Ancient DNA suggests the leading role played by menin the Neolithic disseminationMarie Lacana,b,1, Christine Keyserb, François-Xavier Ricauta, Nicolas Brucatoc, Josep Tarrúsd, Angel Boschd,Jean Guilainee, Eric Crubézya, and Bertrand Ludesb

aLaboratoire d’Anthropologie Moléculaire et Imagerie de Synthèse, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5288, 31073Toulouse, France; bLaboratoire d’Anthropologie Moléculaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5288, Institute of LegalMedicine, University of Strasbourg, 67085 Strasbourg, France; cLanguage and Genetics Department, Max Planck Institute for Psycholinguistics, 6525 XD,Nijmegen, The Netherlands; dMuseu Arqueològic Comarcal de Banyoles, 17820 Catalonia, Spain; and eCollège de France, Centre de Recherche sur laPréhistoire et la Protohistoire de la Méditerranée, École des Hautes Etudes en Sciences Sociales, 31000 Toulouse, France

Edited by Ofer Bar-Yosef, Harvard University, Cambridge, MA, and approved September 29, 2011 (received for review August 9, 2011)

The impact of the Neolithic dispersal on the western Europeanpopulations is subject to continuing debate. To trace and dategenetic lineages potentially brought during this transition and sounderstand the origin of the gene pool of current populations, westudied DNA extracted from human remains excavated in a Span-ish funeral cave dating from the beginning of the fifth millenniumB.C. Thanks to a “multimarkers” approach based on the analysis ofmitochondrial and nuclear DNA (autosomes and Y-chromosome),we obtained information on the early Neolithic funeral practicesand on the biogeographical origin of the inhumed individuals. Noclose kinship was detected. Maternal haplogroups found are con-sistent with pre-Neolithic settlement, whereas the Y-chromosomalanalyses permitted confirmation of the existence in Spain approx-imately 7,000 y ago of two haplogroups previously associatedwith the Neolithic transition: G2a and E1b1b1a1b. These resultsare highly consistent with those previously found in Neolithic indi-viduals from French Late Neolithic individuals, indicating a sur-prising temporal genetic homogeneity in these groups. The highfrequency of G2a in Neolithic samples in western Europe couldsuggest, furthermore, that the role of men during Neolithic dis-persal could be greater than currently estimated.

The Neolithic transition was a crucial step in the history ofEuropean settlement, but the exact modalities of its dis-

semination are still not totally understood. In western Europeparticularly, despite the abundance of archeological data, thereal importance of the Mesolithic substrate and of the Neolithicmigrants in the first farmers’ origin is a crucial point still debatedamong the scientific community (1). In this context, access toancient DNA data seems to be a good way to trace and date thedispersal of European genetic lineages and better understand theorigin of current populations.Presently, few ancient data are available on the Neolithic pe-

riod, and most of them consist of mitochondrial DNA data,which are only informative for the maternal origin. These haverevealed a particularly high frequency of haplogroup N1a, ahaplogroup quite rare currently in central European (2, 3) andin Atlantic coast Neolithic specimens (4), whereas this last wasnever found in southern European samples (5–7). These fur-thermore suggested a probable genetic continuity between an-cient southern Neolithic specimens and current populationslocated in the same areas (6, 7), whereas the ancient centralEuropean plains samples would share a greater affinity with themodern-day Near East and Anatolia (2). The findings deducedfrom the study of maternal genetic lineages seemed consistentwith the archeological evidences of the existence of two distinctroutes of neolithization: one along the central plains of Europeand another along the Mediterranean coasts.Concerning paternal lineages, because of the bad preservation

of nuclear DNA in ancient samples, few analyses have beenperformed to date on the Y-chromosome of Neolithic speci-mens, thus few paternal lineages existing at this period have beencharacterized. The study of only three male specimens associated

with the Linear Pottery Culture, a Neolithic culture found in thecentral European plains (2), and of 22 men buried in a lateNeolithic French necropolis (6) permitted data to be obtainedon the paternal lineages existing before the Cooper and Bronzeage migrations. Interestingly, they all revealed the importance ofthe G2a haplogroup, which is rare in modern European pop-ulations. Of course, these works do not provide a completeoverview of the Neolithic male diffusion. Additionally, no dataare currently available on the paternal lineages existing in theearly Mediterranean Neolithic.In this context, to improve the knowledge of the neolithization

of southwestern Europe, we studied DNA extracted from humanspecimens excavated in the Avellaner cave, an ancient funeralcave of northeastern Spain. According to 14C dating performedon bones and charcoals found in the cavity, this funeral cave wasused during the first part of the fifth millennium B.C. (8), whichcorresponds to the end of the establishment of the Neolithiccultures in Spain (Epicardial Culture). The study of this funeralsite is thus particularly interesting to access directly the genepool of the first farmers in Spain and to understand the partic-ular funeral practices of this transition period, which are stillpoorly understood (9).Because most of bones found in the cave were fragmented and

partially burned, the first challenge of this work was to identifyindividuals buried. Afterward, we analyzed different and com-plementary genetic markers, located on autosomes and Y-chro-mosomal and mitochondrial DNA to characterize any kinshipbetween individuals and to trace their biogeographical origin.Through these data, the main objectives of this work were to

genetically characterize early farmers from northern Spain andto compare the genetic lineages found with those previouslyobtained from Neolithic specimens and those currently presentin European populations, to understand the complexity of theNeolithic dispersal and its heritage in southern Europe.

ResultsAutosomal Results. Of the 27 samples studied, 14 permitted ac-quisition of unambiguous partial or complete autosomal profiles,which can be related to seven individuals (Table 1). Of the sevenindividuals clearly identified, six were male and one was female.No close familial relationship could be highlighted between theseindividuals. Estimation of the nuclear DNA concentration per

Author contributions: M.L. designed research; M.L. performed research; J.T. and A.B.contributed new reagents/analytic tools; M.L. analyzed data; and M.L., C.K., F.-X.R., N.B.,J.G., E.C., and B.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: lacan.marie@netcourrier.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113061108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1113061108 PNAS | November 8, 2011 | vol. 108 | no. 45 | 18255–18259

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sample ranged from below the detection capability of the kit to34.2 pg/μL (Table 1).

Mitochondrial Results. Mitochondrial HVS-I sequences wereobtained for the seven individuals and can be classified into fourdifferent haplotypes (Table 2). All are still frequent in currentEuropean populations (Table S1), and three of them were alsofound in ancient Neolithic samples (Table S2). These haplotypespermitted the determination that the individuals ave01, ave02,and ave06 belonged to K1a, ave04 and ave05 to T2b, ave03 toH3, and ave07 to U5 haplogroups.For all samples, typing of mitochondrial SNPs in the coding

region permitted confirmation of the haplogroup determinationpreviously inferred from the haplotypes (Table 2).

Y-Chromosomal Results. For the six male samples, two completeand four partial Y-STRs haplotypes were obtained (Table 3).They allowed classification of individuals into two different hap-logroups: G2a (individuals ave01, ave02, ave03, ave05, and ave06,which seem to share the same haplotype) and E1b1b1 (individualave07). The four markers chosen to confirm belonging to thesehaplogroups (Y-E1b1b1-M35.1, Y-E1b1b1a1b-V13, Y-G2-M287,and Y-G2a-P15) were typed with a rate of 66%, which permittedconfirmation that four males were G2a and one was E1b1b1a1b(Table 3).Analysis of shared haplotypes showed that the G2a haplotype

found in ancient specimens is rare in current populations: itsfrequency is <0.3% (Table S3). The haplotype of individual ave07is more frequent (2.44%), particularly in southeastern Europeanpopulations (up to 7%). The Ave07 haplotype was also comparedwith current Eb1b1a2 haplotypes previously published (10–14). Itappeared identical at the seven markers tested to five Albanian,two Bosnian, one Greek, one Italian, one Sicilian, two Corsican,and two Provence French samples and are thus placed on thesame node of the E1b1b1a1b-V13 network as eastern, central,and western Mediterranean haplotypes (Fig. S1).

Lactase Persistence Result. The LP-13910-C/T SNP associated withlactase persistence was successfully typed for all ancient samplestested. The mutated position would have appeared during thedissemination of the Linear pottery culture in central Europe(15). All our ancient samples from Spain were homozygous C/Cfor this marker.

DiscussionResults Authenticity. The main difficulty in ancient DNA analysesis to produce authentic data. In this study, drastic precautionspreviously described (6, 16) were taken to avoid contaminations,and a multimarkers approach was used to validate the accuracyof the produced data. For the seven individuals presented here,despite the fact that all of the authenticity criteria could not befully respected, results obtained are in favor of endogenous andreliable outcomes: extraction controls, PCR blanks, and ampli-fications from DNA extracted from sheep or goat remains withhuman primers were always negative. The nuclear DNA quantityrecovered and the inverse relationship between the amplificationefficiency and length of the amplification obtained were char-acteristic of a degraded ancient DNA. Results acquired from thedifferent amplifications and from cloning were always consistentbetween each other, and results of SNP typing were also 100%concordant with mitochondrial and Y-chromosome haplotypespreviously deduced. The absence of the polymorphism associ-ated with the lactase persistence is also coherent with resultspreviously published from ancient Mesolithic and Neolithicsamples (6, 17, 18).

Avellaner Genetic Diversity. Regarding the biogeographical originof Avellaner individuals, mitochondrial and Y-chromosomalTa

ble

1.Consensu

ssh

ort

tandem

repea

t(STR

)au

toso

mal

profilesoftheseve

nan

cien

tAve

llaner

individuals

Nam

eCav

ity

No.of

samples

[DNA]

(ng/μL)

D8S

1179

D21

S11

D7S

820

CSF1P

OD3S

1358

TH01

D13

S317

D16

S539

D2S

1338

D19

S433

vWA

TPOX

D18

S51

AMEL

D5S

818

FGA

Ave

01II

34.62

E-03

13/(14

)30

/31.2

10/11

11/12

18/18

9.3/9.3

8/11

11/11

17/23

12/13

15/16

—17

/20

X/Y

13/13

21/24

Ave

02I

41.39

E-02

11/13

28/32.2

10/12

10/11

15/18

6/(9.3)

10/(11

)8/10

17/19

15/15.2

16/19

8/(11)

16/17

X/Y

11/12

23/26

Ave

03III

38.16

E-03

11/13

28/29

12/12

9/12

17/18

9.3/9.3

8/12

11/12

17/24

12/13

17/19

—14

/18

X/Y

10/11

24/(25

)Ave

04III

1Und

13/(15

)(31.2/32

.2)

(11/11

)(11/11

)16

/(18

)(7/7)

(11)/12

11/14

(24/24

)12

/14

16/16

(8)/9

(16/19

)X/X

13/13

(24/24

)Ave

05I

19.45

E-03

14/14

—(12/13

)(11/11

)18

/18

(7/7)

11/11

(11/11

)(20/24

)(14/14

)(15/16

)(8/8)

(17/21

)X/Y

(11/11

)—

Ave

06II

1Und

(14/15

)29

/29

—11

/12

(14)/17

—(11/11

)(11/11

)(16/20

)(13/13

)(15/15

)(8/8)

12/(14

)X/Y

(11)/13

(20/24

)Ave

07II

18.27

E-03

13/15

28/31

8/10

10/12

15/16

9.3/9.3

11/(12

)(8/11)

17/25

(12/13

)(16/17

)(8/11)

(16/16

)X/Y

10/12

21/22

Dashes

den

ote

that

allelesco

uld

notbeclea

rlyam

plifi

edforthelocu

sin

question.Alle

lesin

paren

theses

werejust

observed

once.C

onsensusallelic

profileswerebuilt

aftertw

oam

plifi

cationsperform

edon

each

DNA

extract.[D

NA],av

erag

equan

tity

ofnuclea

rDNA

obtained

;Und,undetermined

.

18256 | www.pnas.org/cgi/doi/10.1073/pnas.1113061108 Lacan et al.

results suggest different origins of maternal and paternal line-ages. Mitochondrial haplogroups found (U5, K1a, T2b, and H3),which are quite common in western Eurasia, are relatively un-informative for identifying clear genetic affiliations with de-mographic movements. However, they suggested a fairly diverseorigin of Avellaner maternal lineages, consistent with an earlyNeolithic group: a Paleolithic origin from Middle East for hap-logroup K1a, T2b, and U5 and a relation with the postglacialexpansion from southwestern Europe for the H3 haplogroup (19,20). Additionally, haplotypes identical to those assigned to H3,K1a, and T2b haplogroups were previously found in samplesrelated to central and Mediterranean Neolithic cultures (TableS2). Only the U5 haplotype was never found in ancient speci-mens, but it differs at only one position (np 16051) from anotherMesolithic haplotype (21). This finding also observed in otherlate Neolithic samples could indicate a greater diversity of theU5 cluster at the Neolithic period, consistent with an ancientorigin of this lineage (6).Concerning paternal lineages, only two different haplogroups

were identified: G2a and E1b1b1a1b.G2a is common in modern populations of Caucasus (10) but is

quite rare in current western European populations. It repre-sents only approximately 4% of the haplogroups found in theSpanish population (22). It seems nevertheless much morecommon in Neolithic samples because it was previously foundin ancient Neolithic samples from the linear pottery culture inGermany, as well as in late Neolithic French samples (2, 6).Thus, it represents one of the main Y-haplogroups found inNeolithic individuals. Of course, there are still too few data toconfirm the overrepresentation of the G2a haplogroup amongwestern Neolithic populations, but this G2a in early SpanishNeolithic samples is strong evidence of a link previously sug-gested between Neolithic migration and G2a dispersion inEurope (6). Additional G2a haplotypes will, however, be neededto determine whether the G2a found in the male individual as-sociated with dispersal of Neolithic in central Europe and thoselinked with the Mediterranean route shared a same NeolithicMiddle Eastern origin.For E1b1b1a1b, the link between this haplogroup and the

Neolithic expansion could also be made. This haplogroup, whichis the main European clade of haplogroup E, has been describedas having spread into western Europe from the southern Balkans(10, 14, 23), but the exact period at which this expansion wouldbe held is still debated. It has been previously related to severaldemographic events, such as the Neolithic dispersal in directionof the eastern Adriatic (10) or along the Vardar-Morava-Danuberivers into central Europe (24) or the migrations during theBronze age (13, 23, 25). The presence of this haplogroup in anearly Neolithic sample in Spain confirms, therefore, that thismarker may be related to the Mediterranean Neolithic expan-sion, even if it does not permit quantification of the real im-portance of the Neolithization contribution in the spread of thishaplogroup in western Europe. It confirms, furthermore, that theNeolithic dispersal was not a uniform movement from theMiddle East but that it was more probably an arrhythmic phe-nomenon punctuated by rapid expansion phases and periods ofbreaks related to cultural changes like the one previously iden-tified by archeologists in the Balkans area (26–28).

Unusual Neolithic Burial Cave. Because of the fragmented state ofskeletons, few data could be previously obtained from thephysical analysis of remains. Thanks to ancient DNA data, weprove that among the seven individuals clearly identified, sixwere male. No close relationship could be demonstrated, but fiveof the six males buried seem to belong to the same paternallineage, which suggests a funeral recruitment of individuals fromthe same group. According to archaeological evidence, fewburial places were found at the recent phases of the EarlyTa

ble

2.MtD

NA

hap

lotypes

andhap

logroupsinferred

forea

chAve

llaner

specim

en

Nam

eCav

ity

No.of

samples

Mitoch

ondrial

hap

lotypes

H-C70

28T

T2-A

1423

3GT2

B-G

930A

U5-T3

197C

H1-J1-G

3010

AH3-T6

776C

HV-C14

766T

K-A

1055

0GK1-A10

398G

K1A

-C49

7TT-A49

17G

U-A

1230

8GHap

logroup

Ave

01II

316

093C

;16

224C

;16

311C

TA

GT

GT

TG

GT

—G

K1a

Ave

02I

416

093C

;16

224C

;16

311C

TA

GT

GT

TG

GT

AG

K1a

Ave

03III

3CRS

CA

GT

GC

CA

AC

AA

H3

Ave

04III

116

126C

;16

294T

;16

296T

;16

304C

TG

AT

GT

TA

AC

—A

T2b

Ave

05I

116

126C

;16

294T

;16

296T

;16

304C

TG

AT

AG

TT

AA

——

AT2

b

Ave

06II

116

093C

;16

224C

;16

311C

TA

GT

AG

TT

——

TA

GK1a

Ave

07II

116

051G

;16

189C

;16

270T

TA

GC

GT

TA

AC

AG

U5

SNPs

inbold

areva

rian

tsat

concerned

positions.

Lacan et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18257

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Neolithic compared with the putative population density at thisperiod. Epicardial burials were furthermore usually individual innatural cavities like the Gazel cave, Aude, France, or in open-airsites like Ca l’Estrada, Catalonia (1). Collective burials appearedlater during the Late Neolithic period (IV and III millenniaB.C.). In this context, the funeral recruitment of the Avellanercave seems very unusual for the early Neolithic period (1, 29).The absence of any significant grave goods in the cavities is not infavor of funeral recruitment according to potential social elite.According to archaeological hypotheses, it could rather reflecta continuance of funerary rites from the late Mesolithic period(1). The presence of individuals carrying paternal lineages linkedwith the Neolithic expansion in a funeral cave where Mesolithicfuneral practices were used could suggest the maintenance ofthe Mesolithic traditions during the early stages of the westernEurope Neolithic. No genetic data from Mesolithic grave speci-mens are currently available to be compared with the geneticstructure obtained. Further genetic analyses of ancient southernEuropean graves will be necessary to validate this hypothesis andpotentially provide a better idea about the origin of this partic-ular burial.

Avellaner and the Neolithic Transition. All of the results obtainedare concordant with those previously published for ancientNeolithic specimens. The mitochondrial lineages found areconsistent with a genetic continuity in southern Europe, at leastsince Neolithic times, and the absence of the N1a haplogroupwidespread in central Europe confirms furthermore the archae-ological evidence of the existence of two routes of Neolithicdispersal in Europe. Moreover, the paternal lineages character-ized and their current repartition along the Mediterraneancoasts confirm this assumption.The sex-specific diversity observed in Avellaner specimens is

finally broadly similar to that previously observed in the samplesof the Treilles cave, a French funeral cave 2,000 y more recentthan the one presented here. In both studies, the Paleolithicorigin of most of the maternal lineages coupled with a morerecent paternal ancestry as well as the G2a presence could thussuggest a common origin of the two Neolithic groups anda probable genetic continuity in the western Mediterranean areafrom 5000 to 3000 B.C.The high frequency of G2a haplogroup in Neolithic speci-

mens, whereas this haplogroup is very rare in current pop-ulations, also suggests that men could have played a particularlyimportant role in the Neolithic dissemination that is no longervisible today. This would imply that intra-European migrationsrelated to the metal ages may have strongly affected the moderngene pool.

ConclusionThis work offers a direct overview of the genetic lineages of thefirst northern Spanish farmers. It reveals the complexity of theimplementation of agriculture in Spain and the probable highlevel of heterogeneity of the Neolithic dissemination in Europe.It highlights furthermore that maternal and paternal lineagescould have had different histories, which complicates even morethe different scenarios issued on the Neolithic transition inEurope. This study, which was performed on only seven indi-viduals, is of course not sufficient to estimate the real importanceof the arrival of men in the Neolithic transition, and new in-vestigations on ancient southern specimens will be necessary toimprove our knowledge on this crucial period.

Materials and MethodsThe Avellaner cave is a small burial cave located in Catalonia, in northeasternSpain. The main cavity is in fact divided into three independent areas sep-arated by stonewalls, each containing several successive primary or secondaryburials. Skeletons found were not in anatomic connection, which did notTa

ble

3.Y-hap

logroupsinferred

from

Y-STR

hap

lotypes

andNRY-SNPs

typingresu

ltsforthemalesp

ecim

ens

Nam

eCav

ity

No.of

samples

Y-STR

sY-SNPs

DYS4

56DYS3

89IDYS3

90DYS3

89II

DYS4

58DYS1

9DYS3

85DYS3

93DYS3

91DYS4

39DYS6

35DYS3

92GATA

-H4

DYS4

37DYS4

38DYS4

48

Y-

E1b1b

1-M35

.1

Y-

E1b1b

1a1b

-V13

Y-

G2-

M28

7

Y-

G2a

-P1

5Hap

logroups

Ave

01II

315

12(23)

(29)

1815

(14/14

)13

1011

(21)

—12

1610

—G

G—

TG2a

Ave

02I

415

1223

—18

1514

/14

1310

1121

(11)

1216

1022

GG

—T

G2a

Ave

03III

315

1223

2918

1514

/14

1310

1121

1112

1610

(22)

GG

—T

G2a

Ave

05I

115

12(23)

29(18)

(15)

(14/14

)13

(10)

11—

—12

16—

22G

——

TG2a

Ave

06II

115

12—

—18

(15)

—13

(10)

(11)

——

12(16)

——

—G

——

G2a

99.6%

Ave

07II

116

1324

(31)

1613

16/19

13(10)

1122

1110

(14)

1020

CA

—C

E1b1b

1a1b

Dashes

den

ote

that

allelesco

uld

notbeclea

rlyam

plifi

edforthelocu

sin

question.C

onsensusY-STR

profileswerebuilt

aftertw

oam

plifi

cationsfrom

each

DNAex

tract.Alle

lesin

paren

theses

wereobserved

just

once.Fo

rthesample

forwhichtheYhap

logroupco

uld

notbeco

nfirm

edbythetypingofSN

P,thedeterminationofthehap

logroupwas

obtained

solely

from

thehap

lotype.

Thepercentageofprobab

ility

isshownin

thelast

column.SN

Psin

bold

areva

rian

tsat

concerned

positions.

18258 | www.pnas.org/cgi/doi/10.1073/pnas.1113061108 Lacan et al.

permit formal individualization of each individual; however, the minimumnumber of individuals could be estimated at 19 (8). Because of the frag-mentation and the mix of bones within each sepulchral place, we decided towork from teeth still fixed on the four mandible fragments available (two orthree teeth for each fragment), on the most well-preserved scattered teeth,and three femoral shafts. In all, we analyzed 27 human samples taken in thethree cavities, as well as two nonhuman material (two sheep teeth) taken atthe same time with human remains to detect possible contamination eventsduring excavation.

Sample Preparation and DNA Extraction. Bones samples were first abradedwith sterile equipment before UV exposure and grinding into a liquid ni-trogen environment. Teeth were also crushed after decontamination withbleach and UV exposure 30 min on each side. DNA was then extractedaccording to a protocol previously described (30). Four to five extractionswere realized on each sample, according to the powder quantity recovered.

Nuclear DNA Analysis. For at least one extract per sample we determined thenuclear DNA quantity extracted using the Quantifiler Human DNA Quanti-fication Kit (Applied Biosystems).

Autosomal profiles were determined using both AmpFlSTR Identifiler Plusand the MiniFiler PCR Amplification Kits (Applied Biosystems) on a 3500Genetic Analyzer. STRs profiles were analyzed with GeneMapper 4.1 soft-ware. Two amplifications were performed on each DNA extract.

Haplotype Determination. Mitochondrial haplotypes were obtained by thesequencing of 381 base pairs of the HVS-I region of the mtDNA in twooverlapping fragments, as previously described (16). Tomeet the authenticitycriteria, we also cloned the amplicons obtained during the analyses of 5 ofthe 27 samples. Cloning was performed using the pGEM-T Easy Vector sys-tem II kit (Promega), according to the manufacturer protocol. Between 16and 28 clones were analyzed for each sample. All sequences obtained wereused to deduce mitochondrial haplogroups according to the latest mtDNAphylogeny (31).

For male individuals, Y-chromosomal haplotypes were obtained from theanalysis of 17 Y-STRs loci using the AmpFlSTR Yfiler PCR Amplification Kit

(Applied Biosystems). They were used to estimate Y-haplogroups thanks tothe Haplogroup Predictor software (32).

Haplogroup Assignment and Typing of an SNP Associated with LactasePersistence. To clarify the haplogroup status inferred from HVS-I sequencesand Y-chromosomal haplotypes, we analyzed supplemental SNPs localized onthe mitochondrial coding region and the nonrecombining region of the Y-chromosome (NRY). SNP typing was performed using iPLEX Gold technology(Sequenom), which seems to be a very sensitive and effective typing tech-nology for degraded DNA analyzes (30). Two multiplexes containing a totalof 17 SNPs located on mtDNA, the NRY, and the MCM6 gene (SNP associatedwith the lactase persistence) were designed with MassArray Assay designsoftware (version 4.0). The typing reactions were performed twice on twodifferent DNA extracts per sample.

Statistical Analysis. The putative genetic relationships were investigated fromautosomal STR profiles with DNA•VIEW Software (33).

To compare ancient Spanish genetic lineages obtained and those currentin European populations, analyses of shared haplotypes were performedthanks to two personal databases comprising 14,645 mitochondrial HVS-Isequences and 14,166 Y-haplotypes. Mitochondrial profiles obtained werealso compared with ancient Neolithic haplotypes previously published. De-tailed compositions of the different datasets are available in Table S2 andTable S4. To allow maximum comparability among all populations, Y-sharedhaplotype analyses were performed on only seven Y-STRs markers (DYS19,DYS390, DYS391, DYS392, DYS393, DYS389I, and DYS389II).

A haplotype network was generated for NRY haplogroup E-V13 via themedian joining algorithm of Network, version 4.5.1.6. To obtain the mostparsimonious networks the reticulation permissivity was set to zero. Datasetswere preprocessed using the star contraction option in Network, version4.5.1.6 (5). The seven Y-STR loci used were weighted according to the ob-served STR allelic variance, as described by Qamar et al. (34).

ACKNOWLEDGMENTS. We thank Xevi Roura (Museu Comarcal) for accessto the ancient samples, and Angela Gonzalez for her help with Sequenomtechnology.

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Supporting InformationLacan et al. 10.1073/pnas.1113061108

Fig. S1. Median joining network of Y-E1b1b1a1b-V13 haplotypes in current western European populations previously published (n = 215) (1–5) and of theAve07 haplotype.

1. Battaglia V, et al. (2009) Y-chromosomal evidence of the cultural diffusion of agriculture in Southeast Europe. Eur J Hum Genet 17:820e830.2. Caratti S, Gino S, Torre C, Robino C (2009) Subtyping of Y-chromosomal haplogroup E-M78 (E1b1b1a) by SNP assay and its forensic application. Int J Legal Med 123:357e360.3. Di Gaetano C, et al. (2009) Differential Greek and northern African migrations to Sicily are supported by genetic evidence from the Y chromosome. Eur J Hum Genet 17:91e99.4. King RJ, et al. (2011) The coming of the Greeks to Provence and Corsica: Y-chromosome models of archaic Greek colonization of the western Mediterranean. BMC Evol Biol 11:69.5. King RJ, et al. (2008) Differential Y-chromosome Anatolian influences on the Greek and Cretan Neolithic. Ann Hum Genet 72:205e214.

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Table S1. Percentage of each ancient mitochondrial haplotype in current European populations

93C, 224C, 311C CRS 126C, 294T, 296T, 304C 51G, 189C, 270T

Population Total nNo. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)

Middle EastIranians 448 1 0.22 32 7.14 2 0.45 0 0.00Saudi Arabians 702 12 1.71 18 2.56 0 0.00 0 0.00Syrians 118 1 0.85 8 6.78 2 1.69 0 0.00Iraqis 52 1 1.92 0 0.00 0 0.00 0 0.00Druze 356 2 0.56 24 6.74 0 0.00 0 0.00Yemenis 115 0 0.00 3 2.61 0 0.00 0 0.00Kurds 82 1 1.22 12 14.63 0 0.00 0 0.00Dubai 249 2 0.80 4 1.61 0 0.00 0 0.00Palestinians 117 1 0.85 8 6.84 1 0.85 0 0.00Turks 340 3 0.88 33 9.71 0 0.00 0 0.00

Middle East: total 2,579 24 0.93 142 5.51 5 0.19 0 0.00CaucasusWestern Russians 358 0 0.00 45 12.57 10 2.79 0 0.00Other North Caucasus

populations353 4 1.13 23 6.52 6 1.70 0 0.00

Georgians 188 6 3.19 16 8.51 0 0.00 0 0.00Armenians 358 2 0.56 27 7.54 0 0.00 0 0.00Azerbaijanis 48 0 0.00 3 6.25 1 2.08 0 0.00Caucasus: total 1,305 12 0.92 114 8.74 17 1.30 0 0.00

Northwestern EuropeBritish 100 0 0.00 13 13.00 1 1.00 0 0.00French (Brittany) 62 0 0.00 11 17.74 0 0.00 0 0.00French (Normandie) 39 1 2.56 2 5.13 1 2.56 0 0.00French (Limousin) 72 0 0.00 7 9.72 3 4.17 0 0.00French (Var) 37 0 0.00 8 21.62 2 5.41 0 0.00Welsh 92 2 2.17 23 25.00 0 0.00 0 0.00Cornish 92 1 1.09 16 17.39 0 0.00 3 3.26Irish 300 3 1.00 56 18.67 6 2.00 0 0.00Northwestern Europe:

total794 7 0.88 136 17.13 13 1.64 3 0.38

North-Central EuropeGermans 682 5 0.73 96 14.08 15 2.20 8 1.17Danish 38 0 0.00 9 23.68 2 5.26 0 0.00Czechs 262 3 1.15 32 12.21 1 0.38 0 0.00Polish 849 6 0.71 138 16.25 21 2.47 0 0.00Slovakians 335 1 0.30 36 10.75 4 1.19 0 0.00Swiss 224 2 0.89 32 14.29 5 2.23 2 0.89Latvians 299 0 0.00 41 13.71 13 4.35 0 0.00Estonians 149 0 0.00 19 12.75 3 2.01 2 1.34Austrians 99 3 3.03 21 21.21 1 1.01 0 0.00South Tyrol populations 263 1 0.38 50 19.01 3 1.14 0 0.00North-Central Europe:

total3,200 21 0.66 474 14.81 68 2.13 12 0.38

ScandinaviaNorwegians 626 1 0.16 129 20.61 16 2.56 5 0.80Finns 755 2 0.26 95 12.58 6 0.79 1 0.13Scandinavia: total 1,381 3 0.22 224 16.22 22 1.59 6 0.43

Southeastern EuropeBulgarians 30 0 0.00 3 10.00 0 0.00 0 0.00Hungarians 386 5 1.30 43 11.14 8 2.07 0 0.00Bosnians 304 1 0.33 36 11.84 2 0.66 0 0.00Serbians 56 0 0.00 4 7.14 0 0.00 0 0.00Romanians 105 1 0.95 13 12.38 0 0.00 0 0.00Southeastern Europe:

total881 7 0.79 99 11.24 10 1.14 0 0.00

Western MediterraneanNorthern Portuguese 271 5 1.85 60 22.14 3 1.11 0 0.00Central Portuguese 317 7 2.21 64 20.19 10 3.15 0 0.00Southern Portuguese 260 0 0.00 52 20.00 3 1.15 0 0.00

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Table S2. Results of the shared haplotype analysis between Avellaner haplotypes and other ancient Neolithic sequences

Culture Location References Dates Positions n

93C, 224C,311C (no.shared seq)

CRS (no.sharedseq)

126C, 294T,296T, 304C(no. shared

seq)

51G, 189C,270T (no.shared seq)

Gatherer/huntercultures

Germany, Russia,Poland, Lithuania

(1) 13400–2500 BC 15997–16409 20 0 1 0 0

Neolithic, LBK/AVK Austria, Hungry,Germany

(2, 3) 5500–500 BC 15997–16409 42 2 4 2 0

Neolithic Spain (4) 3500–3000 BC 16022–16378 11 0 3 0 0Neolithic, Megalithic Western France (5) 4200 BC 16165–16390 3 0 0 0 0Late Neolithic,Corded WareCulture

Germany (6) 2700–2400 BC 15997–16409 9 0 0 0 0

Late Neolithic,Treilles Culture

France (7) 3030–2890 BC 16009–16390 29 0 6 2 0

Chalcolithic Austria/Italy (8) 3500–3100 BC 16040–16401 1 0 0 0 0

Table S1. Cont.

93C, 224C, 311C CRS 126C, 294T, 296T, 304C 51G, 189C, 270T

Population Total nNo. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)No. shared

seqFq haplotype

(%)

Galicians 135 1 0.74 28 20.74 2 1.48 0 0.00Spanish Catalans 133 2 1.50 23 17.29 0 0.00 0 0.00Andalusians 189 4 2.12 36 19.05 0 0.00 0 0.00Balearic islanders 67 2 2.99 14 20.90 0 0.00 0 0.00Basque Spanish 211 1 0.47 48 22.75 1 0.47 0 0.00Western Mediterranean:

total1,583 22 1.39 325 20.53 19 1.20 0 0.00

Central MediterraneanNorthern Italians 319 3 0.94 45 14.11 3 0.94 0 0.00Central Italians 637 10 1.57 96 15.07 5 0.78 0 0.00Central Southern Italians 199 2 1.01 35 17.59 4 2.01 0 0.00Sicilians 106 0 0.00 30 28.30 2 1.89 0 0.00Corsicans 99 1 1.01 20 20.20 1 1.01 0 0.00Sardinians 234 2 0.85 52 22.22 1 0.43 0 0.00Slovenians 104 1 0.96 14 13.46 2 1.92 0 0.00Croatians 59 1 1.69 5 8.47 2 3.39 0 0.00Central Mediterranean:

total1,757 20 1.14 297 16.90 20 1.14 0 0.00

Eastern MediterraneanMacedonians 237 2 0.84 28 11.81 5 2.11 0 0.00Albanians 84 0 0.00 14 16.67 1 1.19 0 0.00Cretans 200 2 1.00 28 14.00 11 5.50 0 0.00Cypriots 91 3 3.30 8 8.79 0 0.00 0 0.00Greeks 553 3 0.54 57 10.31 3 0.54 2 0.36Eastern Mediterranean:

total1,165 10 0.86 135 11.59 20 1.72 2 0.17

Total 14,645 126 0.86 1946 13.29 194 1.32 23 0.16

1. Bramanti B, et al. (2009) Genetic discontinuity between local hunter-gatherers and central Europe’s first farmers. Science 326:137e140.2. Haak W, et al.; Members of the Genographic Consortium (2010) Ancient DNA from European early neolithic farmers reveals their near eastern affinities. PLoS Biol 8:e1000536.3. Haak W, et al. (2005) Ancient DNA from the first European farmers in 7500-year-old Neolithic sites. Science 310:1016e1018.4. Sampietro ML, et al. (2007) Palaeogenetic evidence supports a dual model of Neolithic spreading into Europe. Proc Biol Sci 274:2161e2167.5. Deguilloux MF, et al. (2010) News from the west: Ancient DNA from a French megalithic burial chamber. Am J Phys Anthropol 5144:108e118.6. Haak W, et al. (2008) Ancient DNA, Strontium isotopes, and osteological analyses shed light on social and kinship organization of the Later Stone Age. Proc Natl Acad Sci USA 105:

18226e18231.7. Lacan M, et al. (2011) Ancient DNA reveals male diffusion through the Neolithic Mediterranean route. Proc Natl Acad Sci USA 108:9788e9791.8. Endicott P, et al. (2009) Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: The singular maternal history of

the Tyrolean Iceman. BMC Genet 10:29.

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Table S3. Percentage of each ancient Y-haplotype in current European populations

G2a E1b1b1a1b

Population (size) No. profiles No. shared haplotype Fq haplotype (%) No. shared haplotype* Fq haplotype (%)

Middle EastIranians 340 0 0 1 0.29Syrians 161 0 0 0 0Druze 283 0 0 38 13.43Palestinians 364 0 0 2 0.55Lebanese 577 2 0.35 14 2.43Turks 663 1 0.15 17 2.56Middle East: total 2,388 3 0.13 72 3.02

CaucasusGeorgians 77 0 0 0 0Armenians 100 0 0 0 0Azerbaijanis 119 0 0 7 5.88Caucasus: total 296 0 0 7 2.36

Northwestern EuropeFrench 100 0 0 0 0Irish 155 0 0 0 0Belgians 113 0 0 0 0Dutch 72 0 0 0 0Northwestern Europe: total 440 0 0 0 0

North-central EuropeGermans 1,414 2 0.14 3 0.21Danish 185 0 0 0 0Czechs 1,750 6 0.34 4 0.23Polish 208 0 0 1 0.48Eastern Slovakians 629 0 0 6 0.95Swiss (Zurich area) 150 0 0 0 0North-central Europe: total 4,336 8 0.18 14 0.32

ScandinaviaNorwegians 1,766 0 0 0 0Swedish 708 1 0.14 1 0.14Scandinavia: total 2,474 1 0.04 1 0.04

Southeastern EuropeHungarians 215 1 0.47 2 0.93Bosnians Herzegovinians 181 0 0 2 1.10Serbians 179 0 0 4 2.23Montenegrins 404 1 0.25 61 15.10Southeastern Europe: total 979 2 0.20 69 7.05

Western MediterraneanNorthern Portuguese 235 0 0 1 0.43Southern Portuguese 78 0 0 0 0Northwestern Spanish 239 0 0 0 0Northeastern Spanish 114 0 0 0 0Southern Spanish 168 1 0.60 0 0Balearic islanders 340 0 0 2 0.59Central Spanish 188 1 0.53 0 0Basque Spanish 115 0 0 0 0Western Mediterranean: total 1,477 2 0.14 3 0.20

Central MediterraneanNorthern Italians 155 2 1.29 0 0Southern Italians 193 0 0 3 1.55Sicilians 491 1 0.20 1 0.20Sardinians 100 0 0 0 0Croatians 200 2 1 0 0Central Mediterranean: total 1,139 5 0.44 4 0.35

Eastern MediterraneanMaltese 50 0 0 0 0Cretans 71 0 0 0 0Cypriots 163 0 0 1 0.61Northern Greeks 183 0 0 5 1.79Central and southern Greeks 73 0 0 0 0Eastern Mediterranean: total 637 0 0 6 0.94

Total 14,166 42 0.30 346 2.44

*All these shared haplotypes were assigned to E1b1b haplogroup thanks to the haplogroup predictor software (1) or were previously described as belonging toE1b1b1a2 or to E1b1b1c haplogroups.

1. Athey W (2005) Haplogroup prediction from Y-STR values using an allele-frequency approach. J Genet Geneal 1:1e7.

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Table S4. Details of modern-day European populations used for comparison

Population (size) No. seq References HVS-I Population (size) No. profiles References Y-STRs

Middle EastIranians 448 (1, 2) Iranians 340 (3, 4)Saudi Arabians 702 (5–7)Syrians 118 (2, 8) Syrians 161 (9)Iraqis 52 (10)Druze 356 (11, 12) Druze 283 (12)Yemenis 115 (13)Kurds 82 (2, 14)Dubai 249 (15)Palestinians 117 (2) Palestinians 364 (9)

Lebanese 577 (16)Turks 340 (17–20) Turks 663 (21, 22)

CaucasusWestern Russians 358 (23, 24)Other North Caucasus

populations353 (2, 11, 20, 25)

Georgians 188 (14, 20, 26) Georgians 77 (4)Armenians 358 (2, 27) Armenians 100 (4)Azerbaijanis 48 (2) Azerbaijanis 119 (3, 4)

Northwestern EuropeBritish 100 (28)French (Brittany, Normandy,

Limousin, Var)62,39,72,37 (29) French 100 (30)

Welsh 92 (31)Cornish 92 (2, 31)Irish 300 (31, 32) Irish 155 (33)

Belgians 113 (34)Dutch 72 (35)

North-Central EuropeGermans 682 (31, 36–40) Germans 1414 (35, 41)Danish 38 (2, 31) Danish 185 (42)Czechs 262 (2, 43) Czechs 1750 (44)Polish 849 (23, 24) Polish 208 (45)Slovakians 335 (27, 46) Eastern Slovakians 629 (47)Swiss 224 (48, 49) Swiss (Zurich area) 150 (50)Latvians 299 (51)Estonians 149 (2, 52, 53)Austrians 99 (54)South Tyrol populations 263 (55)

ScandinaviaNorwegians 626 (2, 56–58) Norwegians 1766 (59)Finns 755 (31, 52, 60-62)

Swedish 708 (63)Southeastern EuropeBulgarians 30 (17)Hungarians 386 (64–66) Hungarians 215 (67)Bosnians 304 (68, 69) Bosnians Herzegovinians 181 (70)Serbians 56 (68) Serbians 179 (71)Romanians 105 (72)

Montenegrins 404 (71)Western MediterraneanNorthern Portuguese 271 (73, 74) Northern Portuguese 235 (75, 76)Central Portuguese 317 (73, 74) Southern Portuguese 78 (75)Southern Portuguese 260 (73, 74)Galicians 135 (74, 77) North Western Spanish 239 (75)Spanish Catalans 133 (78, 79) North Eastern Spanish 114 (75)Andalusians 189 (78, 80, 81) Southern Spanish 168 (75)Balearic islanders 67 (81) Balearic islanders 340 (75, 82)

Central Spanish 188 (75)Basque Spanish 211 (2, 78, 83, 84) Basque Spanish 115 (75)

Central MediterraneanNorthern Italians 319 (85, 86) Northern Italians 155 (87)Central Italians 637 (81, 86, 88, 89)

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Table S4. Cont.

Population (size) No. seq References HVS-I Population (size) No. profiles References Y-STRs

Central Southern Italians 199 (90) Southern Italians 193 (82)Sicilians 106 (91) Sicilians 491 (92, 93)Corsicans 99 (94, 95)Sardinians 234 (81, 96, 97) Sardinians 100 (98)Slovenians 104 (69)Croatians 59 (68) Croatians 200 (99)

Eastern MediterraneanMacedonians 237 (72, 100)Albanians 84 (72, 101)

Maltese 50 (9)Cretans 200 (8, 102) Cretans 71 (103)Cypriots 91 (104) Cypriots 163 (9)Greeks 553 (2, 8, 72, 104, 105) Northern Greeks 280 (106, 107)

Central and southern Greeks 73 (103)

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