the genetic legacy science 2000

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The Genetic Legacy of Paleolithic Homo sapiens sapiens in Extant Europeans: A Y ChromosomePerspectiveAuthor(s): Ornella Semino, Giuseppe Passarino, Peter J. Oefner, Alice A. Lin, SvetlanaArbuzova, Lars E. Beckman, Giovanna de Benedictis, Paolo Francalacci, Anastasia Kouvatsi,Svetlana Limborska, Mladen Marciki, Anna Mika, Barbara Mika, Dragan Primorac, A. SilvanaSantachiara-Benerecetti, L. Luca Cavalli-Sforza and Peter A. UnderhillReviewed work(s):Source: Science, New Series, Vol. 290, No. 5494 (Nov. 10, 2000), pp. 1155-1159Published by: American Association for the Advancement of ScienceStable URL: http://www.jstor.org/stable/3078409 .Accessed: 16/05/2012 07:25

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REPORTSEPORTS

analysis herein, because they are based on eithercompletely sequenced genomes or random sequenc-ing projects.

24. D. Grant, P. Cregan, R. C. Showemaker, Proc. Natl.Acad. Sci. U.S.A. 97, 4168 (2000).

25. This estimate is the average of two surveys based onanalyses of multiple genes in vascular plants (7, 42).

26. L. Skrabanek, K. H. Wolfe, Curr. Opin. Genet. Dev. 8,694 (1998).

27. A. L. Hughes, J. Mol. Evol. 48, 565 (1999).28. A. P. Martin, Am. Nat. 154, 111 (1999).29. P. E. Ahlberg, A. R. Milner, Nature 368, 507 (1994).30. In large genomic databases like those analyzed here,

some sequencing errors may inflate the apparentlevel of divergence, but this error should be indepen-

analysis herein, because they are based on eithercompletely sequenced genomes or random sequenc-ing projects.

24. D. Grant, P. Cregan, R. C. Showemaker, Proc. Natl.Acad. Sci. U.S.A. 97, 4168 (2000).

25. This estimate is the average of two surveys based onanalyses of multiple genes in vascular plants (7, 42).

26. L. Skrabanek, K. H. Wolfe, Curr. Opin. Genet. Dev. 8,694 (1998).

27. A. L. Hughes, J. Mol. Evol. 48, 565 (1999).28. A. P. Martin, Am. Nat. 154, 111 (1999).29. P. E. Ahlberg, A. R. Milner, Nature 368, 507 (1994).30. In large genomic databases like those analyzed here,

some sequencing errors may inflate the apparentlevel of divergence, but this error should be indepen-

dent of S, and in any event, is unlikely to add morethan 0.01 to individual estimates of S. Thus, theimpact of such error on our statistical analysesshould be negligible.

31. This estimate is the average of the results obtained inthree broadly compatible studies, all of which sur-veyed a large number of genes [S. Easteal, C. Collet,Mol. Biol. Evol. 11, 643 (1994); T. Ohta, . Mol. Evol.40, 56 (1995); (7)].

32. A. M. A. Aguinaldo et al., Nature 387, 489 (1997).33. G. M. Rubin t al., Science 287, 2204 (2000).

34. C. Semple, K. H. Wolfe, J. Mol. Evol. 48, 555 (1999).35. C. R. Werth, M. D. Windham, Am. Nat. 137, 515(1991).

36. M. Lynch, A. Force, Am. Nat., in press.

dent of S, and in any event, is unlikely to add morethan 0.01 to individual estimates of S. Thus, theimpact of such error on our statistical analysesshould be negligible.

31. This estimate is the average of the results obtained inthree broadly compatible studies, all of which sur-veyed a large number of genes [S. Easteal, C. Collet,Mol. Biol. Evol. 11, 643 (1994); T. Ohta, . Mol. Evol.40, 56 (1995); (7)].

32. A. M. A. Aguinaldo et al., Nature 387, 489 (1997).33. G. M. Rubin t al., Science 287, 2204 (2000).

34. C. Semple, K. H. Wolfe, J. Mol. Evol. 48, 555 (1999).35. C. R. Werth, M. D. Windham, Am. Nat. 137, 515(1991).

36. M. Lynch, A. Force, Am. Nat., in press.

37. H. A. Orr, Genetics 144, 1331 (1996).38. M. J. D. White, Modes of Speciation (Freeman, San

Francisco, CA, 1978).39. G. Fischer, S. A. James, I. N. Roberts, S. G. Oliver, E.J.

Louis, Nature 405, 451 (2000).40. H. R. Parker, D. P. Philipp, G. S. Whitt, J. Exp. Zool.

233, 451 (1985).41. J. A. Coyne, H. A. Orr, Evolution 51, 295 (1997).42. M. Lynch, Mol. Biol. Evol. 14, 914 (1997).43. This research was supported by NIH grant RO1-

GM36827. We thank A. Force and A.Wagner

forhelpful comments.

22 June 2000; accepted 4 October 2000

37. H. A. Orr, Genetics 144, 1331 (1996).38. M. J. D. White, Modes of Speciation (Freeman, San

Francisco, CA, 1978).39. G. Fischer, S. A. James, I. N. Roberts, S. G. Oliver, E.J.

Louis, Nature 405, 451 (2000).40. H. R. Parker, D. P. Philipp, G. S. Whitt, J. Exp. Zool.

233, 451 (1985).41. J. A. Coyne, H. A. Orr, Evolution 51, 295 (1997).42. M. Lynch, Mol. Biol. Evol. 14, 914 (1997).43. This research was supported by NIH grant RO1-

GM36827. We thank A. Force and A.Wagner

forhelpful comments.

22 June 2000; accepted 4 October 2000

h e e n e t i c L e g a c y o

Paleolithic H o m o s a p i e n s

s a p i e n s in x t a n t Europeans:hromosome Perspective

Ornella Semino,1'2*t Giuseppe Passarino,2'3t Peter J. Oefner,4Alice A. Lin,2 Svetlana Arbuzova,5 Lars E. Beckman,6

Giovanna De Benedictis,3 Paolo Francalacci,7Anastasia Kouvatsi,8 Svetlana Limborska,9 Mladen Marcikiae,'1

Anna Mika,1" Barbara Mika,12 Dragan Primorac,a3A. Silvana Santachiara-Benerecetti,1 L. Luca Cavalli-Sforza,2

Peter A. Underhill2

A genetic perspective of human history in Europe was derived from 22 binarymarkers of the nonrecombining Y chromosome (NRY). Ten lineages account for>95% of the 1007 European Y chromosomes studied. Geographic distributionand age estimates of alleles are compatible with two Paleolithic and oneNeolithic migratory episode that have contributed to the modern Europeangene pool. A significant correlation between the NRY haplotype data and

principal omponentsbased on 95

proteinmarkers was

observed, indicatinghe

effectiveness of NRY binary polymorphisms in the characterization of humanpopulation composition and history.

h e e n e t i c L e g a c y o

Paleolithic H o m o s a p i e n s

s a p i e n s in x t a n t Europeans:hromosome Perspective

Ornella Semino,1'2*t Giuseppe Passarino,2'3t Peter J. Oefner,4Alice A. Lin,2 Svetlana Arbuzova,5 Lars E. Beckman,6

Giovanna De Benedictis,3 Paolo Francalacci,7Anastasia Kouvatsi,8 Svetlana Limborska,9 Mladen Marcikiae,'1

Anna Mika,1" Barbara Mika,12 Dragan Primorac,a3A. Silvana Santachiara-Benerecetti,1 L. Luca Cavalli-Sforza,2

Peter A. Underhill2

A genetic perspective of human history in Europe was derived from 22 binarymarkers of the nonrecombining Y chromosome (NRY). Ten lineages account for>95% of the 1007 European Y chromosomes studied. Geographic distributionand age estimates of alleles are compatible with two Paleolithic and oneNeolithic migratory episode that have contributed to the modern Europeangene pool. A significant correlation between the NRY haplotype data and

principal omponentsbased on 95

proteinmarkers was

observed, indicatinghe

effectiveness of NRY binary polymorphisms in the characterization of humanpopulation composition and history.

Various types of evidence suggest that the

present European population arose from the

merging of local Paleolithic groups and Neo-lithic farmers arriving from the Near East afterthe invention of agriculture in the Fertile Cres-cent (1-5). However, the origin of Paleolithic

European groups and their contribution to the

present gene pool have been debated (6, 7).Assuming no selection, local differentiation oc-curred in isolated and small Paleolithic groupsby drift (8, 9). Range expansions and popula-

tion convergences, which occurred at the end ofthe Paleolithic, were catalyzed by improvedclimate and new technologies and spread the

present genetic characteristics to surroundingareas (8). The smaller effective population sizeof the NRY enhances the consequences of driftand founder effect relative to the autosomes,making NRY variation a potentially sensitiveindex of population composition. Previously,the distribution of two NRY restriction frag-ment length polymorphism (RFLP) markers

suggested Paleolithic and Neolithic contribu-

Various types of evidence suggest that the

present European population arose from the

merging of local Paleolithic groups and Neo-lithic farmers arriving from the Near East afterthe invention of agriculture in the Fertile Cres-cent (1-5). However, the origin of Paleolithic

European groups and their contribution to the

present gene pool have been debated (6, 7).Assuming no selection, local differentiation oc-curred in isolated and small Paleolithic groupsby drift (8, 9). Range expansions and popula-

tion convergences, which occurred at the end ofthe Paleolithic, were catalyzed by improvedclimate and new technologies and spread the

present genetic characteristics to surroundingareas (8). The smaller effective population sizeof the NRY enhances the consequences of driftand founder effect relative to the autosomes,making NRY variation a potentially sensitiveindex of population composition. Previously,the distribution of two NRY restriction frag-ment length polymorphism (RFLP) markers

suggested Paleolithic and Neolithic contribu-

tions to the European gene pool (10). NRY

binary markers (11) representing unique muta-tional events in human history allow a more

comprehensive reconstruction of European ge-netic history.

Twenty-two relevant binary markers [4gathered from the literature and 18 detected

by denaturing high-performance liquid chro-

matography (DHPLC) (12)] were genotypedin 1007 Y chromosomes from 25 different

European and Middle Eastern geographic re-

gions. More than 95% of the samples studiedcould be assigned to haplotypes or clades of

haplotypes defined by just 10 key mutations

(Fig. 1 and Table 1). The frequency distributionof Y chromosome haplotypes revealed heredefines the basic structure of the male compo-nent of the extant European populations and

provides testimony to population history, in-

cluding the Paleolithic period. Two lineages(those characterized by M173 and M170) ap-pear to have been present in Europe since Pa-leolithic times. The remaining lineages entered

tions to the European gene pool (10). NRY

binary markers (11) representing unique muta-tional events in human history allow a more

comprehensive reconstruction of European ge-netic history.

Twenty-two relevant binary markers [4gathered from the literature and 18 detected

by denaturing high-performance liquid chro-

matography (DHPLC) (12)] were genotypedin 1007 Y chromosomes from 25 different

European and Middle Eastern geographic re-

gions. More than 95% of the samples studiedcould be assigned to haplotypes or clades of

haplotypes defined by just 10 key mutations

(Fig. 1 and Table 1). The frequency distributionof Y chromosome haplotypes revealed heredefines the basic structure of the male compo-nent of the extant European populations and

provides testimony to population history, in-

cluding the Paleolithic period. Two lineages(those characterized by M173 and M170) ap-pear to have been present in Europe since Pa-leolithic times. The remaining lineages entered

Europe most likely later during independentmigrations from the Middle East and the Uralsas they are found at higher frequencies and withmore variation of linked microsatellites than inother continents (10-14).

Of the 22 haplotypes that constitute the

phylogeny in Fig. 1 (top), Eul8 and Eul9 char-acterize about 50% of the European Y chromo-somes. Although they share M173, the two

haplotypes show contrasting geographic distri-bution. The frequency of Eul8 decreases fromwest to east, being most frequent in Basques(Fig. 1, bottom, and Table 1). This lineageincludes the previously described proto-Euro-pean lineage that is characterized by the 49a,fhaplotype 15 (10). In contrast, haplotype Eul9,which is derived from the M173 lineage and is

distinguished by M17, is virtually absent inWestern Europe. Its frequency increases east-ward and reaches a maximum in Poland, Hun-

gary, and Ukraine, where Eul8 in turn is virtu-

ally absent. Both haplotypes Eu18 and Eul9share the derived M45 allele. The lineage char-acterized by M3, common in Native Americans

'Dipartimento di Genetica e Microbiologia, Universitadi Pavia, Via Ferrata 1, 27100 Pavia, Italy. 2Depart-ment of Genetics, Stanford University School of Med-icine, 300 Pasteur Drive, Stanford, CA 94305-5120,USA. 3Dipartimento di Biologia Cellulare, Universitadella Calabria, 87030 Rende, Italy. 4Stanford GenomeTechnology Center, 855 California Avenue, Palo Alto,CA 94304, USA. 5lnternational Medico-Genetic Cen-tre, Hospital Nol, 57 Artem Str, 340000 Donetsk,Ukraine. 6Department of Oncology, Pathology andMedical Genetics, University of Umea, S-901 85Umea, Sweden. 7Dipartimento di Zoologia e Antropo-logia Biologica, Universita di Sassari, Via Regina Mar-gherita, 15, 07100 Sassari, Italy. 8Department of Ge-netics, Development and Molecular Biology, AristotleUniversity, 54006 Thessaloniki, Macedonia, Greece.9Institute of Molecular Genetics, Russian Academy of

Sciences, Kurchatov Square, 2, Moscow 123182, Rus-sia. 1Clinical Hospital Center Osijek, Department ofPathology Medical School, J Huttlera 4, 31000 Osijek,Croatia. "Regionalne Centrum Krwiodawstwa i Krwi-olecznictwa w Lublinie-Oddzial w, Zamosciu, ul Le-gionow 10, 22400 Zamosc, Poland. '2SamodzielnyPubliczny Szpital Wojwodzki im. Papieza Jona Pawla IIw, Zamosciu, ul Legionow 10, 22400 Zamosc, Poland.13University Hospital Split, Department of Pediatrics,Laboratory for Clinical and Forensic Genetics, Spi-n6iaeeva 1, 21000 Split, Croatia.

*To whom correspondence should be addressed. E-mail: [email protected] authors contributed equally to this work.

Europe most likely later during independentmigrations from the Middle East and the Uralsas they are found at higher frequencies and withmore variation of linked microsatellites than inother continents (10-14).

Of the 22 haplotypes that constitute the

phylogeny in Fig. 1 (top), Eul8 and Eul9 char-acterize about 50% of the European Y chromo-somes. Although they share M173, the two

haplotypes show contrasting geographic distri-bution. The frequency of Eul8 decreases fromwest to east, being most frequent in Basques(Fig. 1, bottom, and Table 1). This lineageincludes the previously described proto-Euro-pean lineage that is characterized by the 49a,fhaplotype 15 (10). In contrast, haplotype Eul9,which is derived from the M173 lineage and is

distinguished by M17, is virtually absent inWestern Europe. Its frequency increases east-ward and reaches a maximum in Poland, Hun-

gary, and Ukraine, where Eul8 in turn is virtu-

ally absent. Both haplotypes Eu18 and Eul9share the derived M45 allele. The lineage char-acterized by M3, common in Native Americans

'Dipartimento di Genetica e Microbiologia, Universitadi Pavia, Via Ferrata 1, 27100 Pavia, Italy. 2Depart-ment of Genetics, Stanford University School of Med-icine, 300 Pasteur Drive, Stanford, CA 94305-5120,USA. 3Dipartimento di Biologia Cellulare, Universitadella Calabria, 87030 Rende, Italy. 4Stanford GenomeTechnology Center, 855 California Avenue, Palo Alto,CA 94304, USA. 5lnternational Medico-Genetic Cen-tre, Hospital Nol, 57 Artem Str, 340000 Donetsk,Ukraine. 6Department of Oncology, Pathology andMedical Genetics, University of Umea, S-901 85Umea, Sweden. 7Dipartimento di Zoologia e Antropo-logia Biologica, Universita di Sassari, Via Regina Mar-gherita, 15, 07100 Sassari, Italy. 8Department of Ge-netics, Development and Molecular Biology, AristotleUniversity, 54006 Thessaloniki, Macedonia, Greece.9Institute of Molecular Genetics, Russian Academy of

Sciences, Kurchatov Square, 2, Moscow 123182, Rus-sia. 1Clinical Hospital Center Osijek, Department ofPathology Medical School, J Huttlera 4, 31000 Osijek,Croatia. "Regionalne Centrum Krwiodawstwa i Krwi-olecznictwa w Lublinie-Oddzial w, Zamosciu, ul Le-gionow 10, 22400 Zamosc, Poland. '2SamodzielnyPubliczny Szpital Wojwodzki im. Papieza Jona Pawla IIw, Zamosciu, ul Legionow 10, 22400 Zamosc, Poland.13University Hospital Split, Department of Pediatrics,Laboratory for Clinical and Forensic Genetics, Spi-n6iaeeva 1, 21000 Split, Croatia.

*To whom correspondence should be addressed. E-mail: [email protected] authors contributed equally to this work.

www.sciencemag.org SCIENCE VOL 290 10 NOVEMBER 2000www.sciencemag.org SCIENCE VOL 290 10 NOVEMBER 2000 1155155


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(12) and a few Siberian opulations 15), is alsoa derivative f M45. This observation uggeststhat M173 is an ancient Eurasiatic marker hatwas brought by or arose n the group of Homosapiens sapiens who entered Europe and dif-fused from east to west about 40;000 to 35,000years ago (16, 17), spreading he Aurignacculture. This culture also appeared almost si-multaneously n Siberia 17), from which some

M13 YAP-

4064. I .

M2 : M35 :

I I

: :." :o i?

* *

* *

* *

Eul Eu2 Eu3 Eu4 Eu5

RPS4 II

M170 M172

M26

.EuS II

u I-: - I

Eu6--'M l H

REPORTS

groups eventually migrated o the AWe interpret he differentiation i

tribution f haplotypes Eul8 and Eunatures of expansions rom isolatednuclei in the Iberian peninsula and tUkraine, ollowing the Last Glacial(LGM). In fact, during this glac(20,000 to 13,000 years ago), humwere forced o vacate Central Europe

M89

M201 M69

TAT M70

M178

Eu12 MER.'S Eu15

M9

: M1

Fig. 1. (Top) Maximum arsimony hylogeny of the NRY markers ound n Europe nd elsewhere.YAP 32), TAT 14), RPS4 = RPS4YC711T 33)], and 4064 [= SRY4064 34)] were

previouslydescribed. he remaining olymorphisms ere identified with DHPLC 11, 12, 27) and are depositedin the National Center for BioTechnology nformation NCBI)dbSNP database (www.ncbi.nlm.nih.gov/SNP). he phylogeny s rooted with the use of great ape sequences. (Bottom) The 19haplotypes observed Table 1) were pooled nto six classes represented y different olors: Yellowindicates haplotype Eu4; blue includes Eu7 and Eu8, which both involve he M170 mutation; edgroups hree separate haplotypes or reasons explained n the text; pink ncludes haplotypes Eu13and Eu14, which both involve he TAT mutation; and green indicates Eu18 and purple ndicatesEu19,which despite sharing he M173 mutation are distinguished ecause hey represent distinctdichotomy n European hylogeography. he other nine observed haplotypes, which catalog theremaining 5% of the total samples, are shown as black dashed ines and are represented n thewhite sector of relevant pie charts. Three haplotypes, Eu2, Eu5, and Eu21, were not detected. Thepie sectors are proportional o the relative requencies f haplotypes or clades n each population.The two Basque amples have been pooled.

mericas. exception of a refuge in the northern Balkannd the dis- (16). Similar discrete patterns of the flora an119 as sig- fauna n Europe have been attributed o glaciapopulation tion-modulated solation ollowed by dispersahe present from climatic sanctuaries 18). This scenarioMaximum also supported y the finding hat he maximumial period variation or microsatellites inked to Eul9an groups found in Ukraine 19). In turn, the maximume, with the variation for microsatellites inked to 49a

Ht15 and its derivatives and then to the Eu1lineage) s in the Iberian eninsula 19). This iconsistent with the diffusion of M173-markedEul8 from its refuge after he LGM, in agreement with mitochondrial DNA (mtDNA) haplogroup V and some of the H lineages (20

-,--, Haplotype Eul9 has been also observed at sub0124 M3:124

M3 stantial frequency in northern India and Paki-stan (12) as well as in Central Asia (12). Ispread may have been magnified by the expansion of the Yamnaia culture rom the "Kurgaculture" area (present-day southern Ukraine

Eu21 Eu22 into Europe and eastward, resulting in thespread of the Indo-European anguage 21). Aalternative ypothesis of a Middle Eastern orgin of Indo-European anguages was proposeon the basis of archaeological ata (3).

We estimated the age of M173 by usingthe variation of three microsatellites, namelDYS19, YCAIIa, and YCAIIb (22). Athough an estimate of -30,000 years foM173 must be interpreted autiously (23),is consistent with our hypothesis that M173marks the Aurignac settlement in Europe oat least, predates he LGM.

The polymorphism M170 represents nother putative Paleolithic mutation whose age habeen estimated o be -22,000 years (22, 23).With the exception of idiosyncratic distributions indicative of recent gene flow, M170 iconfined o Europe Eu7). The mutation s mostfrequent in central Eastern Europe and alsooccurs in Basques and Sardinians hat haveaccumulated subsequent mutation M26) thadistinguishes Eu8. The closest phylogenetipredecessor s the M89 mutation, rom whichthe most important Middle Eastern lineageoriginated. We propose hat M170 originatedEurope n descendants f men that arrived romthe Middle East 20,000 to 25,000 years agowho have been associated with the Gravettiaculture 16). This migration may have coincided with that of mtDNA haplogroup H to Europe. It has been suggested hat Gravettian nd

Aurignac groupscoexisted for a few thousan

years, maintaining heir dentities despite occasional contacts. During he LGM, Western Europe was isolated rom Central Europe, wheran Epi-Gravettian ulture persisted n the areaof present-day Austria, he Czech Republic, nthe northern Balkans (16). After climatic im-provement, his culture spread north and east(16). This finding is supported y the presenEu7 haplotype distribution. n this scenariohaplotype Eu8 would have originated n thwestern Paleolithic opulation uring he LGM

10 NOVEMBER 000 VOL 290 SCIENCE www.sciencemag.org

1 M45

M173 :h

M17

:.-W.I E

?... I

I

EU16 EU17 bEVUl E-U20

Aginkk. - M

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as local differentiation f the M170 lineage.The frequency and the distribution f haplo-group H across Europe support gene flow be-tween Gravettian nd Western European Aurig-nac groups and suggest differential ender mi-gratory phenomena 24).

The dine of frequencies for haplotypesmarked by M35 (Eu4), M172 (Eu9), M89(EulO), and M201 (Eul 1) decreases rom theMiddle East into Europe. Haplotype Eu4 isphylogenetically distinct from the other threeand defines most European YAP+ chromo-somes. The Eu4 haplotype appears o corre-spond to the previously reported Ht-4, definedby the absence of M2 (25). Comparative eno-typing with the Y chromosome RFLPs 49a,fand 12f2 [(10) and citations herein] revealedthat Eu9 and EulO share he 12f2-derived Kballele, whereas Eull has the ancestral 12f2-10Kb allele. Haplotypes Eu9, EulO, andEul lshare the 49a,f haplotype 8 or its deriva-tives, which are not observed n any of the other16 Eu haplotypes (19), suggesting a sharedcommon

ancestry. Thus,we have

displayedhe

combined requencies f haplotypes Eu9, EulO,and Eull in Fig. 1. By correlation between

REPORTS

Ht-4 - Eu4 and 12f2-8Kb - Eu9 and EulO,the origin of these lineages has been estimatedto be about 15,000 to 20,000 years ago (13). Asimilar date (17,000 years ago) for Eu 1 hasbeen estimated 22, 23). The molecular ge of amutation and its corresponding aplotype mustpredate the demographic migratory event itmarks. The age estimates of these haplotypes,especially considering heir approximation 22,23), cannot distinguish whether they came toEurope before or after he LGM. However, hedecreasing clinal pattern of haplotypes Eu4,Eu9, EulO, and Eul 1 from the Middle East toEurope would not be compatible with the lo-calization of peoples carrying hese Y chromo-somes to refuges during the LGM. If thesehaplotypes were present n Europe before theLGM, we would expect to see a differentiationbetween the European nd Middle Eastern in-eages because of temporal nd spatial solation.Unpublished ata rom a 49a,f system and sev-en short andem repeats STRs) n a large sam-ple of these NRY haplotypes rom Europe andthe Middle East

(19)have revealed hat almost

all the compound haplotypes observed n Eu-rope were included n the smaller ample of the

Middle East 19). A similar esult was observedfor mtDNA haplogroup ,which, although on-sidered Paleolithic, s believed to have beenintroduced o Europe during he Neolithic (6).These observations uggest that the four NRYhaplotypes, as well as mtDNA haplogroup J,had sufficient ime to differentiate n the MiddleEast and then migrate oward Europe n suffi-ciently large numbers o account for most ofthe existing variation. Therefore, haplotypesEu4, Eu9, EulO, and Eu 1 represent he malecontribution f a demic diffusion of farmersfrom the Middle East to Europe. The contribu-tion of the Neolithic farmers o the Europeangene pool seems to be more pronounced longthe Mediterranean oast han n Central Europe.This is evident from Fig. 2, in which we haveplotted he frequencies f haplotypes Eu4, Eu9,Eu10, and Eul 1 against the geographic dis-tances from the Middle East for each pop-ulation. The regression line accounting forMediterranean opulations has a slope that issignificantly different from the other popula-

tions, indicatingthat the diffusion of Neo-

lithic farmers affected Southern more thanCentral Europe.

Table 1. Frequencies in percent) of the haplotypes found in the examined European populations.

Haplotypes*Populationt n

Eul Eu3 Eu4 Eu6 Eu7 Eu8 Eu9 EulO Eu11 Eu12 Eu13 Eu14 Eu15 Eu16 Eu17 Eu18 Eu19 Eu20 Eu21

Andalusian 29 10.3 3.4 6.9 3.4 6.9 3.4 65.5Spanish

Basque 45 2.2 2.2 4.4 2.2 88.9French

Basque 22 9.1 4.5 86.4Catalan 24 4.2 4.2 4.2 8.3 79.2

French 23 8.7 17.4 13.0 4.3 52.2 4.3Dutch 27 3.7 22.2 70.4 3.7German 16 6.2 37.5 50.0 6.2Czech and

Slovakian 45 2.2 15.6 8.9 4.4 2.2 2.2 2.2 35.6 26.7Central-

northernItalian 50 2.0 8.0 14.0 10.0 62.0 4.0

Calabrian 37 2.7 13.5 21.6 10.8 8.0 2.7 5.4 32.4 2.7Sardinian 77 1.3 10.4 1.3 2.6 35.1 5.2 5.2 14.2 1.3 22.1 1.3Croatian 58 6.9 44.8 5.2 1.7 1.7 10.3 29.3Albanian 51 2.0 21.6 19.6 23.5 4.0 2.0 17.6 9.8Greek 76 1.3 22.4 1.3 7.9 21.0 1.3 2.6 1.3 1.3 27.6 11.8Macedonian 20 15.0 20.0 15.0 5.0 10.0 35.0Polish 55 3.6 23.6 16.4 56.4Hungarian 45 8.9 11.1 2.2 2.2 2.2 13.3 60.0Ukrainian 50 4.0 18.0 6.0 4.0 2.0 6.0 2.0 2.0 54.0 2.0

Georgian 6333.3 3.2 30.1 1.6 1.6 1.6 14.3 7.9 6.3

Turkish 30 3.3 13.3 3.3 40.0 3.3 6.6 3.3 3.3 3.3 3.3 6.6 6.6 3.3Lebanese 31 25.8 3.2 3.2 29.0 16.1 3.2 3.2 6.4 9.7Syrian 20 10.0 10.0 5.0 15.0 30.0 5.0 15.0 10.0Saami 24 41.7 41.7 8.3 8.3Udmurt 43 4.7 7.0 4.7 2.3 27.9 4.6 11.6 37.2Mari 46 4.3 6.5 4.3 65.2 6.5 13.0Total 1007

*The aplotypes redefined y he followingmarkers nd he respective erived lleles: ul,M13-C; u3, YAP+, 064-A;Eu4,YAP+, 064-A,M35-C; u6,RPS4-T; u7,M89-T,M170-C;Eu8,M89-T,M170-C,M26-A; u9, M89-T,M172-G; u10, M89-T; u11,M89-T,M201-T; u12,M89-T,M69-C; u13, M89-T,M9-G, AT-C; u14,M89-T,M9-G, AT-C, 178-T; u15,M89-T,M9-G,M70-C; u16, M89-T,M9-G; u17, M89-T,M9-G,M11-G; u18,M89-T,M9-G,M45-A,M173-C; u19, M89-T,M9-G,M45-A,M173-C,M17(delG); u20,M89-T,M9-G,M45-A; u21,M89-T,M9-G,M45-A,M124-T.Haplotypes u2,Eu5, nd Eu22were not observed. tSeveral ampleswere previously escribed 70, 77, 28). Samples ot previouslyexamined ncluded 3 French, 6 Germans, 9 northern talians, 5 Sardinians, 8 Croats, 0 Macedonians rom northern reece, 5 Poles,50 Ukrainians, 0 Syrians, 4 Saami, 3Udmurts, nd 46 Mari.

www.sciencemag.org SCIENCE VOL 290 10 NOVEMBER 2000 1157


5/6

I

a)

I.c

z

'0

>1c:a)23cr)

Europe 14). Neither TAT nor M178 was de-tected in Hungary, where a Uralic language sspoken.

The first two principal components (PC)derived rom the data n Table 1 are shown in

5

4.5

4

3

2

1.5

1

0.5

0

^^--~- ^qL * ? y = -0.3541x + 4.2464- Mediterranean R2= 0.613


6/6

on the extant gene pool from Central Asia

-30,000 years ago. In contrast, Central AsianmtDNA 16223/C haplogroups (I, X, and W)account for only -7% of the contemporarycomposition (26). These discrepancies may bedue in part to the apparent more recent molec-ular age of Y chromosomes relative to otherloci (27), suggesting more rapid replacement of

previous Y chromosomes. Gender-based differ-ential migratory demographic behaviors willalso influence the observed patterns of mtDNAand Y variation (24).

The previously categorized Sardinians,Basques, and Saami outliers (5) share basi-

cally the same Y binary components of theother Europeans. Their peculiar position with

respect to frequency is probably a conse-

quence of genetic drift and isolation. In ad-

dition, our analysis highlights the expansionof the Epi-Gravettian population from thenorthern Balkans.

Almost all of the European Y chromo-somes analyzed in the present study belong to10

lineagescharacterized

by simplebiallelic

mutations. Furthermore, a substantial portionof the European gene pool appears to be of

Upper Paleolithic origin, but it was relocatedafter the end of the LGM, when most of

Europe was repopulated (16).

References and Notes1. P. Menozzi, A. Piazza, L.L.Cavalli-Sforza, cience 201,

786 (1978).2. A. J. Ammerman, L. L. Cavalli-Sforza, The Neolithic

Transition nd the Genetics of Populations n Europe(Princeton Univ. Press, Princeton, NJ, 1984).

3. C. Renfrew, Archaeology and Language; he Puzzle ofIndo-European Origin (Jonathan Cape, London,1987).

4. M. A. Ruhlen, Guide o the World Languages StanfordUniv. Press, Stanford, CA,

1987).5. L. L. Cavalli-Sforza, P. Menozzi, A. Piazza, The Historyand Geography of Human Genes (Princeton Univ.Press, Princeton, NJ, 1994).

6. M. Richards t al., Ann. Hum. Genet. 62, 241 (1998).7. L.Chikhi t al., Proc. Natl. Acad. Sci. U.S.A. 95, 9053

(1998).8. L. L.Cavalli-Sforza, .Menozzi, A. Piazza, Science 259,

639 (1993).9. M. M. Lahr, R. A. Foley, Am. J. Phys. Anthropol. Suppl.

27, 137 (1998).10. 0. Semino, G. Passarino, A. Brega, M. Fellous, A. S.

Santachiara-Benerecetti, m. J. Hum. Genet. 59, 964(1996).

11. P. A. Underhill t al., Nature Genet. 26, 358 (2000).12. P. A. Underhill t al., Genome Res. 7, 996 (1997).13. M. F. Hammer et al., Proc. Natl. Acad. Sci. U.S.A. 97,

6769 (2000).14. T. Zerjal et al., Am. J. Hum. Genet. 60, 1174 (1997).15. J. T. Lell et al., Hum. Genet. 100, 536 (1997).16. M. Otte, in The World at 18000 BP, O. Soffer, C.

Gamble, Eds. (Unwin Hyman, London, 1990), vol. 1,pp. 54-68.

17. R. Klein, Evol. Anthropol. 1, 5 (1992).18. K.J. Willis, R.J. Whittaker, Science 287, 1406 (2000).19. A. S. Santachiara-Benerecetti, npublished data.20. A. Torroni t al., Am. J. Hum. Genet. 62, 1137 (1998).21. M. Gimbutas, n Indo-European nd Indo-Europeans,

G. Cardona, H. M. Hoenigswald, A. M. Senn, Eds.(Univ. of Pennsylvania Press, Philadelphia, PA, 1970),pp. 155-195.

22. The coalescence time for the lineages M173 andMl 70 was calculated on 209 and 73 Y chromosomes,

on the extant gene pool from Central Asia

-30,000 years ago. In contrast, Central AsianmtDNA 16223/C haplogroups (I, X, and W)account for only -7% of the contemporarycomposition (26). These discrepancies may bedue in part to the apparent more recent molec-ular age of Y chromosomes relative to otherloci (27), suggesting more rapid replacement of

previous Y chromosomes. Gender-based differ-ential migratory demographic behaviors willalso influence the observed patterns of mtDNAand Y variation (24).

The previously categorized Sardinians,Basques, and Saami outliers (5) share basi-

cally the same Y binary components of theother Europeans. Their peculiar position with

respect to frequency is probably a conse-

quence of genetic drift and isolation. In ad-

dition, our analysis highlights the expansionof the Epi-Gravettian population from thenorthern Balkans.

Almost all of the European Y chromo-somes analyzed in the present study belong to10

lineagescharacterized

by simplebiallelic

mutations. Furthermore, a substantial portionof the European gene pool appears to be of

Upper Paleolithic origin, but it was relocatedafter the end of the LGM, when most of

Europe was repopulated (16).

References and Notes1. P. Menozzi, A. Piazza, L.L.Cavalli-Sforza, cience 201,

786 (1978).2. A. J. Ammerman, L. L. Cavalli-Sforza, The Neolithic

Transition nd the Genetics of Populations n Europe(Princeton Univ. Press, Princeton, NJ, 1984).

3. C. Renfrew, Archaeology and Language; he Puzzle ofIndo-European Origin (Jonathan Cape, London,1987).

4. M. A. Ruhlen, Guide o the World Languages StanfordUniv. Press, Stanford, CA,

1987).5. L. L. Cavalli-Sforza, P. Menozzi, A. Piazza, The Historyand Geography of Human Genes (Princeton Univ.Press, Princeton, NJ, 1994).

6. M. Richards t al., Ann. Hum. Genet. 62, 241 (1998).7. L.Chikhi t al., Proc. Natl. Acad. Sci. U.S.A. 95, 9053

(1998).8. L. L.Cavalli-Sforza, .Menozzi, A. Piazza, Science 259,

639 (1993).9. M. M. Lahr, R. A. Foley, Am. J. Phys. Anthropol. Suppl.

27, 137 (1998).10. 0. Semino, G. Passarino, A. Brega, M. Fellous, A. S.

Santachiara-Benerecetti, m. J. Hum. Genet. 59, 964(1996).

11. P. A. Underhill t al., Nature Genet. 26, 358 (2000).12. P. A. Underhill t al., Genome Res. 7, 996 (1997).13. M. F. Hammer et al., Proc. Natl. Acad. Sci. U.S.A. 97,

6769 (2000).14. T. Zerjal et al., Am. J. Hum. Genet. 60, 1174 (1997).15. J. T. Lell et al., Hum. Genet. 100, 536 (1997).16. M. Otte, in The World at 18000 BP, O. Soffer, C.

Gamble, Eds. (Unwin Hyman, London, 1990), vol. 1,pp. 54-68.

17. R. Klein, Evol. Anthropol. 1, 5 (1992).18. K.J. Willis, R.J. Whittaker, Science 287, 1406 (2000).19. A. S. Santachiara-Benerecetti, npublished data.20. A. Torroni t al., Am. J. Hum. Genet. 62, 1137 (1998).21. M. Gimbutas, n Indo-European nd Indo-Europeans,

G. Cardona, H. M. Hoenigswald, A. M. Senn, Eds.(Univ. of Pennsylvania Press, Philadelphia, PA, 1970),pp. 155-195.

22. The coalescence time for the lineages M173 andMl 70 was calculated on 209 and 73 Y chromosomes,respectively, by using the variation at YCAIIa, CAIIb,and DYS19 microsatellite loci (28). The variancesrespectively, by using the variation at YCAIIa, CAIIb,and DYS19 microsatellite loci (28). The variances

REPORTS

obtained from these microsatellite data were com-puted with equation 2 from Goldstein et al. (29). Theinitial variance of the CA repeats was considered tobe null. A constant population size of 4500 and ageneration time of 27 years were assumed as sug-gested (29). Mutational rates of 5.6 x 10-4 for YCAIIloci and 1.1 x 10-3 for DYS19 were used [for adiscussion of the mutational rates, see (30)].

23. Many factors confound the estimation of the ages ofbinary mutations based on Y chromosome microsatel-lites. First, he mutation rate of microsatellites s uncer-tain, especially because it is not uniform or all micro-satellites (30). Moreover, here is a difference betweenthe mutation rate measured n pedigrees and the mu-tation rates measured ndirectly hrough phylogeneticanalysis (31). To preserve he comparative if not theabsolute) value of the age estimates, he mutation rateswe used are those most widely accepted currently 30).In addition, t seems that some demographic nd evo-lutionary mechanism reduces he variation of Y chro-mosomes in humans, and as a consequence, datingbased on such data usually ives an underestimate 27).Last, but not least, it is worth noting that the age ofalleles does not correspond o the age of populations,although such estimates can provide nsights.

24. M. T. Seielstad, E. Minch, L L. Cavalli-Sforza, NatureGenet. 20, 278 (1998).

25. M. F. Hammer et al., Genetics 145, 787 (1997).

REPORTS

obtained from these microsatellite data were com-puted with equation 2 from Goldstein et al. (29). Theinitial variance of the CA repeats was considered tobe null. A constant population size of 4500 and ageneration time of 27 years were assumed as sug-gested (29). Mutational rates of 5.6 x 10-4 for YCAIIloci and 1.1 x 10-3 for DYS19 were used [for adiscussion of the mutational rates, see (30)].

23. Many factors confound the estimation of the ages ofbinary mutations based on Y chromosome microsatel-lites. First, he mutation rate of microsatellites s uncer-tain, especially because it is not uniform or all micro-satellites (30). Moreover, here is a difference betweenthe mutation rate measured n pedigrees and the mu-tation rates measured ndirectly hrough phylogeneticanalysis (31). To preserve he comparative if not theabsolute) value of the age estimates, he mutation rateswe used are those most widely accepted currently 30).In addition, t seems that some demographic nd evo-lutionary mechanism reduces he variation of Y chro-mosomes in humans, and as a consequence, datingbased on such data usually ives an underestimate 27).Last, but not least, it is worth noting that the age ofalleles does not correspond o the age of populations,although such estimates can provide nsights.

24. M. T. Seielstad, E. Minch, L L. Cavalli-Sforza, NatureGenet. 20, 278 (1998).

25. M. F. Hammer et al., Genetics 145, 787 (1997).

26. V. Macaulay et al., Am. J. Hum. Genet. 64, 232(1999).

27. P. Shen et al., Proc. Natl. Acad. Sci. U.S.A. 97, 7354(2000).

28. L. Quintana-Murci t al., Ann. Hum. Genet. 63, 153(1999).

29. D. B. Goldstein etal., Mol. Biol. Evol. 13, 1213 (1996).Published erratum appeared in Mol. Biol. Evol. 14,354 (1997).

30. R. Chakraborty, M. Kimmel, D. N. Stivers, L.J. Davison,R. Deka, Proc. Natl. Acad. Sci. U.S.A. 94, 1041 (1997).

31. F. R. Santos et al., Hum. Mol. Genet. 9, 421 (2000).32. M. F. Hammer, Mol. Biol. Evol. 11, 749 (1994).33. A. W. Bergen et al., Ann. Hum. Genet. 63, 63 (1999).34. L.S. Whitfield, J. E. Sulston, P. N. Goodfellow, Nature

378, 379 (1995).35. We thank all the men who donated DNA, K.Kyriakou

for the Syrian samples, H. Cann for the French am-ples, A. Piazza or some of the Italian samples, and G.Brumat or helping us in some blood sample collec-tions. We are also grateful to the anonymous review-ers for their constructive criticisms. Supported byNIH grants GM 28428 and GM 55273 to L.LC.-S. ndby funds from the Italian Ministry of the University"Progetti di ricerca ad interesse Nazionale" and PF"Beni culturali" o A.S.S.-B.

5 April 2000; accepted 25 September 2000

26. V. Macaulay et al., Am. J. Hum. Genet. 64, 232(1999).

27. P. Shen et al., Proc. Natl. Acad. Sci. U.S.A. 97, 7354(2000).

28. L. Quintana-Murci t al., Ann. Hum. Genet. 63, 153(1999).

29. D. B. Goldstein etal., Mol. Biol. Evol. 13, 1213 (1996).Published erratum appeared in Mol. Biol. Evol. 14,354 (1997).

30. R. Chakraborty, M. Kimmel, D. N. Stivers, L.J. Davison,R. Deka, Proc. Natl. Acad. Sci. U.S.A. 94, 1041 (1997).

31. F. R. Santos et al., Hum. Mol. Genet. 9, 421 (2000).32. M. F. Hammer, Mol. Biol. Evol. 11, 749 (1994).33. A. W. Bergen et al., Ann. Hum. Genet. 63, 63 (1999).34. L.S. Whitfield, J. E. Sulston, P. N. Goodfellow, Nature

378, 379 (1995).35. We thank all the men who donated DNA, K.Kyriakou

for the Syrian samples, H. Cann for the French am-ples, A. Piazza or some of the Italian samples, and G.Brumat or helping us in some blood sample collec-tions. We are also grateful to the anonymous review-ers for their constructive criticisms. Supported byNIH grants GM 28428 and GM 55273 to L.LC.-S. ndby funds from the Italian Ministry of the University"Progetti di ricerca ad interesse Nazionale" and PF"Beni culturali" o A.S.S.-B.

5 April 2000; accepted 25 September 2000

Transgene a n d TransposonSilencing in hlamydomonas

reinhardtii y DEAH Box R N

H e l i c a s e

Dancia Wu-Scharf, Byeong-ryoot Jeong, Chaomei Zhang,Heriberto Cerutti*

The molecular mechanism(s) responsible for posttranscriptional gene silencing

and RNA nterference remain poorly understood. We have cloned a gene (Mut6)from the unicellular green alga Chlamydomonas einhardtii hat is required orthe silencing of a transgene and two transposon families. Mut6 encodes aprotein that is highly homologous to RNA helicases of the DEAH-box amily.This protein is necessary for the degradation of certain aberrant RNAs, uch asimproperly processed transcripts, which are often produced by transposons andsome transgenes.

Transgene a n d TransposonSilencing in hlamydomonas

reinhardtii y DEAH Box R N

H e l i c a s e

Dancia Wu-Scharf, Byeong-ryoot Jeong, Chaomei Zhang,Heriberto Cerutti*

The molecular mechanism(s) responsible for posttranscriptional gene silencing

and RNA nterference remain poorly understood. We have cloned a gene (Mut6)from the unicellular green alga Chlamydomonas einhardtii hat is required orthe silencing of a transgene and two transposon families. Mut6 encodes aprotein that is highly homologous to RNA helicases of the DEAH-box amily.This protein is necessary for the degradation of certain aberrant RNAs, uch asimproperly processed transcripts, which are often produced by transposons andsome transgenes.

In plants (1-5) and some fungi (6, 7), theoverexpression r misexpression f transgenescan induce the silencing of homologous se-quences (i.e., transgene and endogenous genesequences) by a process known as posttran-scriptional gene silencing (PTGS). The intro-duction of double-stranded RNA (dsRNA) trig-

gers a similar phenomenon, called RNA inter-ference (RNAi), in a variety of invertebrate andvertebrate species (7-15). The widespread oc-currence of these phenomena in eukaryotessuggests the involvement of one or more ances-

School of Biological Sciences and Plant Science Initia-tive, University of Nebraska-Lincoln, E211 BeadleCenter, Post Office Box 880666, Lincoln, NE 68588,USA.

*To whom correspondence should be addressed. E-mail: [email protected]

In plants (1-5) and some fungi (6, 7), theoverexpression r misexpression f transgenescan induce the silencing of homologous se-quences (i.e., transgene and endogenous genesequences) by a process known as posttran-scriptional gene silencing (PTGS). The intro-duction of double-stranded RNA (dsRNA) trig-

gers a similar phenomenon, called RNA inter-ference (RNAi), in a variety of invertebrate andvertebrate species (7-15). The widespread oc-currence of these phenomena in eukaryotessuggests the involvement of one or more ances-

School of Biological Sciences and Plant Science Initia-tive, University of Nebraska-Lincoln, E211 BeadleCenter, Post Office Box 880666, Lincoln, NE 68588,USA.

*To whom correspondence should be addressed. E-mail: [email protected]

tral mechanisms hat may have evolved to limitthe expression of parasitic elements, such astransposons and viruses (3-8, 10). Althoughseveral models have been proposed o explainPTGS and RNAi (1, 3, 5, 7, 8, 10, 13, 15), andsome genes that are essential or these process-es have been identified (4-6, 8-11), the

molecular machinery responsible for RNArecognition and degradation remains large-ly unexplored.

To gain insight into the molecular mech-anism(s) of PTGS, we have screened for mu-tants defective in this process in Chlamydo-monas reinhardtii. In Chlamydomonas, theRbcS2::aadA::RbcS2 transgene is silenced atboth transcriptional and posttranscriptionallevels (16, 17). This transgene is composedof the coding sequence of the eubacterialaadA gene (conferring spectinomycin resis-

tral mechanisms hat may have evolved to limitthe expression of parasitic elements, such astransposons and viruses (3-8, 10). Althoughseveral models have been proposed o explainPTGS and RNAi (1, 3, 5, 7, 8, 10, 13, 15), andsome genes that are essential or these process-es have been identified (4-6, 8-11), the

molecular machinery responsible for RNArecognition and degradation remains large-ly unexplored.

To gain insight into the molecular mech-anism(s) of PTGS, we have screened for mu-tants defective in this process in Chlamydo-monas reinhardtii. In Chlamydomonas, theRbcS2::aadA::RbcS2 transgene is silenced atboth transcriptional and posttranscriptionallevels (16, 17). This transgene is composedof the coding sequence of the eubacterialaadA gene (conferring spectinomycin resis-

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