Europeans Trace Ancestry To Paleolithic People

Y chromosome data show that living Europeans have deep roots in the region- and researchers say genetic markers may be linked to archaeological cultures

About 8000 years ago, the people living in Franchthi Cave in southern Greece experienced a dramatic change of lifestyle. On the floor of the cave where hunter-gatherers had been dropping stone tools and fishbones for thousands of years, the remains of a new kind of feast appear: traces of wheat, barley, sheep, and goat, which can only be the result of farming and herding animals. Within the next 3000 years, the same abrupt transition ripples through archaeological sites along the shoreline of the Mediterranean, eventually reaching Europe, where settled villages of mud-brick houses appear. "The consequences of the transition were fundamental—village settlement, new beliefs, different social structure," says archaeologist Col-in Renfrew of the University of Cambridge in England. "A behavioral revolution took place."

But which people made that revolutionary European transition? Did farmers move into Europe from the Fertile Crescent in the Middle East, or did local hunter-gatherers learn to trade and farm themselves? And if Neolithic newcomers brought farming technology, did they replace most of the locals, or did those Paleolithic locals survive and become the primary ancestors of modern Europeans?

Now, after years of debate, these questions are being answered not only by ancient remains but also by the genes of living Europeans. In a report on page 1155, an international team reports that a wealth of data from the Y chromosome show that it was the local hunter-gatherers who passed on more of their genes. More than 80% of European men have inherited their Y chromosomes—which are transmitted only from father to son—from Paleolithic ancestors who lived 25,000 to 40,000 years ago. Only 20% of Europeans trace their Y chromosome ancestry to Neolithic farmers. Thus, the genetic template for European men was set as early as 40,000 years ago, then modi fied-but not recast-by the Neolithic farmers about 10,000 years ago.

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Men on the move. Y chromosome data reveal three major migrations into Europe, which researchers tie to known archaeological cultures. At 40,000 years ago (ya), the Aurignacian people moved in (green), followed by the Gravettians 25,000 years ago (blue), and finally the Neolithic farmers (red) 9000 years ago.

These Y chromosome data are "strikingly similar" to new findings on mitochondrial DNA (mtDNA), which is inherited maternal ly, notes evolutionary geneticist Martin Richards of the University ofHuddersfield in England, who led a mtDNA study published in the November issue of the American Jour nal of Human Genetics. "A consensus is emerging on what the genetic data are telling us," says Richards. "After all the debate, this is very exciting and encouraging."

The data from both genetic lineages not only enable researchers to trace the movements of the first farmers, they also paint a remarkably detailed picture of the identity and movements of ancient Europeans. TheY chromosome team, led by geneticists Ornel-la Semino of the University of Pavia in Italy and Giuseppe Passarino of Stanford University, also took the bold step of explicitly connecting genetic and archaeological data—a move that is already drawing some fire. The researchers link two early migrations recorded by the Y chromosome to two Paleolithic cultures, the Aurignacian and Gravettian, each famous for their spectacular art and artifacts (see map). "This paper shows us that molecular genetics is beginning to show us which genetic markers are coordinated with climatic events and population dispersals," says Renfrew.

The earliest glimpse of European genetic origins came from protein markers; more recently, researchers studied the mtDNA of European women. But the results were divided: One group of researchers that included Stanford geneticist L. Luca Cav-alli-Sforza, a co-author of the new Y chromosome study, found similar markers in Europeans and Middle Easterners, which declined from east to west and looked like the signature of the Neolithic expansion. But other researchers proposed that several European genetic markers were too old to have been introduced with the Neolithic newcomers.

The obvious way to reconcile the sometimes heated debate was to look at men's genetic history as recorded on the Y chromosome. By comparing the variations, called polymorphisms or markers, at one site on the chromosome, and the frequency at which those variations occur in different populations, geneticists can sort out which populations are most closely related. They can then build a phylogenetic tree that traces the inheritance of the Y chromosome markers in different populations. And by using average mutation rates, researchers can estimate how long ago particular mutations appeared, thus dating various population splits and movements.

Using samples from 1007 European men, the Y chromosome team got clear results:
Most of the men could be sorted into 10 different Y chromosome variants or haplotypes. The researchers sorted those haplotypes on a phylogenetic tree and used the geographic distributions of modem markers to trace the evolution and spread of the ancient markers. For example, they found that four modern haplotypes, which account for 80% of European men's Y chromosomes, were descended from two now-vanished haplotypes. One, Ml 73, arose more than 40,000 years ago from an even older marker called M45. Apparently M45 was present in men living in Asia, for other descendants of this haplotype are now seen in Siberians and Native Americans. Meanwhile, the descendants of the Ml 73 marker are found at the highest frequency today in Europe. So the researchers conclude that Ml 73 is an ancient Eurasiatic marker that moved into Europe about 35,000 to 40,000 years ago.

The authors note that this is just the time of the advent of the Aurig-nacian, an advanced culture that reached its height in Western Europe about 35,000 years ago and is well-known for its sophisticated rock-art paintings and finely crafted tools of antler, bone, and ivory. Archaeologists have hotly debated whether these people originally came from Europe, Asia, or the Middle East. Now the authors propose that haplotype Ml 73 is the "signature of the Aurignacian," and that these people came from central Asia. If the team is right, then half of modern European men still carry the genetic signature of these ancient artists.

Using similar reasoning, the researchers report that the next wave of migration into Europe, marked by a mutation known as Ml 70, occurred about 22,000 years ago from the Middle East. The authors link this wave to the so-called Gravettian culture, known for its Venus figurines and small, delicate blades, which first appeared in the area that is now Austria, the Czech Republic, and the northern Balkans. But archaeologist Alison Brooks of George Washington University in Washington, D.C., warns that there were many cultures in Europe at these times, such as the Solutrean from Iberia, and that it's risky to link genes to a particular culture.

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Once in Europe, the timing and geographical distribution of markers suggests that Aurignacian people dominated Western and southern Europe, while the Gravettian people thrived in Eastern and Central Europe. But when the climate worsened during the Last Glacial Maximum 24,000 to 16,000 years ago, people carrying the "Aurignacian" marker apparently concentrated in refuges in the Iberian peninsula and the Ukraine. Meanwhile, the Gravettian people apparently moved to the Balkans. After the glaciers retreated, the geneticists say that these people moved out of the refuges and their populations expanded rapidly. That fast expansion is why these markers now account for such a large proportion—80%—of modern Europeans'Y chromosomes.

Finally, another migration occurred maked by four new mutations about 9000 years ago, apparently in men coming from the Middle East. But only about 20% of Europeans have these Neolithic markers.

The authors tie this migration to the spread of farming out of the Fertile Crescent, as seen in the archaeological record. The distribution of markers even suggests something about the route the ancient farmers took: There's more Paleolithic [markers] in the North of Europe than the south and more Neolihic in the south," says Cavalli-Sforza. I believe at least part of the Neolitic people went by boat along the coast.

The new much the same tale, say '"o of European women Paleolithic markers and 'ihic markers—although olithic haplotypes are ' along the Mediterranc, iiding that could reflect the different movement of the sexes. But the mtDNA data also suggest the presence of ice age refuges in Iberia and, to a lesser extent, southern Europe. "This fits completely with the mitochondrial data that show an expansion out of Iberia," says Antonio Torroni, a geneticist at the University of Urbino in Italy who proposed the idea of an Iberian refuge in 1998.

The new Y chromosome data enhance the existing picture, says Renfrew. "The mitochondrial work showed us the way, but the Y is making it even more clear," as the Y chromosome data reveal geographical sources of origin more clearly. This is probably because in many societies women move to join their husband's families, while related men cluster more closely geographically. And because some men have many, many children, they leave more offspring with identical Y chromosomes—and a sharp geographical signal.

But those features also mean that there is less diversity in Y chromosome lineages around the world than in mtDNA, notes Cav-alli-Sforza. That lack of diversity makes dating the Y chromosome mutations more difficult: In their calculations, researchers assume that low genetic diversity means that less time has passed—but instead, men's mating habits might be creating a pool of very similar DNA and swamping the data. That would cause researchers to underestimate the age of genetic and population events.

Some researchers are particularly wary of connecting these roughly dated markers to cultures known from the archaeological record. Although he praises the basic Y chromosome results, "I don't like attaching genetics to archaeological evidence," says Mark Jobling, a geneticist at the University of Leicester in England who also studies the Y chromosome in Europeans. "It appeals to the imagination, but the mutation rates on the Y [and therefore the dating of genetic events] have wide confidence margins."

Cavalli-Sforza agrees that genetic dates have large margins of error. But because even these preliminary dates from different genetic lineages correspond well with each other and with major migrations suggested by the archaeological record, it is hard to resist making the connections. "Genetic dating is in its infancy," says Cavalli-Sforza. "We have to start somewhere. The future will bring new evidence."

-ANN GIBBONS


The Genetic Legacy of Paleolithic Homo sapiens sapiens in Extant Europeans: A Y Chromosome Perspective

Ornella Semino,1-2*! Giuseppe Passarino,2-3! Peter J. Oefner,4 Alice A. Lin,2 Svetlana Arbuzova,5 Lars E. Beckman,6 Ciovanna De Benedictis,3 Paolo Francalacci,7 Anastasia Kouvatsi,8 Svetlana Limborska,9 Miaden Marcikiai,10 Anna Mika,11 Barbara Mika,12 Dragan Primorac,13 A. Silvana Santachiara-Benerecetti,1 L. Luca Cavalli-Sforza,2 Peter A. Underbill2

^ipartimento di Genetica e Microbiologia, Universita di Pavia, Via Ferrata 1, 27100 Pavia, Italy. -'Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA. ^ipartimento di Biologia Cellulare, Universita delta Calabria, 87030 Rende, Italy. *Stanford Genome Technology Center, 855 California Avenue, Palo Alto, CA 94304, USA. international Medico-Genetic Centre, Hospital Nol, 57 Artem Str, 340000 Donetsk, Ukraine. Department of Oncology, Pathology and Medical Genetics, University of Umea; S-901 85 Umea, Sweden. ''Dipartimento di Zoologia e Antropo-logia Biologica, Universita di Sassari, Via Regina Mar-gherita, 15, 07100 Sassari, Italy. ''Department of Genetics, Development and Molecular Biology, Aristotle University, 54006 Thessaloniki, Macedonia, Greece. 'Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Square, 2, Moscow 123182, Russia. ^Clinical Hospital Center Osijek, Department of Pathology 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. "Samodzielny Publiczny Szpital Wojwodzki im. Papieza Jona Pawla II w, Zamosciu, ul Legionow 10, 22400 Zamosc, Poland. "University Hospital Split, Department of Pediatrics, Laboratory for Clinical and Forensic Genetics, Spi-neiaeeva 1, 21000 Split, Croatia.

*To whom correspondence should be addressed. E-mail: semino@>ipvgen.univp.it

f}These authors contributed equally to this work.


A genetic perspective of human history in Europe was derived from 22 binary markers of the nonrecombining Y chromosome (NRY). Ten lineages account for >95% of the 1007 European Y chromosomes studied. Geographic distribution and age estimates of alleles are compatible with two Paleolithic and one Neolithic migratory episode that have contributed to the modern European gene pool. A significant correlation between the NRY haplotype data and principal components based on 95 protein markers was observed, indicating the effectiveness of NRY binary polymorphisms in the characterization of human population composition and history.

Various types of evidence suggest that the present European population arose from the merging of local Paleolithic groups and Neolithic farmers arriving from the Near East after the invention of agriculture in the Fertile Crescent (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 occurred in isolated and small Paleolithic groups by drift (8, 9). Range expansions and population convergences, which occurred at the end of the Paleolithic, were catalyzed by improved climate and new technologies and spread the present genetic characteristics to surrounding areas (8). The smaller effective population size of the NRY enhances the consequences of drift and founder effect relative to the autosomes, making NRY variation a potentially sensitive index of population composition. Previously, the distribution of two NRY restriction fragment length polymorphism (RFLP) markers suggested Paleolithic and Neolithic contributions to the European gene pool (/0). NRY binary markers (11) representing unique muta-tional events in human history allow a more comprehensive reconstruction of European genetic history.

Twenty-two relevant binary markers [4 gathered from the literature and 18 detected by denaturing high-performance liquid chro-matography (DHPLC) (12}} were genotyped in 1007 Y chromosomes from 25 different European and Middle Eastern geographic regions. More than 95% of the samples studied could be assigned to haplotypes or clades of haplotypes defined by just 10 key mutations (Fig. 1 and Table 1). The frequency distribution of Y chromosome haplotypes revealed here defines the basic structure of the male component of the extant European populations and provides testimony to population history, including the Paleolithic period. Two lineages (those characterized by Ml 73 and Ml 70) appear to have been present in Europe since Paleolithic times. The remaining lineages entered Europe most likely later during independent migrations from the Middle East and the Urals as they are found at higher frequencies and with more variation of linked microsatellites than in other continents (10-14}.

Of the 22 haplotypes that constitute the phylogeny in Fig. 1 (top), Eul8 and Eul9 characterize about 50% of the European Y chromosomes. Although they share Ml 73, the two haplotypes show contrasting geographic distribution. The frequency of Eul8 decreases from west to east, being most frequent in Basques (Fig. 1, bottom, and Table 1). This lineage includes the previously described proto-Euro-pean lineage that is characterized by the 49a,f haplotype 15 (Iff). In contrast, haplotype Eul9, which is derived from the Ml 73 lineage and is distinguished by M17, is virtually absent in Western Europe. Its frequency increases eastward and reaches a maximum in Poland, Hungary, and Ukraine, where Eul8 in turn is virtually absent. Both haplotypes Eul8 and Eul9 share the derived M45 allele. The lineage characterized by M3, common in Native Americans (12) and a few Siberian populations (75), is also a derivative ofM45. This observation suggests that Ml 73 is an ancient Eurasiatic marker that was brought by or arose in the group of Homo sapiens sapiens who entered Europe and diffused from east to west about 40,000 to 35,000 years ago (16, 17), spreading the Aurignac culture. This culture also appeared almost simultaneously in Siberia (17), from which some groups eventually migrated to the Americas.

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Fig. 1. (Top) Maximum parsimony phytogeny of the NRY markers found in Europe and elsewhere. YAP (32). TAT (74), RPS4 [= RPS4YC711T (33)}. and 4064 [= SRY4064 (34)] were previously described. The remaining polymorphisms were identified with DHPLC (77,12. 27) and are deposited in the National Center for BioTechnotogy Information (NCBI) dbSNP database (www.ncbi.nlm. nih.gov/SNP). The phytogeny is rooted with the use of great ape sequences. (Bottom) The 19 haplotypes observed (Table 1) were pooled into six classes represented by different colors: Yellow indicates haplotype Eu4; blue includes Eu7 and Eu8, which both involve the M170 mutation; red groups three separate haplotypes for reasons explained in the text; pink includes haptotypes Eu13 and Eu14, which both involve the TAT mutation; and green indicates Eu18 and purple indicates Eu19, which despite sharing the M173 mutation are distinguished because they represent a distinct dichotomy in European phylogeography. The other nine observed haplotypes, which catalog the remaining <5% of the total samples, are shown as black dashed lines and are represented in the white sector of relevant pie charts. Three haplotypes, Eu2, Eu5, and Eu21, were not detected. The pie sectors are proportional to the relative frequencies of haplotypes or clades in each population. The two Basque samples have been pooled.

We interpret the differentiation and the distribution ofhaplotypes Eul8 and Eul9 as signatures of expansions from isolated population nuclei in the Iberian peninsula and the present Ukraine, following the Last Glacial Maximum (LGM). In fact, during this glacial period (20,000 to 13,000 years ago), human groups were forced to vacate Central Europe, with the exception of a refuge in the northern Balkans (16). Similar discrete patterns of the flora and fauna in Europe have been attributed to glacia-tion-modulated isolation followed by dispersal from climatic sanctuaries (18). This scenario is also supported by the finding that the maximum variation for microsatellites linked to Eu19 is found in Ukraine (19). In turn, the maximum variation for microsatellites linked to 49a,f Htl5 and its derivatives (and then to the Eu18 lineage) is in the Iberian peninsula (19). This is consistent with the diffusion ofM173-marked Eu18 from its refuge after the LGM, in agreement with mitochondrial DNA (mtDNA) hap-logroup V and some of the H lineages (20). Haplotype Eu19 has been also observed at substantial frequency in northern India and Pakistan (12) as well as in Central Asia (12). Its spread may have been magnified by the expansion of the Yamnaia culture from the "Kurgan culture" area (present-day southern Ukraine) into Europe and eastward, resulting in the spread of the Indo-European language (21). An alternative hypothesis of a Middle Eastern origin of Indo-European languages was proposed on the basis of archaeological data (3).

We estimated the age of M173 by using the variation of three microsatellites, namely DYS19, YCAIIa, and YCAIIb (22). Although an estimate of —30,000 years for M173 must be interpreted cautiously (23), it is consistent with our hypothesis that M173 marks the Aurignac settlement in Europe or, at least, predates the LGM.

The polymorphism M170 represents another putative Paleolithic mutation whose age has been estimated to be —22,000 years (22, 23). With the exception of idiosyncratic distributions indicative of recent gene flow, M170 is confined to Europe (Eu7). The mutation is most frequent in central Eastern Europe and also occurs in Basques and Sardinians that have accumulated a subsequent mutation (M26) that distinguishes Eu8. The closest phylogenetic predecessor is the M89 mutation, from which the most important Middle Eastern lineages originated. We propose that M170 originated in Europe in descendants of men that arrived from the Middle East 20,000 to 25,000 years ago, who have been associated with the Gravettian culture (76). This migration may have coincided with that of mtDNA haplogroup H to Europe. It has been suggested that Gravettian and Aurignac groups coexisted for a few thousand years, maintaining their identities despite occasional contacts. During the LGM, Western Europe was isolated from Central Europe, where an Epi-Gravettian culture persisted in the area of present-day Austria, the Czech Republic, and the northern Balkans (16). After climatic improvement, this culture spread north and east (76). This finding is supported by the present Eu7 haplotype distribution. In this scenario, haplotype Eu8 would have originated in the western Paleolithic population during the LGM, as local differentiation of the Ml 70 lineage. The frequency and the distribution of haplo-group H across Europe support gene flow between Gravettian and Western European Aurig-nac groups and suggest differential gender migratory phenomena (24).

The cline of frequencies for haplotypes marked by M35 (Eu4), M172 (Eu9), M89 (EulO), and M201 (Eull) decreases from the Middle East into Europe. Haplotype Eu4 is phylogenetically distinct from the other three and defines most European YAP^ chromosomes. The Eu4 haplotype appears to correspond to the previously reported Ht-4, defined by the absence of M2 (25). Comparative geno-typing with the Y chromosome RFLPs 49a,f and 12f2 [(10) and citations therein] revealed that Eu9 and EulO share the 12f2-derived 8Kb allele, whereas Eull has the ancestral 12f2-10Kb allele. Haplotypes Eu9, EulO, and Eull share the 49a,f haplotype 8 or its derivatives, which are not observed in any of the other 16 Eu haplotypes (19), suggesting a shared common ancestry. Thus, we have displayed the combined frequencies of haplotypes Eu9, EulO, and Eull in Fig. 1. By correlation between Ht-4 ^ Eu4 and 12f2-8Kb ^ Eu9 and EulO, the origin of these lineages has been estimated to be about 15,000 to 20,000 years ago (13). A similar date (17,000 years ago) for Eull has been estimated (22, 23). The molecular age of a mutation and its corresponding haplotype must predate the demographic migratory event it marks. The age estimates of these haplotypes, especially considering their approximation (22, 23), cannot distinguish whether they came to Europe before or after the LGM. However, the decreasing clinal pattern of haplotypes Eu4, Eu9, EulO, and Eull from the Middle East to Europe would not be compatible with the localization of peoples carrying these Y chromosomes to refuges during the LGM. If these haplotypes were present in Europe before the LGM, we would expect to see a differentiation between the European and Middle Eastern lineages because of temporal and spatial isolation. Unpublished data from a 49a,f system and seven short tandem repeats (STRs) in a large sample of these NRY haplotypes from Europe and the Middle East (19) have revealed that almost all the compound haplotypes observed in Europe were included in the smaller sample of the Middle East (19). A similar result was observed for mtDNA haplogroup J, which, although considered Paleolithic, is believed to have been introduced to Europe during the Neolithic (6). These observations suggest that the four NRY haplotypes, as well as mtDNA haplogroup J, had sufficient time to differentiate in the Middle East and then migrate toward Europe in sufficiently large numbers to account for most of the existing variation. Therefore, haplotypes Eu4, Eu9, EulO, and Eull represent the male contribution of a demic diffusion of farmers from the Middle East to Europe. The contribution of the Neolithic farmers to the European gene pool seems to be more pronounced along the Mediterranean coast than in Central Europe. This is evident from Fig. 2, in which we have plotted the frequencies of haplotypes Eu4, Eu9, EulO, and Eul 1 against the geographic distances from the Middle East for each population. The regression line accounting for Mediterranean populations has a slope that is significantly different from the other populations, indicating that the diffusion of Neolithic farmers affected Southern more than Central Europe.

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While allelotyping M35 by DHPLC, we found a previously unknown mutation, M178, in 95% of all TAT chromosomes. The latter has been reported to be —4000 years old and marks a recent Uralic migration confined to Northern Europe (14). Neither TAT nor M178 was detected in Hungary, where a Uralic language is spoken.

The first two principal components (PC) derived from the data in Table 1 are shown in Fig. 3. The Udmurts, Mari, and Saami were excluded because they monopolizea the first PC and compressed the rest of the variation because of their high TAT/MI 78 frequency. In the plot, it is possible to see three clusters of distinct geography and culture. The first comprises Basques and Western Europeans, the second Middle Eastern, and the third Eastern European populations from Croatia, Ukraine, Hungary, and Poland. These three geographic clusters correspond to the major glacial refuges and to the region of origin of the farmers' expansion.

The most comprehensive previous survey of the European gene pool has been the PC analysis of 95 autosomal protein polymorphisms (5, 8). We compared the frequency distribution of the major Eu Y chromosome haplotypes with the first three PCs of Europe (Table 2). Because Sardinians were not included in the original PC analysis because of their pronounced outlier phylogenetic status (5), they were also excluded in our correlation analysis. The first PC, which was proposed to reflect the diffusion of Neolithic farmers (5, 8), correlates with Eu4, Eu9, EulO, and Eul 1. The second PC, whose meaning has never been fully assessed (5, 8), is correlated with the spread of Eul 8 from Spain toward Central Europe and, on the opposite pole, with the spread of Uralic TAT/MI 78 (Eul3 and Eul4). The third PC, the meaning of which has been debated (3, 5, 8), correlates to the Ml 7 mutation (Eul 9). The concordance of protein-based PC and NRY data suggests that migration, more than natural selection, has influenced the pattern of NRY variation observed.

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Analyses of mtDNA sequence variation in European populations have been conducted (6, 20). These data suggest that the gene pool has -80% Paleolithic and -20% Neolithic ancestry. Our data support this observation because haplotypes Eu4, Eu9, EulO, and Eul 1 account for —22% of European Y chromosomes. Thus, the mtDNA and Y data corroborate the previous observation that the first PC of the 95 classical polymorphisms accounts for —28% of the overall genetic variation (5, 6). However, some differences exist between the mtDNA and Y data pertaining to the putative Paleolithic components. It has been proposed that mtDNA haplogroup U5 arrived from the Middle East 45,000 years ago (6, 26). We did not detect any corresponding Y haplotypes. Furthermore, most European mtDNA lineages, which account for 60 to 70% of the variation in Europe, have been interpreted as having arrived from the Middle East during the Paleolithic about 25,000 years ago (6). Correspondingly, -20% of contemporary Y lineages characterized by the Ml 70 mutation derive from deep phylogenetic M89 ancestry, consistent with a Middle Eastern Paleolithic heritage. Moreover, the remaining —50% of Y lineages associated with the Ml 73 mutation indicate a major influence on the extant gene pool from Central Asia —30,000 years ago. In contrast. Central Asian mtDNA 16223/C haplogroups (1, X, and W) account for only —7% of the contemporary composition (26). These discrepancies may be due in part to the apparent more recent molecular age of Y chromosomes relative to other loci (27), suggesting more rapid replacement of previous Y chromosomes. Gender-based differential migratory demographic behaviors will also influence the observed patterns of mtDNA and Y variation (24).

The previously categorized Sardinians, Basques, and Saami outliers (5) share basically the same Y binary components of the other Europeans. Their peculiar position with respect to frequency is probably a consequence of genetic drift and isolation. In addition, our analysis highlights the expansion of the Epi-Gravettian population from the northern Balkans.

Almost all of the European Y chromosomes analyzed in the present study belong to 10 lineages characterized by simple biallelic mutations. Furthermore, a substantial portion of the European gene pool appears to be of Upper Paleolithic origin, but it was relocated after the end of the LGM, when most of Europe was repopulated (16).

References and Notes

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2. A. J. Ammerman, L. L. Cavalli-Sforza, The Neolithic Transition and the Genetics of Populations in Europe (Princeton Univ. Press, Princeton, NJ, 1984).

3. C. Renfrew, Archaeology and Language; the Puzzle of Indo-European Origin (Jonathan Cape, London, 1987).

4. M. A. Ruhlen, Cuide to the World Languages (Stanford Univ. Press, Stanford, CA, 1987).

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

6. M. Richards et at.. Ann. Hum. Genet. 62. 241 (1998).

7. L. Chikhi et at.. Proc. Natt. Acad. Sci. U.S.A. 95, 9053 (1998).

8. L. L. Cavalli-Sforza, P. 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, C. Passarino, A. Brega, M. Fellous, A. S. Santachiara-Benerecetti, Am. j. Hum. Genet. 59, 964 (1996).

11. P. A. Underbill et ai. Nature Genet. 26, 358 (2000).

12. P. A. Underbill et ai. Cenome Res. 7, 996 (1997).

13. M. F. Hammer et a/., Proc. Natt. Acad. Sci. U.S.A. 97, 6769 (2000).

14. T. Zerjal et at.. Am. J. Hum. Genet. 60, 1174 (1997).

15. J. T. Leil et a/., Hum. Genet. 100, 536 (1997).

16. M. Otte, in The World at 18000 BP. 0. Softer, 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, unpublished data.

20. A.Torronieta(.,/tm../.Hum. Genet. 62, 1137(1998).

21. M. Cimbutas, in Indo-European and 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 and M170 was calculated on 209 and 73 Y chromosomes, respectively, by using the variation at YCAIIa, YCAIIb, and DYS19 microsatellite loci (28). The variances obtained from these microsatellite data were computed with equation 2 from Goldstein et at. (29). The initial variance of the CA repeats was considered to be null. A constant population size of 4500 and a generation time of 27 years were assumed as suggested (29). Mutational rates of 5.6 X 10-4 for YCAII loci and 1.1 X 10'3 for DYS19 were used [for a discussion of the mutational rates, see (30)].

23. Many factors confound the estimation of the ages of binary mutations based on Y chromosome microsatel-lites. First, the mutation rate of microsatellites is uncertain, especially because it is not uniform for all micro-satellites (30). Moreover, there is a difference between the mutation rate measured in pedigrees and the mutation rates measured indirectly through phylogenetic analysis (37). To preserve the comparative (if not the absolute) value of the age estimates, the mutation rates we used are those most widely accepted currently (30). In addition, it seems that some demographic and evolutionary mechanism reduces the variation of Y chromosomes in humans, and as a consequence, dating based on such data usually gives an underestimate (27). Last, but not least, it is worth noting that the age of alteles does not correspond to the age of populations, although such estimates can provide insights.

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

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

26. V. Macaulay et ai.. Am. j. Hum. Genet. 64, 232 {1999).

27. P. Shen et ai. Proc. Natl. Acad. Sci. U.S.A. S7, 7354 (2000).

28. L, Quintana-Murci et at.. Ann. Hum. Genet. 63, 153 (1999).

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

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

31. F. R. Santos et at.. Hum. Mot. Genet. 9, 421 (2000).

32. M. F. Hammer, Mo(. 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 samples, A. Piazza for some of the Italian samples, and G. Brumat for helping us in some blood sample collections. We are also grateful to the anonymous reviewers for their constructive criticisms. Supported by NIH grants GM 28428 and GM 55273 to LL.C.-S. and by funds from the Italian Ministry of the University "Progetti di ricerca ad interesse Nazionale" and PF "Beni cultural!" to A.S.S.-B.



5 April 2000; accepted 25 September 2000

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