over the sands and far away: interpreting an iberian mitochondrial lineage with ancient western...

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Original Research Article Over the Sands and Far Away: Interpreting an Iberian Mitochondrial Lineage with Ancient Western African Origins ANTONIO F. PARDI ~ NAS, 1 JOS E LUIS MART INEZ, 2 AGUST IN ROCA, 3 EVA GARC IA-VAZQUEZ, 3 AND BEL EN L OPEZ 1 * 1 Departamento de Biolog ıa de Organismos y Sistemas, Universidad de Oviedo, Asturias 33071, Spain 2 Unidad de Ensayos Biotecnol ogicos y Biom edicos, Servicios Cient ıfico-T ecnicos, Universidad de Oviedo, Asturias 33006, Spain 3 Departamento de Biolog ıa Funcional, Universidad de Oviedo, Asturias 33006, Spain Objectives: There is an ongoing effort to characterize the genetic links between Africa and Europe, mostly using lin- eages and haplotypes that are specific to one continent but had an ancient origin in the other. Mitochondrial DNA has been proven to be a very useful tool for this purpose since a high number of putatively European-specific variants of the African L* lineages have been defined over the years. Due to their geographic locations, Spain and Portugal seem to be ideal places for searching for these lineages. Methods: Five members of a minor branch of haplogroup L3f were found in recent DNA samplings in the region of Asturias (Northern Spain), which is known for its historical isolation. The frequency of L3f in this population (1%) is unexpectedly high in comparison with other related lineages in Europe. Complete mitochondrial DNA sequencing of these L3f lineages, as well phylogenetic and phylogeographic comparative analyses have been performed. Results: The L3f variant found in Asturias seems to constitute an Iberian-specific haplogroup, distantly related to lineages in Northern Africa and with a deep ancestry in Western Africa. Coalescent algorithms estimate the minimum arrival time as 8,000 years ago, and a possible route through the Gibraltar Strait. Conclusions: Results are concordant with a previously proposed Neolithic connection between Southern Europe and Western Africa, which might be key to the proper understanding of the ancient links between these two continents. Am. J. Hum. Biol. 00:000–000, 2014. V C 2014 Wiley Periodicals, Inc. Since the first high-resolution population genetics studies it is known that all mitochondrial DNA (mtDNA) lineages of the human species have a deep origin in Africa (Chen et al., 1995; Salas et al., 2002; Scheinfeldt et al., 2010). Many questions underlying that origin have been a matter of great interest for the fields of anthropology and genetics, including the inference of prehistoric population structure (Fadhlaoui-Zid et al., 2011; Rosenberg et al., 2002), regional demographic evolution (Atkinson et al., 2009; Gignoux et al., 2011), ancient intracontinental dispersals and migra- tions (Harich et al., 2010; Quintana-Murci et al., 2008), and the occurrence and dating of the Out-of-Africa event (OOA; Fernandes et al., 2012). The relationship between the mtDNA diversity in Africa and other continents is thus a subject for continuous research, as lineages with a recent African ancestry, mostly included in paragroup L*, can be found worldwide (Behar et al., 2007). As expected, regions where these lineages are more com- mon include the Iberian Peninsula and the Middle East due to their geographical position (Amorim et al., 2005; Gonder et al., 2007), as well as many American countries due to the recent Trans-Atlantic slave trade (Wilson et al., 2012; Zakharia et al., 2009). Regarding the Iberian Penin- sula, the Gibraltar Strait, being only 13 Km of width, is a geographic passage between Africa and Europe that has been permeable at least since the Last Glacial Maximum of around 20,000 years ago (Anderung et al., 2005; Currat et al., 2010; Plaza et al., 2003). This might explain the fre- quencies of some African mtDNA haplogroups at as high as 6% in Iberia (Pino-Yanes et al., 2011), as well as their appearance as far as in Eastern Europe (Malyarchuk and Czarny, 2005; Malyarchuk et al., 2008). Setting a timeframe for the arrival of these lineages is complicated, even when molecular dating is possible (Pereira et al., 2010). From complete mtDNA sequences, and using a global database for comparison, a recent study by Cerezo et al. (2012) determined that the majority (65%) of putatively European-specific L* haplogroups were the consequence of recent introgressions, likely following the routes settled during the Roman Empire and up to the Atlantic slave-trade era. The remaining haplotypes, most of them grouped in uncommon branches of the L1 and L2 haplogroups, would have truly arisen in Europe as sug- gested by their very specific diagnostic mutations, with their ancestors arriving at the Peninsula at the beginning of the Neolithic, as early as 11,000 years ago. In the Iberian Peninsula, as described in other Euro- pean countries such as Italy (Brisighelli et al., 2012) or Russia (Morozova et al., 2012), the spatial distribution of L* haplogroups is not homogenous (Supporting Informa- tion Table S1), being probably affected by geographical, social and historical patterns (Hern andez et al., 2014). Intuitive expectations are that regions in the South should have a relatively high number of African lineages (Casas et al., 2006), while those in the North should show only minor contributions of these haplogroups (Maca- Meyer et al., 2003) or none at all (L opez-Parra et al., Additional Supporting Information may be found in the online version of this article at the publisher’s website. Contract grant sponsor: “Severo Ochoa” FICYT-PCTI Grant, the Astu- rias Regional Government; Contract grant number: BP09038. *Correspondence to: Belen Lopez Martinez, Universidad de Oviedo, Area de Antropolog ıa F ısica, Facultad de Biolog ıa. Dpto. BOS, C/ Cat- edr atico Rodrigo Ur ıa s/n, 33071 Oviedo, Asturias, Spain. E-mail: [email protected] Received 6 December 2013; Revision received 11 July 2014; Accepted 17 July 2014 DOI: 10.1002/ajhb.22601 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). V C 2014 Wiley Periodicals, Inc. AMERICAN JOURNAL OF HUMAN BIOLOGY 00:00–00 (2014)

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Page 1: Over the sands and far away: Interpreting an Iberian mitochondrial lineage with ancient Western African origins

Original Research Article

Over the Sands and Far Away: Interpreting an Iberian Mitochondrial Lineagewith Ancient Western African Origins

ANTONIO F. PARDI ~NAS,1 JOS�E LUIS MART�INEZ,2 AGUST�IN ROCA,3 EVA GARC�IA-VAZQUEZ,3 AND BEL�EN L �OPEZ1*1Departamento de Biolog�ıa de Organismos y Sistemas, Universidad de Oviedo, Asturias 33071, Spain2Unidad de Ensayos Biotecnol�ogicos y Biom�edicos, Servicios Cient�ıfico-T�ecnicos, Universidad de Oviedo, Asturias 33006, Spain3Departamento de Biolog�ıa Funcional, Universidad de Oviedo, Asturias 33006, Spain

Objectives: There is an ongoing effort to characterize the genetic links between Africa and Europe, mostly using lin-eages and haplotypes that are specific to one continent but had an ancient origin in the other. Mitochondrial DNA hasbeen proven to be a very useful tool for this purpose since a high number of putatively European-specific variants of theAfrican L* lineages have been defined over the years. Due to their geographic locations, Spain and Portugal seem to beideal places for searching for these lineages.

Methods: Five members of a minor branch of haplogroup L3f were found in recent DNA samplings in the region ofAsturias (Northern Spain), which is known for its historical isolation. The frequency of L3f in this population (�1%) isunexpectedly high in comparison with other related lineages in Europe. Complete mitochondrial DNA sequencing ofthese L3f lineages, as well phylogenetic and phylogeographic comparative analyses have been performed.

Results: The L3f variant found in Asturias seems to constitute an Iberian-specific haplogroup, distantly related tolineages in Northern Africa and with a deep ancestry in Western Africa. Coalescent algorithms estimate the minimumarrival time as 8,000 years ago, and a possible route through the Gibraltar Strait.

Conclusions: Results are concordant with a previously proposed Neolithic connection between Southern Europeand Western Africa, which might be key to the proper understanding of the ancient links between these two continents.Am. J. Hum. Biol. 00:000–000, 2014. VC 2014 Wiley Periodicals, Inc.

Since the first high-resolution population genetics studiesit is known that all mitochondrial DNA (mtDNA) lineagesof the human species have a deep origin in Africa (Chenet al., 1995; Salas et al., 2002; Scheinfeldt et al., 2010).Many questions underlying that origin have been a matterof great interest for the fields of anthropology and genetics,including the inference of prehistoric population structure(Fadhlaoui-Zid et al., 2011; Rosenberg et al., 2002), regionaldemographic evolution (Atkinson et al., 2009; Gignouxet al., 2011), ancient intracontinental dispersals and migra-tions (Harich et al., 2010; Quintana-Murci et al., 2008), andthe occurrence and dating of the Out-of-Africa event (OOA;Fernandes et al., 2012). The relationship between themtDNA diversity in Africa and other continents is thus asubject for continuous research, as lineages with a recentAfrican ancestry, mostly included in paragroup L*, can befound worldwide (Behar et al., 2007).

As expected, regions where these lineages are more com-mon include the Iberian Peninsula and the Middle Eastdue to their geographical position (Amorim et al., 2005;Gonder et al., 2007), as well as many American countriesdue to the recent Trans-Atlantic slave trade (Wilson et al.,2012; Zakharia et al., 2009). Regarding the Iberian Penin-sula, the Gibraltar Strait, being only 13 Km of width, is ageographic passage between Africa and Europe that hasbeen permeable at least since the Last Glacial Maximum ofaround 20,000 years ago (Anderung et al., 2005; Curratet al., 2010; Plaza et al., 2003). This might explain the fre-quencies of some African mtDNA haplogroups at as highas 6% in Iberia (Pino-Yanes et al., 2011), as well as theirappearance as far as in Eastern Europe (Malyarchuk andCzarny, 2005; Malyarchuk et al., 2008).

Setting a timeframe for the arrival of these lineages iscomplicated, even when molecular dating is possible(Pereira et al., 2010). From complete mtDNA sequences,

and using a global database for comparison, a recent studyby Cerezo et al. (2012) determined that the majority (65%)of putatively European-specific L* haplogroups were theconsequence of recent introgressions, likely following theroutes settled during the Roman Empire and up to theAtlantic slave-trade era. The remaining haplotypes, mostof them grouped in uncommon branches of the L1 and L2haplogroups, would have truly arisen in Europe as sug-gested by their very specific diagnostic mutations, withtheir ancestors arriving at the Peninsula at the beginningof the Neolithic, as early as 11,000 years ago.

In the Iberian Peninsula, as described in other Euro-pean countries such as Italy (Brisighelli et al., 2012) orRussia (Morozova et al., 2012), the spatial distribution ofL* haplogroups is not homogenous (Supporting Informa-tion Table S1), being probably affected by geographical,social and historical patterns (Hern�andez et al., 2014).Intuitive expectations are that regions in the Southshould have a relatively high number of African lineages(Casas et al., 2006), while those in the North should showonly minor contributions of these haplogroups (Maca-Meyer et al., 2003) or none at all (L�opez-Parra et al.,

Additional Supporting Information may be found in the online versionof this article at the publisher’s website.

Contract grant sponsor: “Severo Ochoa” FICYT-PCTI Grant, the Astu-rias Regional Government; Contract grant number: BP09038.

*Correspondence to: Belen Lopez Martinez, Universidad de Oviedo,�Area de Antropolog�ıa F�ısica, Facultad de Biolog�ıa. Dpto. BOS, C/ Cat-edr�atico Rodrigo Ur�ıa s/n, 33071 Oviedo, Asturias, Spain. E-mail:[email protected]

Received 6 December 2013; Revision received 11 July 2014; Accepted 17July 2014

DOI: 10.1002/ajhb.22601Published online 00 Month 2014 in Wiley Online Library

(wileyonlinelibrary.com).

VC 2014 Wiley Periodicals, Inc.

AMERICAN JOURNAL OF HUMAN BIOLOGY 00:00–00 (2014)

Page 2: Over the sands and far away: Interpreting an Iberian mitochondrial lineage with ancient Western African origins

2009). However, African lineages do occur in northern Ibe-ria, some of them being shared with southern regions(Alvarez et al., 2010), while others appearing as rare out-liers (Cerezo et al., 2012).

A remarkable case is the north-western coastal regionof Asturias, considered by historical sources to have suf-fered a certain demographic isolation until the 19th–20thcenturies (Infiesta, 2005) and a low overall input of medie-val Arab emigration (Ruiz de la Pe~na, 1979). Recently,both facts were suggested in an analysis of the mitochon-drial hypervariable segment 1 (HVS1) of an ancestry-controlled sample of 429 volunteers (Pardi~nas et al.,2012b). Among the seven individuals from L* lineagesthat were found, five could be ascribed to the uncommonhaplogroup L3f1b4a, which was later supported by fullcontrol-region sequencing (Pardi~nas et al., 2012a).

This haplogroup was first described in a South Africanindividual, with related haplotypes scattered across theAfrican continent reaching even the Middle East (Beharet al., 2008; Cerny et al., 2009). Its ancestor, L3f, seems tohave arisen in Eastern Africa around 50,000 years ago,and shows three branches well-defined in terms of theirdistribution inside the African continent, where they canamount to 10% of the local mtDNA frequency (Soareset al., 2012). By contrast, they are extremely rare outsideAfrica with the exception of African-Americans (Cernyet al., 2009). So far, and to our knowledge, L3f lineageshave been only sporadically reported in European popula-tions, most of them based in control-region motifs andaccounting to one or two samples per study (Casas et al.,2006; Irwin et al., 2007; Karachanak et al., 2012; Prietoet al., 2011; Turchi et al., 2008). Thus, the five members ofa very particular branch of this haplogroup found in Astu-rias provide a quite exceptional opportunity to delve deeperinto the molecular characteristics of this mtDNA lineage.This examination will elucidate the details and timing ofits entry into Iberia, and expand our knowledge of the his-torical genetic exchanges between Africa and Europe.

MATERIALS AND METHODS

This study was approved by the Research and EthicsCommittees of the Central University Hospital of Astu-rias and the University of Leon (Spain). All sample donorsgave written informed consent prior to their recruitment,according to the Spanish Law for Biomedical Research(Law 14/2007–July 3rd).

Full mtDNA amplification and sequencing

Samples used for the study were collected for a previousstudy using buccal-swabs and a Chelex-extraction proto-col (Pardi~nas et al., 2012a,b), after which they werenamed AST022, AST227, AST233, AST347 and AST577.All five had been ascribed to haplogroup L3f1b4a onthe basis of their control-region motifs, which showedtwo different haplotypes separated by two mutations:16209C-16223T-16311C-16355T-16519C-73G-189G-200G-263G-309.1C-315.1C-523delA-524delC (AST022) and16209C-16223T-16311C-16519C-73G-189G-200G-263G-315.1C-523delA-524delC (all the others). The five sampledonors came from different towns, although two of themwere from the same region inside Asturias. Nevertheless,ancestor birthplaces and surnames reported during thesampling process showed no family connections betweenany of them up to two generations ago.

In this study, the five samples were chosen for completemtDNA sequencing using the revised protocol by Ramoset al. (2009, 2011). In this procedure, the mtDNA genomeis divided in nine overlapping fragments, using oligonu-cleotides specifically designed to prevent the co-amplification of nuclear insertions of mitochondrial origin(NUMTs). This step, coupled with double-strand amplifi-cation for each fragment using internal and flanking pri-mers, minimizes several sources of incorrect base-calls onthe resulting data (Parson and Bandelt, 2007; Yao et al.,2008). The protocol could not be completed on the AST233sample due to the low DNA quantity available in thesource material, and its partial sequence was not used infurther analyses. The other four samples yielded high-quality complete sequences. All the sequencing reactionswere carried out in an ABI PRISM 3730xl Genetic Ana-lyzer by the Sequencing Service of the Scientific-TechnicalServices (SCTs) of the University of Oviedo.

Haplogroup assignment

The newly generated complete mtDNA sequences of theAsturian samples were analyzed with the softwaremtPHYL v4.015 (Eltsov and Volodko, 2012) to assess theirdifferences to the revised Cambridge Reference Sequence(rCRS). Afterwards, HaploGREP (Kloss-Brandst€atteret al., 2011) was used to obtain an updated haplogroupassignment. For referencing purposes, version 16 of themtDNA phylogenetic tree was consulted (van Oven andKayser, 2009).

Phylogeny estimation

For estimating the phylogenetic position of the Asturiansamples, a total of 113 complete mtDNA sequences per-taining to paragroup L3f* were downloaded from Gen-bank, as described in Supporting Information Table S2.The software suite BEAST v1.80 (Drummond et al., 2012)was used to compute a Bayesian tree, using the partitionmodel and mutation rates described by Fu et al. (2013).Best fitting mutation models for our dataset in each of thepartitions were calculated using the Bayesian Informa-tion Criterion (BIC) implemented in the jModeltest v2software (Darriba et al., 2012). The BEAST analysis wasrun with a Monte-Carlo-Markov-Chain (MCMC) of40,000,000 steps on two different coalescent models,assuming constant or expanding population size. Likeli-hood of both models was compared after the analysis bythe Bayes Factor procedure implemented in the softwareTracer v.1.6 (Suchard et al., 2001). After comparison, theexpanding model obtained a slightly better statistical sup-port (BF 5 1.966), and thus it was used for dating and fur-ther analyses. Its maximum clade credibility (MCC) treewas obtained with the TreeAnnotator tool using a credibil-ity cutoff of 95% and a burn-in of 2,500 trees out of thetotal 10,000.

Phylogeographic reconstruction

To estimate the geographic diffusion and divergence ofbasal L3f1b lineages directly related to the Asturian sam-ples, we ran the dispersal-extinction-cladogenesis model(DEC; Ree and Smith, 2008) as implemented in the RASPv2.1 software (Yu et al., 2013). This is a particular case ofa maximum likelihood ancestral-state estimator algo-rithm (Pagel, 1999), that can be used in phylogenies forwhich the geographic origins of each terminal node are

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known and where the nodes might have undergone rangeexpansions or contractions in the past. The main assump-tion is that the range of a species (or lineage), in theabsence of divergence, can change by either dispersal(expansion) or contraction (extinction). When divergenceoccurs after a range expansion, one daughter species willinherit a part of its ancestor range, with the other inherit-ing the remainder, and the coupling of this process withevolutionary bifurcations allows it to be traced backwardsthrough the phylogeny (Ree et al., 2005). For this analy-sis, the complete MCC tree generated by BEAST wasused. Locations for the Genbank sequences were obtainedfrom the literature (Supporting Information Table S2)and divided in seven geographic regions (East Africa,Central Africa, West Africa, South Africa, North Africa,Middle East, and Europe). As the DEC model evaluatesthe available range dispersal possibilities using a regionalconnectivity parameter, this was set to one for geographi-cally adjacent locations and to zero otherwise, as recom-mended to reduce computational burden and increaseaccuracy (Ree and Smith, 2008).

Demographic history assessment

Historical population growth trends were inferred inBEAST with the extended Bayesian Skyline procedure(Drummond et al., 2005) using a subset of the originalL3f* dataset that included only sequences from paragroupL3f1b*, including the reported Asturian data. For thisanalysis, a Monte-Carlo-Markov-Chain (MCMC) with40,000,000 steps was used, and the whole procedure wasrun in the public CIPRES server (Miller et al., 2010).Effective population sizes were calculated from the sky-line plot (eBSP) considering a generation time of 25 years(Fenner, 2005).

African continental-wide frequency assessment

For an estimation of the frequency of L3f1b* in the Afri-can continent and other geographical regions of interest,the forensic EMPOP database release 11 (Parson andD€ur, 2007) was surveyed using the control-region motif ofthe Asturian sequences without the poly-C stretches ofthe hypervariable segment 2 (HVS2). We used this data-base because of its stringent quality-control criteria andavailability of data from scarcely sampled countries (Car-racedo et al., 2010). Frequency estimations for Africanregions (Northern, Central, Eastern, Western), the MiddleEast and Southern Europe were performed using thePN 1 1 method included in the database interface (Roewerand Parson, 2013).

RESULTS

Phylogenetic analyses

After complete mtDNA sequencing, 28 nucleotide dif-ferences with the rCRS were found in the coding regionof all four samples. Twenty-seven of them clearly definedhaplogroup L3f1b, according to HaploGREP. This resultupdates and corrects the previous assignment toL3f1b4a, which was an artifact caused by a mutation atposition 16292 shared between both lineages, whichmakes the control-region unspecific for discriminatingbetween them. The remaining coding mutation, whichwas termed “local private” by HaploGREP, is the synony-mous transition G8994A. It seems to have occurred sev-eral times during mitochondrial evolution, being found

in different lineages and constituting even one of theclassical diagnostic mutations of haplogroup W (Macau-lay et al., 1999; Pike et al., 2010). This mutation was alsopresent in the AST233 partial sequence, confirming thatall five Asturian samples are members of the same line-age. Complete mtDNA genomes were submitted toGenbank (accession numbers KF011502, KF011503,KJ959229, and KJ959230).

The obtained sequences were compared with other pub-lished L3f samples, with the most frequent haplotypebeing identical to JQ703621.1, found also in Spain (Beharet al., 2012). Interestingly, the maternal ancestor of theJQ703621.1 donor comes from Leon, a neighboring regionto Asturias (Doron M. Behar, personal communication).These sequences seem to form a distinctive lineagetogether with AST022, which differs from them in thecontrol-region insertion 309.1C and transition C16355T,both commonly considered “speedy” mutation hotspots(Fig. 1; Bandelt et al., 2002). The resulting haplogroupwas termed L3f1b6 and will be included in a futurerelease of Phylotree (Mannis van Oven, personal commu-nication), and its median coalescent age was estimated tobe of 3,138 years (95% HPD: 715–6,650). Additionally, the

Fig. 1. Phylogeny of Spanish L3f1b6 lineages based in differencestowards the rCRS. The four lineages from Asturias are includedtogether with the JQ703621.1 haplotype collected in Leon (North-western Spain) by Behar et al. (2008). Haplogroup names are shownin red. Underlined mutations are back-mutations and those in bluebelong to the control-region.

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Bayesian phylogeny showed that the most recent commonancestor (MRCA) of L3f1b6 with the nearest clade (Sup-porting Information Fig. S1), which includes Levantine(EU092741.1) and Iberian (JN214434.1) sequences, canbe dated to 8,217 years ago (95% HPD: 5,441–13,012). TheNetwork analysis did not show any clear geographicalgrouping for L3f1b6 or its closest branches, as it does forother clades (Fig. 2), but highlighted a star-like patternfor the whole L3f1b* cluster, which is the typical signa-ture of a past demographic expansion (Donnelly, 1996).

Phylogeographic analyses

As the information revealed by the geographicaladscription of each sample is, by itself, insufficient toresolve the origin of the clade from which the Asturianlineages arose, the DEC procedure was used to estimatean origin for each ancestral node (Supporting Informa-tion Table S3). Through this method, a Western Africanorigin could be inferred for the entire L3f1b* paragroup(Fig. 3; node 0), in which several dispersal and vicarianceevents during recent history have led to a continent-widedistribution. This origin hypothesis is also in agreementwith the frequency patterns found in EMPOP using theL3f1b6 control region motif, which could be presumablyshared with other L3f1b* lineages (Table 1). As for thefinding of the L3f1b6 haplogroup in Spain, a migrationfrom and through Northern Africa, as pointed out by theDEC (Fig. 3: nodes 11, 12), seems the most parsimoniousexplanation, given the lack of similar findings in Europe.Temporally, this arrival would fall within a broad10,000–20,000 years interval, one compatible with atimeframe shown by the eBSP as a steady growth ineffective population size for the whole L3f1b* paragroup(Fig. 4).

DISCUSSION

After complete mtDNA characterization, the AsturianL3f1b6 lineages seem to be part of a putatively Iberian(perhaps even Southern European) haplogroup. Takinginto account the maternal ancestry of the JQ703621.1sequence, we can explain the relatively high �1% fre-quency in Asturias as a by-product of the historical isola-tion of the Iberian north, as has been noted for otherhaplogroups affected by the resulting genetic drift (Garciaet al., 2011; Pardi~nas et al., 2012b). Nevertheless,although a recent migration cannot be completely dis-carded, regional African frequencies suggest an ancientorigin for this lineage, even considering the isolationeffect. Based on the coalescent estimates, we canplace such an origin in Northern Africa around at least

Fig. 2. Network median-joining analysis of complete mtDNAgenomes of L3f* samples, centered on the L3f1b* paragroup. Branchlength is proportional to number of substitutions. Poly-C stretches inHVS1 and HVS2 have not been taken into account to avoid excessivecomplexity and reticulation. Spanish L3f1b6 haplotypes are situatedin the nodes marked by a red arrow. Geographic affiliation of allnodes is color-coded (Orange: Europe; Red: North Africa; Purple: Mid-dle East; Green: West Africa; Blue: Central Africa; Yellow: EastAfrica; Brown: South-Africa, White: USA/Unknown; Gray: Inferredhaplotype).

Fig. 3. DEC reconstruction of the geographic origin of the L3f1b*paragroup, with most likely geographic affiliations indicated insidethe nodes as a single location or a combination of two (A: CentralAfrica; B: East Africa; C: West Africa; D: South Africa; E: NorthAfrica; F: Middle East; G: Europe). Less likely states for each nodeand their probabilities are shown in Supporting Information TableS3. Note that the L3f1b6 sequences are identical with exception ofAST022, and thus the bifurcations on their nodes reflect phylogeneticuncertainty.

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8,000 years ago, possibly associated with the east-westpopulation movements that shaped the mitochondrial pro-file of both North Africa and the Levant (Fadhlaoui-Zidet al., 2004, 2011). From there, an even deeper originwould be in Western Africa, where its radiation mighthave followed other L3 clades, which are known to haveexpanded from here during the advantageous climaticconditions that spanned several millennia (Brooks et al.,2005; Soares et al., 2012).

As a cautionary note, coalescent and arrival times can-not be related in a simple manner for a given lineage, astheir relationship is highly dependent on the structureand characteristics of the population in which the lineagearose (Oppenheimer, 2012). Phylogeographic methods,even though they can offer useful insights, are not devoidof methodological problems, especially when large-scalepatterns are to be interpreted (Barbujani and Goldstein,2004; Jobling, 2012). Thus, one can reasonably regard coa-lescent dates only as a rough estimate of the minimumarrival time of a founder lineage (Pereira et al., 2010).This would place our inferred transcontinental migrationin the Early Neolithic, when several pastoralist societiesof southern Spain and Northern Africa showed similarcultural traits and human contacts resulting from herdingand fishing expeditions might have taken place over theGibraltar Strait (Anderung et al., 2005; Straus, 2001).

However, is also necessary to consider that in scenarioswhich combine demographic and range expansions, such

as those described here, as divergence times might beunderestimated by the action of founder effects on geneticdrift. This phenomenon has recently been termed “M-buffer” by Boivin et al. (2013), and is based on the highinitial frequency of a founder haplotype, which causes itspersistence in the population for a high number of genera-tions. As this is translated in the lack of observable muta-tions in respect to other related haplotypes, its effect indating is akin to a “pause” of the molecular clock. This hasbeen originally inferred for the expansion of haplogroupM in Asia, a well-known founder effect coupled to a demo-graphic expansion (Macaulay et al., 2005), and the samephenomenon may possibly have acted in particularbranches of L3f1b* during its continental and transconti-nental dispersal. Were this to have happened in theL3f1b6 lineage, the minimum boundary for its Iberianarrival might have to be pushed backwards, probably todates near the �15,000 years ago shown in the eBSP anal-yses as the start of the demographic growth for the wholeparagroup. Taking these findings into account, thebroader timeframe described in the present work corre-sponds to a period of fluid population connectivitybetween Africa and Europe, as shown by other studies onmtDNA (Achilli et al., 2005; Cerezo et al., 2012;Malyarchuk et al., 2008).

CONCLUSIONS

The finding of a highly scarce branch of haplogroup L3fin Northern Spain has provided us with an illustration ofthe complex profile of African mitochondrial DNA intro-gression into the Iberian Peninsula. While pre-NeolithicEuropean-specific lineages had been previously inferredfor paragroups L1* and L2*, they were less clear for L3*,a lineage known for its rapid and successful dispersalthroughout Africa (Soares et al., 2012). In the presentwork we have shown that, together with the ItalianL3d1b1a (Cerezo et al., 2012; Soares et al., 2009), there isanother strong candidate for an ancient European L3*clade, which unexpectedly belongs to a rare branch of theL3f haplogroup, thus far unknown outside of Spain. Both

Fig. 4. Bayesian Skyline plot of L3f1b* lineages assuming a generation time of 25 years.

TABLE 1. Frequency of the control-region motif of the most frequentL3f1b6 haplotype (73G, 189G, 200G, 263G, 315.1C, 523DEL,

524DEL, 16209C, 16223T, 16311C, and 16519C), in the EMPOP r11database

Region EMPOP samples Frequency (95%CI)

Southern Europe 1711 0.06% (0.01–0.33)Middle East 1025 0.10% (0.02–0.55)Northern Africa 914 0.11% (0.02–0.62)Eastern Africa 551 0.18% (0.03–1.02)Central Africa 244 0.41% (0.07–2.28)Western Africa 191 0.52% (0.09–2.90)

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the arrival timeframe and the location of the ancestralclades are in agreement with the findings of Cerezo et al.(2012) for L1b1a8 and L2a1k, which pointed towardsWestern Africa as the source of a possible prehistoricalmigration to Southern Europe. If the magnitude of thismigration and the demography of the population involvedin it are to be assessed, further surveys and analyses ofuniparental lineages with African origins will have to beundertaken in regional and local European populations.

ACKNOWLEDGMENTS

This is a contribution from the Marine AnthropoceneResearch group of the Marine Observatory of Asturias.The authors are grateful to Antonio S�anchez Palacio forhis assistance during experimental design; to Dr. BruceWinney for his assistance with amplification and sequenc-ing protocols; to Dr. Doron M. Behar for additional dataregarding the Spanish JQ703621.1 sample; and to Dr.Mannis van Oven for his helpful comments and advicetowards including the L3f1b6 lineage in Phylotree.

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