al 2005) Onlyanother ancient population the sixth to second century BCIberians (Sampietro et al 2005) showed a comparablemitochondrial diversity much lower than in all modernsamples and in the Etruscans
Genetic variation in Sardinia appears limited across timeas well Six haplotypes observed in the Nuragic sample arestill present in the modern Ogliastra and Sardinia samplesand they include not only the widespread CRS (whose fre-quency is higher in moderns as well as in ancient Sardinianscompared to other samples) but also rarer sequences suchas those that we labelled AL07 SE02 ST16 SE01-ST30and CA14-SE13 Three such sequences AL07 ST16 andCA14-SE13 also occur in North Africa their presence inboth modern and Nuragic Sardinia suggest the eVects ofcommon ancestry or ancient gene Xow rather than those ofgene Xow in historical times In particular modern peoplefrom Ogliastra share three haplotypes with Nuragic peoplefrom the nearby localities of Perdasdefogu and Seulo iehalf of both the modern (83175) and the ancient (1223)sequences (Table 4) On the contrary the modern popula-tion of Gallura appears genetically distant from bothancient and modern Sardinian samples in agreement withthe eVects of relatively recent immigration from continental
Italy known to have aVected Gallura more than Central andSouthern Sardinia (Morelli et al 2000)
The other pre-Roman Italic population described so farat the genetic level the Etruscans (Vernesi et al 2004)showed very diVerent patterns of mtDNA diversity TheEtruscans appear more closely related to modern Tuscansthan to any other European or Near Eastern population butonly 2 of 23 diVerent Etruscan sequences were found tohave an exact match in the modern Tuscan populationsincluding some that were selected for their supposed Etrus-can origins (Achilli et al 2007) The diVerence with NuragicSardinia (where shared sequences are seven out of ten) ishighly signiWcant (2 = 1321 1 dof P lt 0001) Plausibleexplanations for the low resemblance between Etruscansand modern Tuscans include loss of haplotypes by geneticdrift andor extensive immigration possibly but not neces-sarily related to the social changes that occurred after theRoman assimilation (Belle et al 2006) or in modern timesBy contrast the results of the present study show a muchclearer genealogical continuity in Sardinia presumablyreXecting the islandrsquos higher degree of isolation
Despite this clear genealogical continuity there arediVerences between ancient and modern Sardinians nota-bly in their levels of internal diVerentiation much higher inthe latter and very high at the allele-frequency level (Cappello
Fig 3 Multi-dimensional scaling Squares ancient populations cir-cles North Africa and the Near East diamonds Europe triangles Asiaand the Caucasus Population codes as follows Algerians (AL) Cen-tral Asia (AS) Catalans (CA) Druze (DR) Egyptians (EG) Etruscans(ET) Ancient Iberians (IB) Ingushians (IG) Moroccan Arabs (MO)Near Easterners (NE) Nuragic Sardinians (SN) Sardinians Ogliastra(SO) The circle includes all other populations namely AbazinianAdygei Armenians Austrians Azerbaijan Basques Belgium Bela-rus British Bulgarians Chechenians Cherkessians Cornwell Danes
Darginians Estonians Finns French Galicians Germany GeorgiansSardinians Gallura Germans North Germans South Icelanders Ital-ians Ladin and German speakers Italians South Italians TuscanyKarelians Kurds Moroccan Berbers Dutch Norwegians PortugueseRussians Saami Italians Sicily Sardinians Swedes Medieval Span-iards Spaniards Spaniards South Swiss Sirians Tunisians BerbersTurks Volga-Finns Welsh The total number of individuals consid-ered is 4875
MDS
mtDNA HVRI - 59 European populations
Fst (Kimura 2P gamma a value=026) Stress=019
Dim 1
Dim
2
AL
AS
CA
DR
EG
ET
IG
MO
NE
TB
SN
All other populations(including IB)
SO
123
439 403 xxxxJournal Article MS Code
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LE TYPESET CP DISK
Hum Genet
491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543
544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543
544
545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607
UNCORRECTED PROOF
et al 1996) A previous genetic analysis excluded pre-Bronze-Age expansions similar to those that occurred incontinental Italy and other European regions (Barbujaniet al 1995) Although mitochondrial and autosomal varia-tion are not necessarily correlated this Wnding suggests thatSardinian populations diVerentiated recently probablybecause of local immigration and population subdivisionConsistent with a recent diVerentiation is the fact that zonesof increased genetic change in Sardinia often correspond tolinguistic boundaries which cannot be more than2000 years old (Barbujani and Sokal 1991) The simplestway to reconcile these Wndings with the archaeological evi-dence of demographic growth in the Late Bronze Age is toenvisage the expansion of a small group of geographicallydispersed but maternally related individuals The succes-sive subdivision in geographically and linguistically diVer-entiated isolates enhanced the evolutionary eVects ofgenetic drift
In the multidimensional scaling of Fig 3 NuragicSardinians cluster with the majority of the European popu-lations Given the small sample size inevitable in ancientDNA studies it is at present impossible to infer their evo-lutionary relationships from mtDNA aYnities Neverthe-less in relation with ancient samples Nuragic Sardiniansappear more related to the Iberians than to the Etruscanswhose position in the graph is eccentric Three data pointsare not enough for a robust generalisation However onecan at least conclude that Sardinians and Iberians show agreater genealogical continuity with the Bronze-Ageinhabitants of the same regions than the Tuscans To betterunderstand the processes leading to these diVerences itwill be necessary to genetically characterise people wholived in those areas between 2000 years ago and the pres-ent time At present a study of paternal genealogies inHolland shows that gene Xow in the last 3 centuriesresulted in a massive population displacement (Manniet al 2005) We do not know to what extent this Wndingcan be generalised to other European populations and tomaternal genealogies although female migration is knownto be higher than male migration in most human societies(Seielstad et al 1998 Dupanloup et al 2003) Howeverthe Dutch data raise the possibility that mildly isolatedpopulations such as those of Holland and Tuscany haverecently experienced drastic demographic changes a pro-cess that has aVected only marginally more isolatedregions such as Sardinia
GENBANK accession numbers for the ancient Sardiniansequences are This paper was supported by funds from theItalian Ministry of the Universities (PRIN 2006) and byfunds from the Universities of Ferrara and Florence Wethank Antonio Torroni for a thorough preliminary discus-sion of the results of this study and three anonymous refer-ees for several useful suggestions
References
Achilli A Rengo C Battaglia V Pala M Olivieri A Fornarino SMagri C Scozzari R Babudri N Santachiara-Benerecetti ASBandelt HJ Semino O Torroni A (2005) Saami and Berbersmdashanunexpected mitochondrial DNA link Am J Hum Genet 76883ndash886
Achilli A Olivieri A Pala M Metspalu E Fornarino S Battaglia VAccetturo M Kutuev I Khusnutdinova E Pennarun E Cerutti NDi Gaetano C Crobu F Palli D Matullo G Santachiara-Benerec-etti S Cavalli-Sforza LL Semino O Villems R Bandelt HJPiazza A Torroni A (2007) Mitochondrial DNA variation ofmodern Tuscans supports the Near Eastern origin of EtruscansAm J Hum Genet 80759ndash768
Bandelt HJ Forster P Sykes BC Richards MB (1995) Mitochondrialportraits of human populations using median networks Genetics141743ndash753
Barbujani G Bertorelle G Capitani G Scozzari R (1995) Geographi-cal structuring in the mtDNA of Italians Proc Natl Acad Sci USA929171ndash9175
Barbujani G Goldstein D (2004) Africans and Asians abroad Geneticdiversity in Europe Annu Rev Genomics Hum Genet 5119ndash150
Barbujani G Sokal RR (1990) Zones of sharp genetic change in Eu-rope are also linguistic boundaries Proc Natl Acad Sci USA871816ndash1819
Barbujani G Sokal RR (1991) Genetic population structure of Italy IIPhysical and cultural barriers to gene Xow Am J Hum Genet48398ndash411
Belle EM Ramakrishnan U Mountain JL Barbujani G (2006) Serialcoalescent simulations suggest a weak genealogical relationshipbetween Etruscans and modern Tuscans Proc Natl Acad Sci USA1038012ndash8017
Cappello N Rendine S GriVo R Mameli GE Succa V Vona GPiazza A (1996) Genetic analysis of Sardinia I Data on 12 poly-morphisms in 21 linguistic domains Ann Hum Genet 60125ndash141
Caramelli D Lalueza-Fox C Vernesi C Lari M Casoli A Mallegni FChiarelli B Dupanloup I Bertranpetit J Barbujani G BertorelleG (2003) Evidence for a genetic discontinuity between Neander-tals and 24000-year-old anatomically modern Europeans ProcNatl Acad Sci USA 1006593ndash6597
Casas MJ Hagelberg E Fregel R Larruga JM Gonzalez AM (2006)Human mitochondrial DNA diversity in an archaeological site inal-Andalus genetic impact of migrations from North Africa inmedieval Spain Am J Phys Anthropol 131539ndash551
Cavalli-Sforza LL Piazza A (1993) Human genomic diversity inEurope a summary of recent research and prospects for the fu-ture Eur J Hum Genet 13ndash18
Cooper AR Poinar H (2000) Ancient DNA do it right or not at all Sci-ence 2891139
Di Rienzo A Wilson AC (1991) Branching pattern in the evolutionarytree for human mitochondrial DNA Proc Natl Acad Sci USA881597ndash1601
Dupanloup I Pereira L Bertorelle G Calafell F Prata MJ Amorim ABarbujani G (2003) A recent shift from polygyny to monogamyin humans is suggested by the analysis of worldwide Y-chromo-some diversity J Mol Evol 5785ndash97
ExcoYer LG Laval S Schneider (2005) Arlequin ver 30 an inte-grated software package for population genetics data analysisEvol Bioinform Online 147ndash50
Fraumene C Petretto E Angius A Pirastu M (2003) Striking diVeren-tiation of sub-populations within a genetically homogeneous iso-late (Ogliastra) in Sardinia as revealed by mtDNA analysis HumGenet 1141ndash10
Gilbert MTP Hansen AJ Willerslev E Rudbeck L Barnes I LynnerupN Cooper A (2003) Characterization of genetic miscoding lesionscaused by postmortem damage Am J Hum Genet 7148ndash61
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK
Hum Genet
608609610611612613614615616617618619620621622623624625626627628629630631632633634
635636637638639640641642643644645646647648649650651652653654655656657658659660661
UNCORRECTED PROOF
Handt O Krings M Ward RM Paumlaumlbo S (1996) The retrieval of ancienthuman DNA sequences Am J Hum Genet 59368ndash376
Hofreiter M Serre D Poinar HN Kuch M Paumlaumlbo S (2001) AncientDNA Nat Rev Genet 2353ndash359
Manni F Toupance B Sabbagh A Heyer E (2005) New method forsurname studies of ancient patrilineal population structures andpossible application to improvement of Y-chromosome samplingAm J Phys Anthropol 126214ndash228
Morelli L Grosso MG Vona G Varesi L Torroni A Francalacci P(2000) Frequency distribution of mitochondrial DNA haplo-groups in Corsica and Sardinia Hum Biol 72585ndash595
Nei M Kumar S (2000) Molecular evolution and phylogeneticsOxford University Press Oxford
Piazza A Cappello N Olivetti E Rendine S (1988) A genetic historyof Italy Ann Hum Genet 52203ndash213
Poinar HN Hoss M Bada JL Paumlaumlbo S (1996) Amino acid racemiza-tion and the preservation of ancient DNA Science 272864ndash866
Poinar HN Stankiewicz BA (1999) Protein preservation and DNA re-trieval from ancient tissues Proc Natl Acad Sci USA 968426ndash8431
Quintana-Murci L Veitia R Fellous M Semino O Poloni ES (2003)Genetic structure of Mediterranean populations revealed byY-chromosome haplotype analysis Am J Phys Anthropol121157ndash171
Rosenberg NA Pritchard JK Weber JL Cann HM Kidd KK Zhivo-tovsky LA Feldman MW (2002) Genetic structure of human pop-ulations Science 2982381ndash2385
Sampietro ML Caramelli D Lao O Calafell F Comas D Lari MAgusti B Bertranpetit J Lalueza-Fox C (2005) The genetics ofthe pre-Roman Iberian Peninsula a mtDNA study of ancient Ibe-rians Ann Hum Genet 69535ndash548
Seielstad MT Minch E Cavalli-Sforza LL (1998) Genetic evidence fora higher female migration rate in humans Nat Genet 20278ndash280
Semino O Passarino G Oefner PJ Lin AA Arbuzova S Beckman LEDe Benedictis G Francalacci P Kouvatsi A Limborska S Marci-kiae M Mika A Mika B Primorac D Santachiara-BenerecettiAS Cavalli-Sforza LL Underhill PA (2000) The genetic legacyof Paleolithic Homo sapiens sapiens in extant Europeans a Ychromosome perspective Science 2901155ndash1159
Serre D Langaney A Chech M Teschler-Nicola M Paunovic MMennecier P Hofreiter M Possnert G Paabo S (2004) No evi-dence of Neandertal mtDNA contribution to early modern hu-mans PLOS Biol 20001ndash0005
Tenesa A Wright AF Knott SA Carothers AD Hayward C AngiusA Persico I Maestrale G Hastie ND Pirastu M Visscher PM(2004) Extent of linkage disequilibrium in a Sardinian sub-iso-late sampling and methodological considerations Hum MolGenet 1325ndash33
Vernesi C Caramelli D Dupanloup I Bertorelle G Lari M CappelliniE Moggi-Cecchi J Chiarelli B Castri L Casoli A Mallegni FLalueza-Fox C Barbujani G (2004) The Etruscans a population-genetic study Am J Hum Genet 74694ndash704
Webster GS (1996) A prehistory of Sardinia 2300ndash500 BC SheYeldAcademic Press SheYeld
123
439 403 xxxxJournal Article MS Code
Dispatch 5707 No of Pages 10
LE TYPESET CP DISK