doi: 10.1111/j.1469-1809.2007.00421.x a perspective on the

doi: 10.1111/j.1469-1809.2007.00421.x

A Perspective on the History of the Iberian GypsiesProvided by Phylogeographic Analysis of Y-ChromosomeLineages

A. Gusmao1, L. Gusmao1,∗, V. Gomes1, C. Alves1, F. Calafell2, A. Amorim1,3 and M. J. Prata1,3

1Ipatimup, Instituto de Patologia e Imunologia da Universidade do Porto. R. Dr. Roberto Frias, s/n. 4200-465 Porto. Portugal2Unitat de Biologia Evolutiva, Departament de Ciencies Experimentals i de la Salut, Universitat Pompeu Fabra. 08003 Barcelona,Catalonia, Spain3Faculty of Sciences, University of Porto, Pr. Gomes Teixeira, 4099-002 Porto. Portugal

Summary

The European Gypsies, commonly referred to as Roma, are represented by a vast number of groups spread across many

countries. Although sharing a common origin, the Gypsy groups are highly heterogeneous as a consequence of genetic

drift and different levels of admixture with surrounding populations. With this study we aimed at contributing to the

knowledge of the Roma history by studying 17 Y-STR and 34 Y-SNP loci in a sample of 126 Portuguese Gypsies.

Distinct genetic hallmarks of their past and migration route were detected, namely: an ancestral component, shared by

all Roma groups, that reflects their origin in India (H1a-M82; ∼17%); an influence from their long permanence in the

Balkans/Middle-East region (J2a1b-M67, J2a1b1-M92, I-M170, Q-M242; ∼31%); traces of contacts with European

populations preceding the entrance in the Iberian Peninsula (R1b1c-M269, J2b1a-M241; ∼10%); and a high proportion

of admixture with the non-Gypsy population from Iberia (R1b1c-M269, R1-M173/del.M269, J2a-M410, I1b1b-M26,

E3b1b-M81; ∼37%). Among the Portuguese Gypsies the proportion of introgression from host populations is higher than

observed in other groups, a fact which is somewhat unexpected since the arrival of the Roma to Portugal is documented

to be more recent than in Central or East Europe.

Keywords: Portuguese Gypsies, Gypsy diaspora, Roma, Y-chromosome lineages, Y-SNP haplogroups, Y-STR haplo-

types

Introduction

Portugal is the westernmost region reached by the Gypsydiaspora after the Roma people arrived in Europe 600–700 years ago (Liegeois, 1989; Fraser, 1998; Kendrick &Puxon, 1998). The establishment of Gypsy groups in Por-tugal is recorded since the second half of the 15th centuryand at present they are estimated to amount to 30–50 thou-sand individuals. The Portuguese Gypsies are likely to bea branch of the group that crossed the Pyrenees as earlyas the first quarter of the 15th century and rapidly spreadthroughout Spain and Portugal. Contrary to many otherRoma, the Iberian Gypsies, known as Gitanos in Spainand Ciganos in Portugal, are non-Romani-speakers. Af-

∗Corresponding Author: Leonor Gusmao, Ipatimup Rua Dr.Roberto Frias, s/n 4200-465 Porto, Portugal. Tel: +351 22 5570700;Fax: +351 22 5570799; E-mail address: [email protected]

ter entering Iberia they progressively lost the original lan-guage, and while nowadays many Gitanos still speak Calo,which is basically Spanish with a large amount of Romaniloan words, the Ciganos from Portugal speak Portuguesewith the Calo just being a reminiscent reference language(Fraser, 1998).

The Roma in Portugal, as indeed the Roma elsewhere,are a transnational genetic isolate which fulfil the prop-erties that make genetic isolates an interesting resource ingenetic epidemiology; namely, they have reduced geneticdiversity and increased linkage disequilibrium (Gonzalez-Neira et al. personal communication). The Roma thereforepresent a particular genetic disease spectrum, with someprevalent diseases almost absent, others specific to Gypsies,and others with private Roma mutations (for a review,see Kalaydjieva et al. 2001b). Presently in Portugal, Gypsycommunities are spread all over the country and representindeed a conspicuous component of the Portuguese social

C© 2008 The AuthorsJournal compilation C© 2008 University College London

Annals of Human Genetics (2008) 72,215–227 215

A. Gusmao et al.

and demographic landscape. Despite that, little is knownabout the Portuguese Gypsies. How are they related withother Gypsy groups? To what extent interaction with otherPortuguese contributed to change the founder gene pool?What signs do they still retain from the complex historythat pre-dated their entrance in Iberia?

Such questions cannot be satisfactorily answered byapplying conventional historical approaches, because theproper sources are almost non-existent. As happens withother Roma groups, the Ciganos from Portugal lack a writ-ten history of their own and the documentary sources fromnon-Gypsies (Coelho, 1996) are limited to episodic refer-ences.

The absence of records constitutes a major complicationin Gypsy studies and explains why the most instructiveevidences about the original homeland of the proto-Gypsies have resulted from linguistics and genetics. Lin-guistics has provided compelling evidence of an ancestralorigin in the Indian subcontinent (Liegeois, 1989; Fraser1998; Kendrick & Puxon, 1998). Evidence for this hypoth-esis has been consistently found in genetic analyses (e.g.Gresham et al. 2001; Morar et al. 2004; Malyarchuk et al.2006), which, although essentially centred in non-IberianGypsies, have further revealed a particular high level ofsub-structuring among Gypsy groups, this is in sharp con-trast to the surrounding relatively uniform European pop-ulations. Small population size, strong drift effects, limitedinter-group gene flow and differential admixture with thehost populations, seem to have acted together leading tostrong genetic differentiation between Gypsy groups. Thismeans that no group of Gypsies can be representative ofother groups, which consequently makes the group thebasic unit of genetic research, as it is of social organisation(Fraser, 1998). In view of this and in order to address thequestions raised above about the Portuguese Ciganos, inthis study we firstly examined a sample of 126 unrelatedGypsy males with a high-resolution Y-chromosome STRand SNP typing strategy; secondly we exploited the resultsin the context of previously published data on other Gypsygroups and respective host populations, and finally we triedto unfold layers of genetic male lineages in the PortugueseGypsies to retrieve information about major events in theirdemographic history.

Material and Methods

Sample Collection

Blood samples were taken under informed consent from 126unrelated Gypsy males, from 18 different communities in 11 dif-ferent Portuguese districts. Personal inquiries were made to eachindividual in order to avoid close kinship. Genomic DNA wasextracted according to standard phenol-chloroform method.

Y-STR Typing

Seventeen Y-STR loci (DYS19, DYS385, DYS389I, DYS389II,DYS390, DYS391, DYS392, DYS393, DYS437, DYS438,DYS439, DYS448, DYS456, DYS458, DYS635 and GATAH4.1) were amplified using the AmpFlSTR Yfiler PCR Am-plification Kit (AB Applied Biosystems), following the manufac-turers’ instructions. Genotypes were produced with a 310 Ge-netic Analyser (AB Applied Biosystems), and by comparison toreference sequenced ladders provided with the kit. The nomen-clature was according to the ISFG recommendations (Gusmaoet al. 2006) and alleles for GATA H4.1 locus were named byadding nine repeats to the allele numbering of the typing kit(Mulero et al. 2006).

Y-SNP Typing

Thirty-four Y-SNP markers were hierarchically typed, in order todefine the major male lineages (Figure 1). All samples were typedfor the biallelic markers SRY1532, M213, M9, M70, M22, Tat,92R7, M173 and P25, as previously described by Brion et al.(2005). For the remaining SNPs, the following typing schemewas used:

- M201, M170, 12f2, M26, M62 and M172 were typed in samplescarrying the M213 mutation and lacking M9, as in Brion et al.(2005);- M96, M35, M78, M81, M123 and M34 were typed in samplescarrying only the SRY1532 mutation, as previously described byBrion et al. (2005);- M242 was tested in one individual that fell within haplogroupP (xR1). This mutation was typed by SNaPshot according toBlanco-Verea et al. (2006);- M269, M18 and M73 were screened in all individuals withinR1∗ haplogroup. M18 and M73 were typed by SNaPshot usingthe primers described in Brion et al. (2005). M269 was typed byRFLP (Supplementary Table S1);- F∗ (xGIJK) individuals were screened for M69, M52, M82and Apt mutations in a newly implemented multiplex SNaPshotreaction (Supplementary Table S1);- Samples carrying M172 were tested for M410, M67, M92,M12 and M241 using also SNaPshot methods (SupplementaryTable S1).

The YCC (2002) and Jobling & Tyler-Smith (2003) nomen-clature was updated according to Cruciani et al. (2004); Senguptaet al. (2006); Semino et al. (2004) and Cinnioglu et al. (2004).

Data Analysis

Haplotype diversity, mean number of pairwise differences andpairwise genetic distances (Rst, for STR-haplotypes and Fst forSNP-haplogroups) were calculated using the Arlequin softwarever. 3.01 (Excoffier & Scheinder, 2005). In genetic distanceanalyses, DYS385 was not considered, and the number of re-peats in DYS389I was subtracted from DYS389II. Haplogroupfrequencies were estimated by direct counting.

216 Annals of Human Genetics (2008) 72,215–227 C© 2008 The AuthorsJournal compilation C© 2008 University College London

A Perspective on the History of the Iberian Gypsies

Figure 1 Phylogenetic network of Y-SNP haplogroups, indicating the biallelic markers typed in the present

work and their absolute frequencies in a sample of 126 Portuguese Gypsies. See Adams et al. (2006) for details

on P25 recurrence.

The YHRD - Y Chromosome Haplotype Reference Database(Willuweit & Roewer, 2007), URL: www.ystr.org, release22 from August 10, 2007, was used for haplotype matchanalysis.

Phylogenetic median-joining networks of Y-STR haplotypesinside haplogroups were constructed using Network 4.2.0.0(www.fluxus-technology.com).

Results and Discussion

Y-STR Haplotype Diversity

The analysis of 17 Y-STR loci in the Portuguese Gypsiesrevealed 52 different haplotypes yielding a proportion of

42% of distinct lineages (Supplementary Table S2). Out ofthese, 38 were found only once (73.1% of unique haplo-types) while the remaining 14 were shared by two or moremales. Three of the shared haplotypes were very well rep-resented (H18-7.9%; H21-15.9% and H24-13.5%) reach-ing unusually high values in comparison to those foundin most populations for lineages defined by such a highnumber of Y-STRs (e.g. Alves et al. 2007; Turrina et al.2006; Berger et al. 2005; Soltyszewski et al. 2007). In mostEuropean populations haplotype diversity is higher than99.9%, in contrast with the low value now detected in thePortuguese Gypsies (0.9437 ± 0.0107).

Previous studies (Gresham et al. 2001; Zaharova et al.2001; Ploski et al. 2002; Nagy et al. 2007; Furedi et al.



A. Gusmao et al.

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Figure 2 Correlation between genetic and geographic distances in Roma and non-Roma populations.

1999; Pericic et al. 2005a; Kalaydjieva et al. 2001a) al-ready had assessed Y-STR diversity in other Roma groups(see Table 1) using, however, very distinct sets of loci. Onlyseven Y-STRs were common to all studies, namely DYS19,DYS389 I, DYS389 II, DYS390, DYS391, DYS392 andDYS393. In view of that, comparisons with Roma andtheir host populations (which were available for all exceptSlovakia) were based exclusively upon those seven loci.Haplotype diversity in the Portuguese Gypsies, using thesemarkers, was now 0.9097 ± 0.0121, fitting well in therange of values observed in other Roma groups, whichvaried between a minimum of 0.8430 ± 0.0218 in Bul-garian Gypsies and a maximum of 0.9200 ± 0.0373 inSpanish Gypsies, both studied by Gresham et al. (2001). Y-STR diversity in each Gypsy group was remarkably lowerthan in any non-Gypsy host population where diversitieswere always higher than 0.98.

The reduced diversities in all Gypsy groups are signs ofdrift effects acting in their pool of Y-STR lineages andsuch effects can be explained by the small population sizeof the Roma groups, together with the deeply rooted en-dogamy that characterises the Gypsy people (Kalaydjieva etal. 2001b; Gresham et al. 2001).

Pairwise Genetic Distance Analysis

Pairwise genetic affinities between Gypsy and non-Gypsyhost populations were evaluated through Rst distances.On average, the highest genetic distances were observedin comparisons involving a Gypsy and a non-Gypsypopulation (Table 1), with all values reaching statisticalsignificance. Among the non-Gypsy pairs, absence of dif-ferentiation was only detected between Spain/Portugal,Bulgaria/Macedonia and Bulgaria/Hungary, which de-notes an Y-STR sub-structuring pattern visibly influencedby geographical distance. Geography does not seem to playsuch a linear role in the inter-group differentiation be-tween Gypsies. In fact the Spanish Gypsies neither differedfrom their Iberian neighbours, the Portuguese Gypsies, nor

from the distant Lithuanian or Slovakian groups. Accord-ingly, while genetic distances in the European host popula-tions were found to be correlated with geographic distance(Figure 2), in agreement with the general picture widelyreported for Europe (Rosser, 2000), the same was not ob-served in the Gypsy groups (r2 = 0.0499; P = 0.2529;Figure 2) as previously reported by Gresham et al. (2001)in a study restricted to Gypsies from Bulgaria, Spain andLithuania.

The overall structure of the distance matrix can bevisualised by means of multidimensional scaling (MDS,Fig. 3), in which Roma and non-Roma groups are clearlyseparated. Such separation is statistically significant: the dif-ference between groups accounted for 14.8% of the to-tal genetic variance (as computed with AMOVA; p =0.001), and was larger than that within groups (8.0%,p < 0.001). Differentiation among Roma (Fst = 8.71%,p < 0.000) was larger than among non-Roma (Fst =7.70%, p < 0.000). The high genetic distances observed be-tween Roma groups pinpoints the fast differentiation thatGypsy populations experienced during their dispersion inEurope.

Y-SNP Haplogroup Variation

In the Portuguese Gypsies, 17 different Y-SNP haplogroupswere detected (Figure 1) from which five accounted for88% of their chromosomes, namely R1b1c-M269, J2a1b-M67, J2a-M410, H1a and I-M170.

Comparing the Y-SNP profile of Gypsy and non-GypsyPortuguese populations (Beleza et al. 2003) highly signifi-cant differences were observed (Fst = 0.152; P < 0.001).What mainly differentiates them is the low prevalence ofR1b1c in non-Gypsies (∼60% against 27%), the muchhigher frequency of J lineages (∼40% versus ∼10%) as wellas the absence of H haplogroup in non-Gypsies, whereasin the Gypsy sample it reached a proportion nearing 17%.

Furthermore, although the number of different hap-logroups in both samples is similar, the frequency


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Table 1.

distribution in Gypsies was much less monotonous than innon-Gypsies, as indicated by haplogroup diversities that are0.8367 ± 0.0138 and 0.6520 ± 0.0206, respectively. Thehigh haplogroup diversity in Gypsies can be explained byadmixture during the history of the group, which can besimplistically outlined as encompassing three main phases:the period beyond the early establishment and fragmenta-tion of founder groups in Europe, possibly in the Balkanregion (since it represented the fons et origo of Roma in Eu-rope; Fraser 1998), the fast journey towards Western Eu-rope, and the arrival in Iberia and subsequent settlement inPortugal.

However, haplogroup diversity does not reflect otherrelevant demographic processes, such as founder effects,although they are quite evident when comparing levelsof diversity within haplogroups assessed from STR vari-ability. Dramatically low diversities characterise most ofthe haplogroups present more than once in the sample(Supplementary Table S2), being particularly clear within(i) J2a∗ (xJ2a1b)-M410, in which 20 out of 21 chromo-somes share exactly the same 16-STR haplotype; (ii) the25 J2a1b∗ (xJ2a1b1)-M67 lineages, where 17 individualsbelong to the same STR background and 5 belong to adifferent one; or (iii) I∗ (xI1b1b)-M170, with 10 out of 12individuals sharing a given haplotype.

Phylogeographical Analyses

Haplogroup HHaplogroup H, defined by the M69 mutation (Underhillet al. 2001), possibly arose in the boundaries of present-

day India (Sengupta et al. 2006) where it is widespreadthroughout the entire subcontinent, reaching the highestfrequencies in Central and Southern India. Being a majorhaplogroup in all European Roma but absent in their hostpopulations, it provides clear genetic evidence of the an-cestral geographical origin of the proto-Gypsies (Greshamet al. 2001; Kalaydjieva et al. 2001a).

Haplogroup H was one of the most represented inour sample (16.7%). All individuals inside this haplogroupwere H1a, carrying both the M52 and M82 mutations.Figure 4A depicts the network constructed after recover-ing the STR information currently available on H1 chro-mosomes of different Gypsy groups (Gresham et al. 2001;Kalaydjieva et al. 2001a). It shows a star-shaped networkwhere an outstandingly high number of chromosomes liein a modal haplotype, shared by all Gypsy groups, and theremaining differing from the modal by no more than twomolecular steps. Taking together all Gypsy H1 chromo-somes, the internal STR diversity was remarkably reducedwith a haplotype diversity of 0.398 ± 0.049 and a meanvariance across the seven STR loci of 0.033. This patternof STR diversity within H1 is a clear indication that aprofound bottleneck happened in the most distant past ofthe Gypsy people, suggesting that, in full accordance withGresham et al. (2001), a remarkably small number offounders must have been present in the ancestral groupfrom which new Roma populations emerged.

When searching for matches with the Gypsy H1 modalhaplotype (DYS19∗15, DYS389I∗14, DYS389II∗30,DYS390∗22, DYS391∗10, DYS392∗11 and DYS393∗12)it was possible to observe that it was shared by two tribalgroups from Southern India, being rather common among



Figure 4 Median-joining networks of Y STR haplotypes within haplogroups H1, J2a1b∗ (xJ2a1b1), I and R1∗ of Gypsy

populations. For each haplogroup the area of the circles is proportional to the number of individuals; the smallest area is

equivalent to one individual. Within the pie-charts, orange refers to Portuguese, blue to Bulgarian (Gresham et al. 2001 and

Kalaydjieva et al. 2001a), yellow to Spanish, and red to Lithuanians (Gresham et al. 2001). Arrows assign lineages possessing

the Dinaric modal haplotype (DMH) or the Nordic modal haplotype (NMH). ∗ identifies the Portuguese individual

belonging to I1b12.

one of them (Kivisild et al. 2003). Additionally, four exactmatches for the Gypsy H1 haplotype extended to threemore markers (DYS437∗14, DYS438∗9 and DYS439∗11)were also found among individuals belonging to castesof Indian warriors, two of them from the North (Zerjalet al. 2007). These findings illustrate how difficult it is toaddress the questions: which specific Indian region andsocial stratum did the first Gypsies arise? The widespread,although uneven, distribution of the Gypsy H1 modalhaplotype in different Indian populations (either castes or

tribes from North or South), means that it is not possibleto exclude any scenario for the Indian source populationof the first wave of migration.

Portuguese Gypsies preserved a low proportion of Hlineages in comparison to other Roma from Europe (17%against ∼80% in the Bulgarians, for instance).

Haplogroup JOver one third of the Portuguese Gypsy males be-long to haplogroup J2, being distributed in five different



A. Gusmao et al.

subgroups. Of the two subgroups defined by mutationM67, J2a1b∗ (xJ2a1b1) was very well represented account-ing for ∼20% of the Gypsy males. J2a1b∗ was also com-monly observed in the Roma from Bulgaria, Lithuania andSpain (Gresham et al. 2001).

The network produced with the STR haplotypes withinJ2a1b∗ lineages (Figure 4B) was very similar to the oneobtained for haplogroup H1. However, it is worth notic-ing the almost opposite gradient of distribution of H1and J2a1b∗ between the Bulgarian Gypsies, on one side,and the Portuguese, Spanish and Lithuanian, on the other.The frequency of J2a1b∗ in the latter three groups variesbetween approximately 21% and 33%, whereas in theBulgarian Roma it just attains 9%. Adding to these findingsthe non-detection of J2a1b∗ (xJ2a1b1) among the Macedo-nian Roma (Pericic et al. 2005b), it seems likely that thehaplogroup was not integrated in the ancestral gene poolof the first proto-Roma migrating from India, and thatlater on, during the fragmentation of the Roma settledin the Balkan region (which gave rise to the groups thatcontinued to move west and northward in Europe), strongdrift effects operated successively, enhancing the frequencyof J2a1b∗ (xJ2a1b1) in a group that would turn out to bea common founder of both Iberian and North EuropeanRoma.

The spatial distribution pattern of the haplogroup inEurasia makes it likely that indeed the proto-Roma alreadycarried J2a1b∗ (xJ2a1b1) when they first arrived in Europe.Like other subgroups of the J2a-M410 cluster, its presencein Eurasia is widely recognised to be associated with the de-mographic spread of the Neolithic farmers. In India, M67lineages were found to be almost virtually absent (Zerjalet al. 2007; Sengupta et al. 2006) although two isolatedoccurrences have been reported (Kivisild et al. 2003). Theregion where their frequency peaks is South Caucasus, butthey have been observed from the Middle East throughPakistan and also diffused throughout Europe. Moreover,since higher frequencies and variances have been registeredin Europe and Turkey than in the Middle East, it has beensuggested that M67 chromosomes arrived in Europe fromTurkey (Semino et al. 2004) probably following an Aegeanroute (Di Giacomo et al. 2004).

Having this in mind and knowing from historical sourcesthat Turkey and Greece represented open doors for the in-flux of Roma into the Balkan region, it seems quite admis-sible that the introduction of J2a1b∗ (xJ2a1b1) in the Gypsygene pool occurred prior to their arrival in the Balkans,maybe in the Turkish region where the Gypsies settled fora long time before entering Europe, either crossing theBosphorus or following an Aegean route.

Besides M67, one isolated chromosome further har-boured M92, thus falling into J2a1b1. This haplogroup

was also observed at low frequencies in the Bulgarian andSpanish Gypsies (Gresham et al. 2001) with the STR back-ground of two of the Bulgarian males differing just onemolecular step from the Portuguese J2a1b1 lineage. J2a1b1has been predominantly found in the Northern Mediter-ranean from Turkey westward (Di Giacomo, 2004), havingan overall geographical distribution and history of disper-sion quite similar to J2a1b∗ (xJ2a1b1). Thus, an identicalexplanation can be argued for the introduction of bothlineages in the Gypsies, though not followed in the case ofJ2a1b1 by the strong drift effects that drove the distributionof J2a1b∗ (xJ2a1b1).

Another very common J lineage in the Portuguese Gyp-sies was J2a∗ (xJ2a1b), reaching 16.7%. It is characterised bythe absence of M67 and the presence of the very recentlydiscovered mutation M410 (Sengupta et al. 2006). It seemsextremely unlikely that such lineages were present amongthe Romani groups studied by Gresham et al. (2001), sincewith their typing strategy only five out of 252 samples werenot assigned to a specific haplogroup and these five sam-ples had microsatellite backgrounds very distinct from theJ2a∗ (xJ2a1b) chromosomes now detected. As the patternof geographical distribution of J2a∗ (xJ2a1b) is still incipi-ently known, in order to address the question of its originamong the Portuguese Gypsies we recruited the STR in-formation anchored in those lineages and we conducted ahaplotype match analysis using the YHRD. Based on the‘minimal haplotype+SWGDAM core set’ of the database(11 STR loci) we only observed a full match in a world-wide population sample of 23,979 haplotypes, in a set of221 populations, and the match was with a Portuguesesample. This led us to presume that admixture with Por-tuguese non-Gypsies was responsible for the introductionof the haplogroup in the Gypsy gene pool and again a strongfounder effect extraordinarily raised its frequency. Interest-ingly, 20 of the 21 J2a∗(xJ2a1b) chromosomes shared thesame 16 STR haplotype which was only one molecularstep distant from the first. Given that the founder haplo-type appeared in Gypsy communities spread throughoutPortugal, the introduction event must have occurred soonafter the arrival of the first Gypsies in the country.

Each of the remaining two J lineages detected, J2b-M12and J2b1a-M241, were observed only once in the sample.Consulting the YHRD, we found eleven full matches withthe STR haplotype harboured by the J2b chromosomein individuals from Romania, Macedonia, Bosnia, Albaniaand Italy. However, the same haplotype did not match anyof the Romani samples contained in the database, namelyfrom Hungary (N = 224), Bulgaria (N = 81), Turkey(N = 111), and Slovakia (N = 63). We can admit thatsome J2b-M12 lineages were assimilated in the Balkanregion.



Concerning J2b1a-M241, several haplotypes molecu-larly related with the Portuguese Gypsy J2b1a were ob-served disseminated in Western European populations anda Pyrenean sample showed a full match to it. Therefore, thismight have been incorporated during their passage throughWestern Europe.

Haplogroup IThe M170 mutation defining haplogroup I was carriedby 10.3% of Portuguese Gypsy males. The clade containsa variety of lineages restricted to Europe and almost ab-sent elsewhere including in the Near-East and Middle-East.In Europe, where the haplogroup arose in middle UpperPalaeolithic times (Semino et al. 2000; Rootsi et al. 2004),two distinct areas of very high incidence are observed,one located around Scandinavia and the other around theDinaric Alps in the Balkan region. These two foci of fre-quency are due to different I sub-clades, namely I1a andI1b∗ , whose defining mutations were not assessed in thisstudy. Besides M170, only M26 was additionally tested andthe sub-clade it defines, I1b1b, was found in one out the 13I chromosomes. The Portuguese Gypsy lineage classifiedas I1b1b had a STR background quite distinct from othersbut differing by just two steps from a chromosome of aSpanish Gypsy, which may also be I1b1b. Western Europeis the region where I1b1b peaks and where the definingmutation, M26, most possibly arose in a population fromIberia/South France (Rootsi et al. 2004). A match anal-ysis with the extended haplotype revealed that the Por-tuguese I1b1b chromosome fully matched six records inthe YHRD, all from Portugal or Spain. We can safely con-clude that the introgression of I1b1b in the Iberian Gypsiesoccurred after the Pyrenees crossing.

Gresham et al. (2001) showed that M170 lineages werepresent in Roma from Bulgaria (∼25%), Spain (∼15%) andLithuania (∼5%), Pericic et al. (2005b) also found themamong the Macedonian Romani (∼5%).

In the YHRD, for the most frequent haplotype insideI∗ (xI1b1b), we found 20 identical haplotypes. Fifteen werein Eastern Europe, eight of them in Gypsy populationsfrom Slovakia and Hungary, supporting their possible in-corporation in the Balkan region.

The phylogenetic network reconstructed with the STRdata within I chromosomes from Gypsy individuals is pre-sented in Figure 4C and shows that the most common ofthe two Portuguese I∗ (xI1b1b) haplotypes is also the mostfrequent among the Bulgarian Roma. Lineages molecu-larly close to this modal haplotype are found in Gypsiesfrom Bulgaria, Portugal and Spain. A substantial propor-tion of Bulgarian haplotypes fall in a second molecularcluster to which the single Lithuanian I chromosome alsobelongs. Interestingly, while the haplotypes within this last

cluster fit in or are molecularly related to the known Di-naric modal haplotype (16–24-11–11-13 at DYS19, 390,391, 392, 393), which is an STR motif strongly associatedwith the sub-cluster I1b (Barac et al. 2003; Rootsi et al.2004), the haplotypes within the most represented clusterbelong to, or are molecularly related to, the Nordic modalhaplotype (14–23-10-11-13 at the same STR loci), whichin turn has been connected to the sub-cluster I1a (this isonly moderately observed in the Balkans). Admitting thatboth clusters were incorporated into the Roma gene poolby admixture events occurring in the Balkan region, it isnoteworthy that in Romani people the two clusters occurat inverted proportions compared to their host populations,a finding that can easily be explained by drift effects, butwhich also testifies how these events can lead to misinter-pretation when addressing the origin of specific lineages.

Haplogroup R1bThe second most common Y clade in the Portuguese Gyp-sies was R1, defined by M173, and detected in 36 indi-viduals (∼29%), 35 of them holding the derivative muta-tion M269 that characterises the sub-clade R1b1c. In onesample it was not possible to type M269, becauseof a large deletion of at least 3.5 Mb affecting thischromosome (see haplotype H33, classified as R1

∗-

M173/del.M9/del.M269; Supplementary Table S2). Nev-ertheless, this sample haplotype profile is identical to themost frequent haplotype inside R1b1c haplogroup.

R1 was found in all Roma groups with variable fre-quency: 3.5% in Macedonians (Pericic et al. 2005b),4.4% in Bulgarians; 10% in Lithuanians, 22.2% in Spanish(Gresham et al. 2001). The gradient of distribution withinGypsy groups overlaps quite well with the global clinalpattern of R1 across Europe where the Palaeolithic R1bsubgroup of this paragroup occurs significantly more fre-quently in Western populations, reaching 60% frequencyin Portugal (Beleza et al. 2006). The Iberian Peninsula isthought to be the region of origin of the major deriva-tive R1b1c as well as of its expansion after the Last GlacialMaximum (Semino et al. 2001; Cinnioglu et al. 2004). Inagreement with the R1 distribution pattern, the Gypsiesfrom Spain and Portugal present the highest frequenciesof these chromosomes, and most likely a large proportionof them were essentially acquired by admixture with theIberian surrounding populations. Two lines of evidencelead to this interpretation. Firstly, the absence of founderlineages being transversal to all Roma groups, as shown inFigure 4D, where the network of the STR haplotypes an-chored in Gypsy R1 is depicted. We can observe a clusterof haplotypes molecularly related that contains chromo-somes from Portuguese, Spanish and Lithuanian Roma.The remaining haplotypes are very divergent and occur



A. Gusmao et al.

erratically in distinct Roma populations. This suggests thatR1 chromosomes were incorporated independently in dif-ferent Roma and that diversification in loco within eachgroup was of minor relevance in the context of the GypsyR1 diversity.

The other evidence comes from the haplotype matchanalysis. Out of 35 STR haplotypes within R1b1c chro-mosomes, 28 showed complete coincidence within Euro-pean populations in the YHRD, in a total of 183 matches.From those, only 14 matches were with individuals fromEastern European populations and none was with any ofthe 479 haplotypes from Gypsy populations contained inthe database. The other 169 coincidences were dissemi-nated in Western Europe but nearly 38% were with Iberianhaplotypes, 57% of which inside Portugal.

These results led us to conclude that the majority ofR1b1c lineages were assimilated after the arrival in Iberia.

Other Lineages

Three chromosomes were positive for M35, a mutationthat defines haplogroup E3b. This haplogroup was not de-tected among the Lithuanian or Spanish Gypsies, but waspresent in 4.4% of Bulgarian Roma and reached 29.8% inthe Macedonian Romani. At least the two M35 chromo-somes from the Portuguese Gypsies that additionally hadM81 (E3b1b), do not seem to belong to the array of E3blineages of the Roma from the Balkans. M81 was absent inthe Macedonian Romani, and although not typed in theBulgarian Gypsies, their E3b chromosomes had STR hap-lotypes quite different from the Portuguese E3b1b. Thishaplogroup was likely introduced in Iberia during the Is-lamic occupation (Bosch et al. 2001; Cruciani et al. 2004)being present in ∼6% of the Portuguese non-Gypsy pop-ulation (Beleza et al. 2006). Searching in YHRD withthe extended haplotype, one of the chromosomes fullymatched 12 records out of which eight were from Portugaland one from Spain, seeming to imply that the lineageswere introduced into the Gypsies already in Iberia.

Concerning the third E3b chromosome, it belonged toE3b1a-M78, with a distribution that encompasses Northand Eastern Africa, all of Europe, and the Middle East.In YHRD only two full matches were observed with bothchromosomes from non-Gypsies, one from Poland and an-other from Germany.

A single male was found carrying the M242 mutation,belonging to haplogroup Q. This mutation originated incentral Asia, although it is also been detected at low fre-quencies in India, Pakistan (Sengupta et al. 2006), Iran(Regueiro et al. 2006) and Turkey (Cinnioglu et al. 2004).Apart from its presence in Turkey, this mutation is virtu-ally absent in Europe, which supports its acquisition by the

Gypsies before entrance in Europe. This mutation was notdetected in a sample of 57 Macedonian Romani, and thelack of resolution in other studies on Gypsy groups doesnot allow confirmation of its presence among them.

E∗ (xE3b1)-M96, G-M201, and K2-M70 were observedonce each in the Portuguese Gypsy sample. They were notfound in other Roma and all are relatively uncommon inEurope. No matches were observed for these lineages. Likein other European populations, they are present in Portugalat low frequencies (Beleza et al. 2006).

Conclusions

In this work we studied the Y-chromosome diversity ofPortuguese Gypsies, aiming at deepening previous knowl-edge about the population structure of the Roma inEurope.

Combining a large amount of both SNP and STR in-formation, it was possible to unfold layers of informationkept in the Portuguese Gypsy male gene pool, enablingan extensive reconstruction of the major episodes in theirdemographic history.

The Ancestral Component Shared by the

European Roma

In the Portuguese Ciganos four lineages were detected, H1a,I∗ (xI1b1b), J2a1b∗ (xJ2a1b1), and J2a1b1, which are widelydistributed in Roma groups. The sharing of this set of lin-eages, usually uncommon in European non-Gypsy popu-lations, is a genetic link among Gypsies that clearly bearwitness to the common origin of all Roma in Europe.Two other lineages, J2b∗ (xJ2b1a) and Q, could have alsobeen incorporated into the Gypsies prior to their entranceinto Europe. On the whole, the proportion retained froman ancient gene pool already present in the Balkan Roma(before the fragmentation event and westward dispersionthat occurred during the 15th century) amounted to about48.4% of the Portuguese Gypsy chromosomes.

The Passage from the Balkans to Iberia

During the short period spanning the exodus from theBalkans to the entrance into Iberia, the frequency of lin-eage R1b1c might have been considerably enriched. R1∗

was present at 28.6% frequency in the Portuguese Gyp-sies, but only at 2%, 4% and 10% in Macedonian, Bulgar-ian and Lithuanian Roma, respectively, which implies thatabout 18.6% of R1b1c lineages can be attributed to Iberianinflux. Actually, out of the 35 STR-haplotypes within theR1b1c Portuguese Gypsy chromosomes, 23 had previously



been detected in non-Gypsy Iberians. Assuming that these23 lineages were integrated into the Gypsy gene pool al-ready in Iberia, we can estimate that ∼18.3% (23 in 126)of chromosomes in Gypsies resulted from R1b1c admix-ture in Iberia, in full conformity with that obtained whenthe Lithuania Gypsies were used as reference. Admittingthis, the remaining proportion of the stock of PortugueseGypsies’ R1b1c, together with J2b1a, roughly 10.3%, mayhave been acquired in Europe, mainly during the journeytowards Iberia. Despite the supposedly short lasting passagefrom the Balkans to Iberia, interaction with surroundingpopulations was nonetheless enough to leave visible im-prints in the genetic composition of the Portuguese Gyp-sies.

Acquisitions after Arrival in Iberia

R1b1c and J2a∗ (xJ2a1b) are the two lineages among thePortuguese Ciganos that provide the best clues to the ex-tent of admixture that occurred in Iberia. Within Iberiait is impossible to discriminate between the Portugueseand Spanish contributions since the Iberian Y-chromosomelandscape is quite homogeneous.

We have estimated the proportion of R1b chromosomesof Iberian descendent to be ∼18.3%.

We also hypothesized an Iberian origin for J2a∗ (xJ2a1b),encompassing 16.7% of the Portuguese Ciganos, and forI1b1b and E3b1b, observed at the residual frequency of2.4%.

Summing up all contributions of probable Iberian ori-gin, we suggest 37.4% as the minimum estimate of theadmixture proportion between Gypsies and non-Gypsy in-dividuals in Iberia.

A number of other lineages detected in the PortugueseGypsies, namely E∗ (xE3b1), E3b1a, G, J2b1a and K2, to-taling ∼4.0%, probably introgressed during their passagefrom the Balkans to Iberia or after arrival in Iberia. Whilstfully absent in the Balkan Roma, all are found dispersedat low or moderate frequencies throughout Europe, it istherefore difficult to infer where they were absorbed intothe Gypsy gene pool.

We have previously estimated that 23 R1b1c chromo-somes were of Iberian origin, and that they are representedby ten different 17-STR haplotypes. Pairwise comparisonsbetween these ten haplotypes revealed that (with a singleexception) out of 45 possible comparisons the observeddifferences were always higher than four (between four to13 repeats). Therefore, we can say that at least nine R1b1cIberian chromosomes have been incorporated in Gypsies.Because this haplogroup exists in 60% of the non GypsyIberian males (Beleza et al. 2006), we can extrapolate that,from the remaining 40% of the Iberian non-Gypsy gene

pool, influxes of roughly six additional non R1b1c lineagesmight also have occurred. From these, four were alreadyconsidered as Iberian influx (J2a, I1b1b and E3b1b). Theother expected lineages can belong to any of the tails ofhaplogroups, E∗ (xE3b1), G, J2b1a or K2, referred to above,since they are also present in Iberia.

Summarizing, the emergence of the Portuguese Gyp-sies was the result of a complex process stretching over avaguely known history of migration, multiple fragmenta-tion and interaction with other populations. In the currentstudy some progress was made towards disentangling a littlemore of their past. Emphasising a few, it seems now possi-ble to refute the hypothetical relevance of a second routeof entrance of Gypsies into Europe through North Africa,at least from the male point of view, as we did not find ge-netic evidence supporting any considerable North Africaninfluence.

We can also contest the common idea that the Por-tuguese Gypsies (and Gypsies in general) are an extremelyclosed group. The minimum estimate of around 37% as theproportion of male admixture that had already occurred inIberia is in clear contradiction with the stereotype. Thishigh rate of lineage assimilation means that through adop-tion of a socio-cultural style of life, many people becameand are becoming Gypsy (and vice-versa). More than re-flecting a biological/linguistic entity, the term “Gypsy”must be perceived as a social construct in a continuallychanging environment.

Despite the high rate of gene pool renewal, the processdid not erase signatures of the strong endogamous natureof the Portuguese Gypsy society. All Gypsy groups sharethis structural tradition and all of them experienced iden-tical and peculiar population histories. Along time, bothdemography and culture acted together providing the con-ditions for profound and continuous reshuffling of previ-ous genetic compositions. Eventually, new founder effectsinside old founder effects under a continuous corridor ofgene influx will progressively attenuate the existing geneticsigns of their origin. As this and previous works demon-strate, at the moment, genetic studies can still contributeto partially recover that past.

Acknowledgements

We thank all DNA donors and those, which made possible thecontacts with Gypsy communities in Portugal, including ACIMEand Cruz Vermelha Internacional. This work was partially sup-ported by Fundacao para a Ciencia e a Tecnologia, through POCI(Programa Operacional Ciencia e Inovacao 2010) and projectPTDC/ANT/70413/2006. The primers used to type M242 mu-tation were kindly provided by Maria Brion (Institute of LegalMedicine, Santiago de Compostela, Spain).



A. Gusmao et al.

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Supplementary Material

The following material is available for this article online:

Table S1. Multiplex H, J2 and M269. Details on sequencesof PCR and SBE primers used, and typing conditionsTable S2. Y-chromosome haplotypes in 126 Gypsies fromPortugal. District codes are: Aveiro (Av); Lisboa (Li); Vianado Castelo (VC); Braga (B); Porto (P); Braganca (Bn);Vila Real (VR); Viseu (Vi); Coimbra (Co); Guarda (Gu);Castelo Branco (CB); Leiria (Le); Santarem (Sa); Setubal(Se); Portalegre (Po); Evora (Ev); Beja (Be); Faro (Fa); Fun-chal (Fu).

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-1809.2007.00421.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsiblefor the content or functionality of any supplementarymaterials supplied by the authors. Any queries (other thanmissing material) should be directed to the correspondingauthor for the article.

Received: 15 October 2007

Accepted: 20 October 2007



doi: 10.1111/j.1469-1809.2007.00421.x a perspective on the

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