Su et al. Modern Human Migration in Eastern Asia 1719 combinant part of the y chromosome allow the recon- derhill, P Shen, A. A Lin, L. Jin, G. Passarino, W.H. struction of intact haplotypes, which are not likely to Yang, E. Kauffman, E.S. Dietrich, J. Kidd, S.Q. Mehdi be eroded by recombination and recurrent mutation and T. Jenkins, R S. Wells, M. T Seielstad, M. Ibrahim, P. which are, therefore, highly informative for tracing an- Francalacci, J. Bertranpetit, R. W. Davis, L. L. Cavalli cient human migrations. Given the fact that Y chro- Sforza, and P. J. Oefner, unpublished data), since they mosomes have a smaller effective population size are polymorphic in the individuals of eastern-Asian or- than do autosomes, Y-chromosome-specific polymor- igin, in the screening set used. For genotyping, an allelic- phic markers are probably the best genetic tool to study specific PCR assay was used for Y-chromosome biallelic early human migrations as bottleneck events that are markers. For each Y locus, two allele-specific primers often associated with such migrations become more pro- were designed to recognize two different alleles at this nouns locus(the primer sequences are available on request) In this study, a set of Y-chromosome biallelic and mi- After PCR, the products were visualized through agarose crosatellite markers were used to examine the genetic gel electrophoresis. In addition, three Y-chromosome mi structure of eastern-Asian populations. The Y-chromo- crosatellite loci-DYS389, DYS390, and DYS391 some haplotype distribution in extant eastern-Asian were also typed as described by Kayser et al. ( 1997) populations was used to reconstruct ancient migration The phylogenetic tree for the Y-chromosome haplotypes patterns within this region was constructed on the basis of the parsimony rule, and multifurcation was introduced to accommodate equally Material and Methods DNA Sample Statistica/ Method From worldwide populations, we collected 925 male To estimate the age of M122C haplotypes in the Han DNA samples, of which 739 were from eastern-Asian Chinese, we used the equation t=-N In(1-V/N m) populations. The collection of DNA samples from mem- We derived this equation from the single- step mutation bers of 21 Chinese ethnic-minority populations was model for a haploid population, assuming that popu- done with the coordination of the Chinese Human Ge- lation size (where N is the effective population size whe ected toa y Project. The Chinese Han samples were stayed constant, that V is the variance of repeat numbers n persons living in 22 provincial areas in the population, and that m is the mutation rate. If ose geographic origins were assigned according to the the population undergoes a strong bottleneck event fol birthplaces of their four grandparents. In addition, sam- lowed by a rapid population expansion, it can be shown ples obtained in previous projects were analyzed, in- that this formula is still approximately valid. A widely cluding those from 3 northeast-Asian(Buryat, Korean, accepted estimation of the effective population size of and Japanese)and 5 southeast-Asian( Cambodian, Thai, modern humans is 5,000-10,000, suggested by Taka Malaysian, Batak, and Javanese) populations and from hata(1993). Given a relatively smaller genetic diversity an additional 12 non-Asian populations (3 from Africa, in Asia compared with that seen in Africa, a value of 3 from America, 2 from Europe, and 4 from Oceania) 2,000 is a drastic overestimation of the effective popu (fig. 1 and table 1) lation size for males in eastern Asia. For the level of variance observed in 160 individuals in this study, 750 Genotyping and Phylogenetic-Tree Construction is the minimum integer allowed for the effective popu a total of 19 Y-chromosome biallelic loci were lation size, for the variance observed careened. Seven of them were from previous reports (Vollrath et al. 1992; Underhill et al. 1996, 1997a; Kay. Results ser et al. 1997), including M3(C-T mutation), M (A-G mutation), M7(CG mutation), M9(CG mu- In all the individuals studied for the 19 Y-chromosome tation), M15 (9-bp insertion), M17(1-bp deletion), and biallelic markers, 17 Y haplotypes were obtained. The DYS287(YAP). The other 12 single-nucleotide poly- frequency distribution of Y haplotypes in all the pop morphisms are being first reported here, including M4.5 ulations is listed in table 1. Under the parsimony as (G-A mutation), M50(TC mutation), M88(AG sumption, no recurrent mutations were observed, and a mutation), M89(CT mutation), M95(CT mutation), phylogenetic tree was constructed for the 17 Y haplo M103(C-T mutation), M110(T-+C mutation), Mlll types, in which H1 was considered as the ancestor hap- (4-bp deletion), M119(AC mutation), M120(T-C lotype because of its appearance in chimpanzee(fig. 2) mutation), M122(T-C mutation), and M134 (1-bp de- The H1 and H2 haplotypes are relatively ancient, ap letion). The 19 markers used in this study were selected pearing in both African and non-African populations, from 166 biallelic Y-chromosome markers(P. A. Un- implying that the occurrence of the mutation definingSu et al.: Modern Human Migration in Eastern Asia 1719 combinant part of the Y chromosome allow the recon￾struction of intact haplotypes, which are not likely to be eroded by recombination and recurrent mutation and which are, therefore, highly informative for tracing an￾cient human migrations. Given the fact that Y chro￾mosomes have a smaller effective population size than do autosomes, Y-chromosome–specific polymor￾phic markers are probably the best genetic tool to study early human migrations as bottleneck events that are often associated with such migrations become more pro￾nounced. In this study, a set of Y-chromosome biallelic and mi￾crosatellite markers were used to examine the genetic structure of eastern-Asian populations. The Y-chromo￾some haplotype distribution in extant eastern-Asian populations was used to reconstruct ancient migration patterns within this region. Material and Methods DNA Samples From worldwide populations, we collected 925 male DNA samples, of which 739 were from eastern-Asian populations. The collection of DNA samples from mem￾bers of 21 Chinese ethnic-minority populations was done with the coordination of the Chinese Human Ge￾nome Diversity Project. The Chinese Han samples were collected from persons living in 22 provincial areas whose geographic origins were assigned according to the birthplaces of their four grandparents. In addition, sam￾ples obtained in previous projects were analyzed, in￾cluding those from 3 northeast-Asian (Buryat, Korean, and Japanese) and 5 southeast-Asian (Cambodian, Thai, Malaysian, Batak, and Javanese) populations and from an additional 12 non-Asian populations (3 from Africa, 3 from America, 2 from Europe, and 4 from Oceania) (fig. 1 and table 1). Genotyping and Phylogenetic-Tree Construction A total of 19 Y-chromosome biallelic loci were screened. Seven of them were from previous reports (Vollrath et al. 1992; Underhill et al. 1996, 1997a; Kay￾ser et al. 1997), including M3 (CrT mutation), M5 (ArG mutation), M7 (CrG mutation), M9 (CrG mu￾tation), M15 (9-bp insertion), M17 (1-bp deletion), and DYS287 (YAP). The other 12 single-nucleotide poly￾morphisms are being first reported here, including M45 (GrA mutation), M50 (TrC mutation), M88 (ArG mutation), M89 (CrT mutation), M95 (CrT mutation), M103 (CrT mutation), M110 (TrC mutation), M111 (4-bp deletion), M119 (ArC mutation), M120 (TrC mutation), M122 (TrC mutation), and M134 (1-bp de￾letion). The 19 markers used in this study were selected from 166 biallelic Y-chromosome markers (P. A. Un￾derhill, P. Shen, A. A. Lin, L. Jin, G. Passarino, W. H. Yang, E. Kauffman, F. S. Dietrich, J. Kidd, S. Q. Mehdi, T. Jenkins, R. S. Wells, M. T. Seielstad, M. Ibrahim, P. Francalacci, J. Bertranpetit, R. W. Davis, L. L. Cavalli￾Sforza, and P. J. Oefner, unpublished data), since they are polymorphic in the individuals of eastern-Asian or￾igin, in the screening set used. For genotyping, an allelic￾specific PCR assay was used for Y-chromosome biallelic markers. For each Y locus, two allele-specific primers were designed to recognize two different alleles at this locus (the primer sequences are available on request). After PCR, the products were visualized through agarose gel electrophoresis. In addition, three Y-chromosome mi￾crosatellite loci—DYS389, DYS390, and DYS391— were also typed as described by Kayser et al. (1997). The phylogenetic tree for the Y-chromosome haplotypes was constructed on the basis of the parsimony rule, and multifurcation was introduced to accommodate equally parsimonious topologies. Statistical Method To estimate the age of M122C haplotypes in the Han Chinese, we used the equation t = 2N ln(1 2 V/N m). e e We derived this equation from the single-step mutation model for a haploid population, assuming that popu￾lation size (where Ne is the effective population size) stayed constant, that V is the variance of repeat numbers in the population, and that m is the mutation rate. If the population undergoes a strong bottleneck event fol￾lowed by a rapid population expansion, it can be shown that this formula is still approximately valid. A widely accepted estimation of the effective population size of modern humans is 5,000–10,000, suggested by Taka￾hata (1993). Given a relatively smaller genetic diversity in Asia compared with that seen in Africa, a value of 2,000 is a drastic overestimation of the effective popu￾lation size for males in eastern Asia. For the level of variance observed in 160 individuals in this study, 750 is the minimum integer allowed for the effective popu￾lation size, for the variance observed. Results In all the individuals studied for the 19 Y-chromosome biallelic markers, 17 Y haplotypes were obtained. The frequency distribution of Y haplotypes in all the pop￾ulations is listed in table 1. Under the parsimony as￾sumption, no recurrent mutations were observed, and a phylogenetic tree was constructed for the 17 Y haplo￾types, in which H1 was considered as the ancestor hap￾lotype because of its appearance in chimpanzee (fig. 2). The H1 and H2 haplotypes are relatively ancient, ap￾pearing in both African and non-African populations, implying that the occurrence of the mutation defining