letters to nature 22. Begun, D& Walker, A in The Narickotome Homo erectus Stedetom(eds Walker, A Leakey, R) researchers in many field culture is thus of great interest to 21. Riscutia, C in Paleoanthropology, Morphology and Paleoecology (ed. Tuttle, R. H)373-375(Mouton, expansion process of H According to the historical records, the Hans were descended R. LHuman paleontological evidence relevant to language behavior. Hum. Neurobiol 2, from the ancient Huaxia tribes of northern China, and the Han culture(that is, the language and its associated cultures)expanded 24. Holloway, R. L The Indonesian Homo erectus brain endocasts revisited. Am. /. Phys Anthropol 55, into southern China-the region originally inhabited by the 25 Sartono, S&Tyler, D E A New Homo erectus Skll from Sangiran, Arvar: am Annoumcement 1-4 southern natives, including those speaking Daic, Austro-Asiatic (Intern. Conf. Human Paleoecol LIPPI, Jakarta, 1993). nd Hmong-Mien languages-in the past two millennia". Studies 26. Rak Y.& Rensburg. B. Kebara 2 neanderthal pelvis: first look at a complete inlet. Am. J. Phys. on classical genetic markers and microsatellites show that the a.73,227-231(1987) [ cortical bone and dental Han people, like East Asians, are divided into two genetically namel by computed tomography: applications and problems. Am I. Phys. Anthropol. 91, 469-484 differentiated groups, northern Han and southern Han, separated 28. Hohne, K.H. et al. A new representation of knowledge concerning human anatomy and function. approximately by the Yangtze river. Differences between thes Natere Med. 1, 506-511(19 groups in terms of dialect and customs have also been noted o npanzee, with special reference to growth of brain, eruption of Such observations seem to support a mechanism involving rimarily cultural diffusion and assimilation(the cultural diffusion 30. Schultz, A H in Contributions to Embryology no 170(ed, Schultz, A.H. )1-63(Camegie Institution of model)in Han expansion towards the south. However, the sub Washington Publication no 518, Washington DC, 1940). stantial sharing of Y-chromosome and mitochondrial lineages SupplementaryInformationaccompaniesthepaperonwww.nature.com/nature between the two groups.and the historical records describing the expansion of Han people' contradict the cultural diffusion Acknowledgements We are grateful to the following individuals for their assistance in accessing model hypothesis of Han expansion. In this study, we aim to collections and their advice and comments during the preparation of this paper: S. Anton, examine the alternative hypothesis; that is, that substantial popu L P Bocquet-Appel, J. Braga, G Brauer, M. Braun, P Darlu, M. Haas, M. von Harling, C. He er,S Paabo, F. Renoult, M. Richards, Ph. Rightmire, F. Schrenk, ation movements occurred during the expansion of Han culture HSick,ESpoor,TStriano, ITreil, w.Wendelen and V Zeitoun. This research was supported by ( the demic diffusion model) grants from CNRS and by the Max Planck Society. To test this hypothesis, we compared the genetic profiles of southern Hans with their two parental population groups: northern Competing interests statement The authors deare that they have no competing financial Hans and southern natives, which include the samples of Daic, Hmong-Mien and Austro-Asiatic speaking populations currently Correspondence and requests for materials should be addressed to J.J.H. (hublineeva.mpg. de residing in China, and in some cases its neighbour Ing countries. Genetic variation in both the non-recombining region of the Y chromosome(NRY) and mitochondrial DNA (mtDNA)S- were surveyed in 28 Han populations from most of the China(see Fig. I and Supplementary Table 1 for details On the paternal side, southern Hans and northern Hans share similar frequencies of Y-chromosome haplogroups(Supplementary Genetic evidence supports demic Table 2), which are characterized by two haplogroups M122-C mutations(O3-M122 and O3e-M134)that are prevalent diffusion of han culture in almost all Han populations studied(mean and range: 53.8%6 -71%0; 54.2%, 35-74%6, for northern and southern Hans, respect ively). Haplogroups carrying MI19-C(O1* and Olb)and/or Bo Wen, Hui ui, Daru Lu, Xiufeng Song, Feng Zhang, Yungang He, M95-T(O2a* and O2a1)(following the nomenclature of the Y Jianzhong Jin', Wei Huang, Ranjan Deka, Bing Su, Jingze Tan Chromosome Consortium)which are prevalent in southern Feng Li, Yang Gao, Xianyun Mao, Liang Zhang,, Ji Qia natives, are more frequent in southern Hans(19%,3-42%)than Ranajit Chakraborty &LiJin in northern Hans (5%, 1-10%). In addition, haplogroups O1b-M110, O2al-M88 and O3d-M7, which are prevalent in tg and Center for Anthropological southern natives " 7, were only observed in some southern Hans Studies, School of life Sciences and Morgan-Tan International Center for Li Sciences, Fudan University, Shanghai 200433, China 4%on average), but not in northern Hans. Therefore, the contri- bution of southern natives in southern hans is limited if we assume Chinese National Human Genome Center, Shanghai 201203, China Center for Genome Information, Department of Environmental Heai that the frequency distribution of Y lineages in southern natives niversity of Cincinnati, Cincinnati, Ohio 45267, USA presents that before the expansion of Han culture that started "Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of 000 yr ago. The results of analysis of molecular variance Zoology, the Chinese Academy of Sciences, Kunming 650223, China (AMOVA) further indicate that northern Hans and southern Hans are not significantly different in their Y haplogroups The spread of culture and language in human populations is (FST=0.006, P>0.05), demonstrating that southern Hans bear explained by two alternative models: the demic diffusion model, a high resemblance to northern Hans in their male lineages which involves mass movement of people; and the cultural On the maternal side, however, the mtDNA haplogroup distri- diffusion model, which refers to cultural impact between popu- bution showed substantial differentiation between northern Hans lations and involves limited genetic exchange between them. The and southern Hans(Supplementary Table 3). The overall frequen mechanism of the peopling of Europe has long been debated, a cies of the northern East Asian-dominating haplogroups(A, C,D, key issue being whether the diffusion of agriculture and language G, M8a, Yand Z)are much higher in northern Hans(55%, 49-64%) from the Near East was concomitant with a large movement of than are those in southern Hans(36%, 19-52%) In contrast, the farmers. Here we show, by systematically analysing Y-chromo- frequency of the haplogroups that are dominant lineages(B, F, R9a, some and mitochondrial DNA variation in Han populations, that R9b and N9a)in southern natives 4Is is much higher in southern the pattern of the southward expansion of Han culture is (55%, 36-72%)than it is in northern Hans(33%, 18-42%) consistent with the demic diffusion model, and that males played Northern and southern Hans are significantly different in their larger role than females in this expansion. The Han people, who mtDNA lineages(FsT=0.006, P< 10 ). Although the Fsr values all share the same culture and language, exceed 1.16 billion(2000 between northern and southern Hans are similar for mtDNA census), and are by far the largest ethnic group in the world. The and the Y chromosome, Fsr accounts for 56% of the total among e2004NaturePublishingGroupNatuReIvoL431116SePtEmbEr2004www.nature.com/nature
© 2004 NaturePublishingGroup 21. Riscutia, C. in Paleoanthropology, Morphology and Paleoecology (ed. Tuttle, R. H.) 373–375 (Mouton, The Hague, 1975). 22. Begun, D. & Walker, A. in The Nariokotome Homo erectus Skeleton (eds Walker, A. & Leakey, R.) 326–358 (Springer, Berlin, 1993). 23. Holloway, R. L. Human paleontological evidence relevant to language behavior. Hum. Neurobiol. 2, 105–114 (1983). 24. Holloway, R. L. The Indonesian Homo erectus brain endocasts revisited. Am. J. Phys. Anthropol. 55, 503–521 (1981). 25. Sartono, S. & Tyler, D. E. A New Homo erectus Skull from Sangiran, Java: an Announcement 1–4 (Intern. Conf. Human Paleoecol., LIPPI, Jakarta, 1993). 26. Rak, Y. & Arensburg, B. Kebara 2 neanderthal pelvis: first look at a complete inlet. Am. J. Phys. Anthropol. 73, 227–231 (1987). 27. Spoor, C. F., Zonneveld, F. W. & Macho, G. A. Linear measurements of cortical bone and dental enamel by computed tomography: applications and problems. Am. J. Phys. Anthropol. 91, 469–484 (1993). 28. Ho¨hne, K. H. et al. A new representation of knowledge concerning human anatomy and function. Nature Med. 1, 506–511 (1995). 29. Zuckerman, S. Age-changes in the chimpanzee, with special reference to growth of brain, eruption of teeth, and estimation of age; with a note on the Taung ape. Proc. Zool. Soc. Lond. 1, 1–42 (1928). 30. Schultz, A. H. in Contributions to Embryology no 170 (ed. Schultz, A. H.) 1–63 (Carnegie Institution of Washington Publication no 518, Washington DC, 1940). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We are grateful to the following individuals for their assistance in accessing collections and their advice and comments during the preparation of this paper: S. Anton, J. P. Bocquet-Appel, J. Braga, G. Bra¨uer, M. Braun, P. Darlu, M. Haas, M. von Harling, C. Hemm, J.-L. Kahn, C. Lefe`vre, W. van Neer, S. Pa¨a¨bo, F. Renoult, M. Richards, Ph. Rightmire, F. Schrenk, H. Sick, F. Spoor, T. Striano, J. Treil, W. Wendelen and V. Zeitoun. This research was supported by grants from CNRS and by the Max Planck Society. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to J.J.H. (hublin@eva.mpg.de). .............................................................. Genetic evidence supports demic diffusion of Han culture Bo Wen1,2, Hui Li1 , Daru Lu1 , Xiufeng Song1 , Feng Zhang1 , Yungang He1 , Feng Li1 , Yang Gao1 , Xianyun Mao1 , Liang Zhang1 , Ji Qian1 , Jingze Tan1 , Jianzhong Jin1 , Wei Huang2 , Ranjan Deka3 , Bing Su1,3,4, Ranajit Chakraborty3 & Li Jin1,3 1 State Key Laboratory of Genetic Engineering and Center for Anthropological Studies, School of Life Sciences and Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, China 2 Chinese National Human Genome Center, Shanghai 201203, China 3 Center for Genome Information, Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267, USA 4 Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China ............................................................................................................................................................................. The spread of culture and language in human populations is explained by two alternative models: the demic diffusion model, which involves mass movement of people; and the cultural diffusion model, which refers to cultural impact between populations and involves limited genetic exchange between them1 . The mechanism of the peopling of Europe has long been debated, a key issue being whether the diffusion of agriculture and language from the Near East was concomitant with a large movement of farmers1–3. Here we show, by systematically analysing Y-chromosome and mitochondrial DNA variation in Han populations, that the pattern of the southward expansion of Han culture is consistent with the demic diffusion model, and that males played a larger role than females in this expansion. The Han people, who all share the same culture and language, exceed 1.16 billion (2000 census), and are by far the largest ethnic group in the world. The expansion process of Han culture is thus of great interest to researchers in many fields. According to the historical records, the Hans were descended from the ancient Huaxia tribes of northern China, and the Han culture (that is, the language and its associated cultures) expanded into southern China—the region originally inhabited by the southern natives, including those speaking Daic, Austro-Asiatic and Hmong-Mien languages—in the past two millennia4,5. Studies on classical genetic markers and microsatellites show that the Han people, like East Asians, are divided into two genetically differentiated groups, northern Han and southern Han6,8, separated approximately by the Yangtze river9 . Differences between these groups in terms of dialect and customs have also been noted10. Such observations seem to support a mechanism involving primarily cultural diffusion and assimilation (the cultural diffusion model) in Han expansion towards the south. However, the substantial sharing of Y-chromosome and mitochondrial lineages between the two groups11,12 and the historical records describing the expansion of Han people5 contradict the cultural diffusion model hypothesis of Han expansion. In this study, we aim to examine the alternative hypothesis; that is, that substantial population movements occurred during the expansion of Han culture (the demic diffusion model). To test this hypothesis, we compared the genetic profiles of southern Hans with their two parental population groups: northern Hans and southern natives, which include the samples of Daic, Hmong-Mien and Austro-Asiatic speaking populations currently residing in China, and in some cases its neighbouring countries. Genetic variation in both the non-recombining region of the Y chromosome (NRY) and mitochondrial DNA (mtDNA)13–16 were surveyed in 28 Han populations from most of the provinces in China (see Fig. 1 and Supplementary Table 1 for details). On the paternal side, southern Hans and northern Hans share similar frequencies of Y-chromosome haplogroups (Supplementary Table 2), which are characterized by two haplogroups carrying the M122-C mutations (O3-M122 and O3e-M134) that are prevalent in almost all Han populations studied (mean and range: 53.8%, 37–71%; 54.2%, 35–74%, for northern and southern Hans, respectively). Haplogroups carrying M119-C (O1* and O1b) and/or M95-T (O2a* and O2a1) (following the nomenclature of the Y Chromosome Consortium) which are prevalent in southern natives, are more frequent in southern Hans (19%, 3–42%) than in northern Hans (5%, 1–10%). In addition, haplogroups O1b-M110, O2a1-M88 and O3d-M7, which are prevalent in southern natives17, were only observed in some southern Hans (4% on average), but not in northern Hans. Therefore, the contribution of southern natives in southern Hans is limited, if we assume that the frequency distribution of Y lineages in southern natives represents that before the expansion of Han culture that started 2,000 yr ago5 . The results of analysis of molecular variance (AMOVA) further indicate that northern Hans and southern Hans are not significantly different in their Y haplogroups (FST ¼ 0.006, P . 0.05), demonstrating that southern Hans bear a high resemblance to northern Hans in their male lineages. On the maternal side, however, the mtDNA haplogroup distribution showed substantial differentiation between northern Hans and southern Hans (Supplementary Table 3). The overall frequencies of the northern East Asian-dominating haplogroups (A, C, D, G, M8a, Yand Z) are much higher in northern Hans (55%, 49–64%) than are those in southern Hans (36%, 19–52%). In contrast, the frequency of the haplogroups that are dominant lineages (B, F, R9a, R9b and N9a) in southern natives12,14,18 is much higher in southern (55%, 36–72%) than it is in northern Hans (33%, 18–42%). Northern and southern Hans are significantly different in their mtDNA lineages (FST ¼ 0.006, P , 1025 ). Although the FST values between northern and southern Hans are similar for mtDNA and the Y chromosome, FST accounts for 56% of the total amongletters to nature 302 NATURE | VOL 431 | 16 SEPTEMBER 2004 | www.nature.com/nature
letters to nature Figure 1 Geographic distribution of sampled populations Shown are the three waves of one-sixth of the southen population at that time)occurred during the westem Jin Dynasty orth-to-south migrations according to historical record. The identifications of populations (AD 265-316); the second migration, more extensive than the first, took place during the are given in Supplementary Table 1. Populations 1-14 are northen Hans, and 15-28 are Tang Dynasty (AD 618-907); and the third wave, including-5 million immigrants, southern Hans. The solid, dashed and dotted arrows refer to the first, second and occurred during the Southen Song Dynasty (AD 1127-1279) third waves of migrations, respectively. The first wave involving 0.9 million(approximately population variation for mt DNA but only accounts for 18% for the Table 1 Northern oportion in southern Hans mtDNA component a is consistent with the observation Population For the NRY, almost all Han populations cluster together in the Ant/bE(=se. m) Mpk based on the distribution of the haplogroups in Han populations MgE(±se.m) 0868±0.119 0816±0.214 upper right-hand part of Fig. 2a. Northern Hans and southern Fuijian natives are separated by the second principal component(PC2)and Guangdong 0.677+0.121 0238±024 0543±0.1 0.451±0.263 0212 natives but are much closer to northern Hans(northern Han, Hubei 0.981±0.1 0.946±0.26 0.58+ 0.01; southern Han, 0.46+ 0.03; southern native, Hunan .732±0219 0.565±0.2g -0.32+ 0.05), implying that the southern Hans are paternally Jiang natives. In contrast,for mtDNA, northern Hans and southern natives yinnan similar to northern Hans, with limited influence from southern shanghe 0819±008 0750±0.1 9150.376±0.221 are distinctly separated by PC2(Fig. 2b), and southern Hans are Yunnan located between them but are closer to southern natives(northern Zhejang Han 0.56+0.02: southern Han 0.09+0.06: southern native Average. 819 0.500 0.23+0.04), indicating a much more substantial admixture in M s and reie to the statistics descr bed n rets i and Th, respectively f he standard error outhern Hans' female gene pool than in its male counterpart populations of the southem Hans. It was assumed that the allele frequency ern Hans and southern natives )in southern Hans was estimated by and the genetic exchange between nor hern ha s amd soe hern atares has been iie in. for single-locus data(Table 1). The estimations of the admixture underestimated and is without proper adjustment. The damic expansion of Han would have pefficient(M, proportion of northern Han contribution)from the been more pronounced than was observed in tris study. wo methods are highly consistent (for the Y chromosome, r=0.922, P<0.01; for mtDNA, r=0.970, P<0.01). For the Y hromosome,all southern Hans showed a high proportion of gene pool (M BE: 0. 56= 0.24 [0.15,0.95] MRH: 0.50+0. 26[0.07, northern Han contribution(MBE: 0.82+0.14, range from 0.54 to 0.91)). The contribution of northern Hans to southern Hans is 1;MRH: 0.82+0.12, range from 0.61 to 0.97)(see refs 20 and 19 for significantly higher in the paternal lineage than in the maternal definitions of MBE and MRH, respectively) indicating that males lineage collectively (t-test, P <0.01)or individually(1l out of 13 from the northern Hans are the primary contributor to the gene populations for MBE, and 13 out of 13 populations for Mrh pool of the southern Hans. In contrast, northern Hans and southern P<0.01, assuming a null binomial distribution with equal male natives contributed almost equally to the southern Hans'mtDNA and female contributions), indicating a strong sex-biased popu NatuReiVoL431116SepTemBer2004www.nature.com/nature02004naTurepUblishingGroup
© 2004 NaturePublishingGroup population variation for mtDNA but only accounts for 18% for the Y chromosome. A principal component analysis is consistent with the observation based on the distribution of the haplogroups in Han populations. For the NRY, almost all Han populations cluster together in the upper right-hand part of Fig. 2a. Northern Hans and southern natives are separated by the second principal component (PC2) and southern Hans’ PC2 values lie between northern Hans and southern natives but are much closer to northern Hans (northern Han, 0.58 ^ 0.01; southern Han, 0.46 ^ 0.03; southern native, 20.32 ^ 0.05), implying that the southern Hans are paternally similar to northern Hans, with limited influence from southern natives. In contrast, for mtDNA, northern Hans and southern natives are distinctly separated by PC2 (Fig. 2b), and southern Hans are located between them but are closer to southern natives (northern Han, 0.56 ^ 0.02; southern Han, 0.09 ^ 0.06; southern native, 20.23 ^ 0.04), indicating a much more substantial admixture in southern Hans’ female gene pool than in its male counterpart. The relative contribution of the two parental populations (northern Hans and southern natives) in southern Hans was estimated by two different statistics19,20, which are less biased than other statistics for single-locus data21 (Table 1). The estimations of the admixture coefficient (M, proportion of northern Han contribution) from the two methods are highly consistent (for the Y chromosome, r ¼ 0.922, P , 0.01; for mtDNA, r ¼ 0.970, P , 0.01). For the Y chromosome, all southern Hans showed a high proportion of northern Han contribution (MBE: 0.82 ^ 0.14, range from 0.54 to 1; MRH: 0.82 ^ 0.12, range from 0.61 to 0.97) (see refs 20 and 19 for definitions of MBE and MRH, respectively) indicating that males from the northern Hans are the primary contributor to the gene pool of the southern Hans. In contrast, northern Hans and southern natives contributed almost equally to the southern Hans’ mtDNA gene pool (MBE: 0.56 ^ 0.24 [0.15, 0.95]; MRH: 0.50 ^ 0.26 [0.07, 0.91]). The contribution of northern Hans to southern Hans is significantly higher in the paternal lineage than in the maternal lineage collectively (t-test, P , 0.01) or individually (11 out of 13 populations for MBE, and 13 out of 13 populations for MRH: P , 0.01, assuming a null binomial distribution with equal male and female contributions), indicating a strong sex-biased popuTable 1 Northern Han admixture proportion in southern Hans Population Y Chromosome mtDNA MBE (^s.e.m) MRH MBE (^s.e.m) MRH ............................................................................................................................................................................. Anhui 0.868 ^ 0.119 0.929 0.816 ^ 0.214 0.755 Fujian 1 0.966 0.341 ^ 0.206 0.248 Guangdong1 0.677 ^ 0.121 0.669 0.149 ^ 0.181 0.068 Guangdong2 ND ND 0.298 ^ 0.247 0.312 Guangxi 0.543 ^ 0.174 0.608 0.451 ^ 0.263 0.249 Hubei 0.981 ^ 0.122 0.949 0.946 ^ 0.261 0.907 Hunan 0.732 ^ 0.219 0.657 0.565 ^ 0.297 0.490 Jiangsu 0.789 ^ 0.078 0.821 0.811 ^ 0.177 0.786 Jiangxi 0.804 ^ 0.113 0.829 0.374 ^ 0.343 0.424 Shanghai 0.819 ^ 0.087 0.902 0.845 ^ 0.179 0.833 Sichuan 0.750 ^ 0.118 0.713 0.509 ^ 0.166 0.498 Yunnan1 1 0.915 0.376 ^ 0.221 0.245 Yunnan2 0.935 ^ 0.088 0.924 0.733 ^ 0.192 0.645 Zhejiang 0.751 ^ 0.084 0.763 0.631 ^ 0.180 0.540 Average 0.819 0.819 0.560 0.500 ............................................................................................................................................................................. MBE and MRH refer to the statistics described in refs 20 and 19, respectively. The standard error of MBE was obtained by bootstrap with 1,000 replications. The proportions of contribution from northern Hans were estimated using northern Hans and southern natives as the parental populations of the southern Hans. It was assumed that the allele frequency in the southern natives remained unchanged before and after the admixture, which started about 2,000 yr ago, and the genetic exchange between northern Hans and southern natives has been limited. In fact, the gene flow from northern Hans to southern natives has been larger than that from southern natives to northern Hans; therefore, the level of admixture presented in this table is underestimated and is without proper adjustment. The demic expansion of Han would have been more pronounced than was observed in this study. Figure 1 Geographic distribution of sampled populations. Shown are the three waves of north-to-south migrations according to historical record. The identifications of populations are given in Supplementary Table 1. Populations 1–14 are northern Hans, and 15–28 are southern Hans. The solid, dashed and dotted arrows refer to the first, second and third waves of migrations, respectively. The first wave involving 0.9 million (approximately one-sixth of the southern population at that time) occurred during the Western Jin Dynasty (AD 265–316); the second migration, more extensive than the first, took place during the Tang Dynasty (AD 618–907); and the third wave, including ,5 million immigrants, occurred during the Southern Song Dynasty (AD 1127–1279). letters to nature NATURE | VOL 431 | 16 SEPTEMBER 2004 | www.nature.com/nature 303
letters to nature lation admixture in southern Hans. The proportions of northern Our genetic observation is thus in line with the historical accounts. Han contribution(M)in southern Hans showed a clinal geographic The massive movement of the northern immigrants led to a change pattern, which decreases from north to south. The Ms in southern in genetic makeup in southern China, and resulted in the demo- Hans are positively correlated with latitude(r=0.569, P0.05), because the difference of Ms in the paternal Hans, southern Hans and southern natives also contributed to lineage among southern Hans is too small to create a statistically the admixture which shaped the genetic profile of the extant ificant trend We provide two lines of evidence supporting the demic diffusion hypothesis for the expansion of Han culture. First, almost all Han Methods populations bear a high resemblance in Y-chromosome haplogroup Sample distribution, and the result of principal component analysis indi- Blood samples of 871 unrelated anonymous individuals from 17 Han populations cated that almost all Han populations form a tight cluster in their y collected across china. Genomic DNA was extracted by the phenol-chloroform me chromosome. Second, the estimated contribution of northern hans mtDNA variation, the final sample sizes for analysis expanded to 1. 289 individuals to southern Hans is substantial in both paternal and maternal (23 Han populations)for the Y chromosome and 1, 119 individuals(23 Han populations) lineages and a geographic cline exists for mtDNA. It is noteworthy sur lementary Tab se mp.ls encompass most of the provinces in China (Fig. I greater contribution to the Y-chromosome than the mtDNA from Genetic markers northern Hans to southern Hans. A sex-biased admixture pattern Thirteen bi-allelic y r,YAPM15,M130,M89,M9,M122,M134 was also observed in Tibeto-Burman-speaking populations M119, M110, M95, M88, M45 and M120 were typed by polymerase chain reaction- According to the historical records, there were continuous south- restriction-fragment length polymorphism methods". These markers are highly ward movements of Han people due to warfare and famine in the informative in East Asians" and define 13 haplogroups following the YChromosome north, as illustrated by three waves of large-scale migrations(Fig. 1). The HvS-I of mtDNA and eight coding region variations, 9-bp deletion, 10397Alu Aside from these three waves, other smaller southward migrations 5176 Alul, 4831 Hhal, 13259 Hincll, 663 Haelll, 12406 Hpal and also occurred during almost all periods in the past two millennia. sequenced and genotyped as in our previous report".Both the HVS-I motif and the g region variations were used to infer haplogroups following the phylogeny of East HM+ DAC X A-A△sH▲N Data analysis opulation relationship was investigated by principal component analysis, which was conducted using mtDNA and Y-chromosome haplogroup frequencies and SPSS10.0 sted by AMOVA using ARLEQUIN software". ADMIX 2.0(ref. 27) software were used to estimate the level of admixture of the northern Hans EADMIXa and we were careful to minimize b s East Asia. In this alysis, the average haplogroup frequencies(for Y-chromosome or mtDNA markers, -2.0 northern parental population. The frequency of southern natives was estimated by the erage of three groups including Austro-Asiatic(NRY, 6 populatie (NRY, 18 populations; mtDNA, 14 populations). The geographic pattern of Han ien populations), Daic(NRY, 22 populations; mtDNA, Il populations)and Hmong populations was revealed by the linear regression analysis of admixture proportion against the latitudes of samples". Received 28 April; accepted 20 July 2004; doi: 10.1038/nature02878. PC1(48.5 2. Sokal, R, Oden, N L& wilson, C Genetic evidence for the spread of agriculture in Europe by demic 3. Chikhi, L etaL Y genetic data support the Neolithic demic diffusion model. Proc. Natl Acd. Sci. U/SA 9.11008-11013(202) 1. Fei, X.T. The Pattern of Diversity in Unity of the Chinese Nation( Central Univ for Nationalities Press, 0.4 5. Ge, J.X. Wu, S D. Chao, S 1. Zhongguo yimin shi( The Migration History of Chime)(Fujian People's 6. Zhao, T.M.&Lee, T D Gm and Km allotypes in 74 Chinese populations a hypothesis of the origin of Chinese nation. Hur. Ger 7. Du, R E, Xiao, CI& Cavalli-Sforza. LL Genetic distances calculated on gene frequencies of 38 lod 8. Chu, L Y et al. Genetic relationship of populations in Chima. Proc Natl Acad. Sci. USA 95, 9. Xiao, C I et aL Principal component analysis of gene frequencies of Chinese populations. Sai. Chin 43,472-81(2000 Il. Su, B et al Y chromosome haplotypes reveal prehistorical migrations to the Himalayas. Hum. Genet. 107.582-590(2000 12. Yao, Y G. ef al Phylogeographic differentiation of mitochondrial DNA in Han Chinese. Arm. /Hunn Gent.70,635-651(20 0.20.30.40.50.60.7080.9 Cavalli-Sforza, LL& Feldman, M. w. The application of molecular genetic approaches to the study Pc1(40.1% 4. Wallace, D. C, Bron, M. D. Lott, M. T Nucleotide mitochondrial DNA variation in human Figure 2 Principal comp evolution and disease. Gene 238, 211-230(1999) haplogroup frequency plot. a, b, Plots are of Y-chromosome (a) and mtDNA(b) 15. Underhill.p. A. et al Y chromosome sequence variation and the history of human populations.Nature ion groups: H-M, Hmong-Mien; DAC, Daic: A-A, Austro- Gemt.26358-361(200 Asiatic: SH. southern Hi northem Han Jobling. M. A& Tyler-Smith, C. The human Y chromosome an evolutionary marker comes of age. e2004NaturePublishingGroupNatuReIvoL431116SePtEmbEr2004www.nature.com/nature
© 2004 NaturePublishingGroup lation admixture in southern Hans. The proportions of northern Han contribution (M) in southern Hans showed a clinal geographic pattern, which decreases from north to south. The Ms in southern Hans are positively correlated with latitude (r 2 ¼ 0.569, P , 0.01) for mtDNA, but are not significant for the Y chromosome (r 2 ¼ 0.072, P . 0.05), because the difference of Ms in the paternal lineage among southern Hans is too small to create a statistically significant trend. We provide two lines of evidence supporting the demic diffusion hypothesis for the expansion of Han culture. First, almost all Han populations bear a high resemblance in Y-chromosome haplogroup distribution, and the result of principal component analysis indicated that almost all Han populations form a tight cluster in their Y chromosome. Second, the estimated contribution of northern Hans to southern Hans is substantial in both paternal and maternal lineages and a geographic cline exists for mtDNA. It is noteworthy that the expansion process was dominated by males, as is shown by a greater contribution to the Y-chromosome than the mtDNA from northern Hans to southern Hans. A sex-biased admixture pattern was also observed in Tibeto-Burman-speaking populations22. According to the historical records, there were continuous southward movements of Han people due to warfare and famine in the north, as illustrated by three waves of large-scale migrations (Fig. 1). Aside from these three waves, other smaller southward migrations also occurred during almost all periods in the past two millennia. Our genetic observation is thus in line with the historical accounts. The massive movement of the northern immigrants led to a change in genetic makeup in southern China, and resulted in the demographic expansion of Han people as well as their culture. Except for these massive population movements, gene flow between northern Hans, southern Hans and southern natives also contributed to the admixture which shaped the genetic profile of the extant populations. A Methods Samples Blood samples of 871 unrelated anonymous individuals from 17 Han populations were collected across China. Genomic DNA was extracted by the phenol-chloroform method. By integrating the additional data obtained from the literatures on the Y chromosome and on mtDNA variation, the final sample sizes for analysis expanded to 1,289 individuals (23 Han populations) for the Y chromosome and 1,119 individuals (23 Han populations) for mtDNA. These samples encompass most of the provinces in China (Fig. 1 and Supplementary Table 1). Genetic markers Thirteen bi-allelic Y-chromosome markers, YAP, M15, M130, M89, M9, M122, M134, M119, M110, M95, M88, M45 and M120 were typed by polymerase chain reactionrestriction-fragment length polymorphism methods11. These markers are highly informative in East Asians23 and define 13 haplogroups following the Y Chromosome Consortium nomenclature24. The HVS-1 of mtDNA and eight coding region variations, 9-bp deletion, 10397 AluI, 5176 AluI, 4831 HhaI, 13259 HincII, 663 HaeIII, 12406 HpaI and 9820 HinfI were sequenced and genotyped as in our previous report22. Both the HVS-1 motif and the coding region variations were used to infer haplogroups following the phylogeny of East Asian mtDNAs18. Data analysis Population relationship was investigated by principal component analysis, which was conducted using mtDNA and Y-chromosome haplogroup frequencies and SPSS10.0 software (SPSS Inc.). The genetic difference between northern and southern Hans was tested by AMOVA25, using ARLEQUIN software26. ADMIX 2.0 (ref. 27) and LEADMIX21 software were used to estimate the level of admixture of the northern Hans and southern natives in the southern Han populations, using two different statistics19–20. The selection of parental populations is critical for appropriate estimation of admixture proportion28,29 and we were careful to minimize bias by using large data sets across East Asia. In this analysis, the average haplogroup frequencies (for Y-chromosome or mtDNA markers, respectively) of northern Hans (arithmetic mean of 10 northern Hans) were taken for the northern parental population. The frequency of southern natives was estimated by the average of three groups including Austro-Asiatic (NRY, 6 populations; mtDNA, 5 populations), Daic (NRY, 22 populations; mtDNA, 11 populations) and Hmong-Mien (NRY, 18 populations; mtDNA, 14 populations). The geographic pattern of Han populations was revealed by the linear regression analysis of admixture proportion against the latitudes of samples1,3. Received 28 April; accepted 20 July 2004; doi:10.1038/nature02878. 1. Cavalli-Sforza, L. L., Menozzi, P. & Piazza, A. The History and Geography of Human Genes (Princeton Univ. Press, Princeton, 1994). 2. Sokal, R., Oden, N. L. & Wilson, C. Genetic evidence for the spread of agriculture in Europe by demic diffusion. Nature 351, 143–145 (1991). 3. Chikhi, L. et al. Y genetic data support the Neolithic demic diffusion model. Proc. Natl Acad. Sci. USA 99, 11008–11013 (2002). 4. Fei, X. T. The Pattern of Diversity in Unity of the Chinese Nation (Central Univ. for Nationalities Press, Beijing, 1999). 5. Ge, J. X., Wu, S. D. & Chao, S. J. Zhongguo yimin shi (The Migration History of China) (Fujian People’s Publishing House, Fuzhou, China, 1997). 6. Zhao, T. M. & Lee, T. D. Gm and Km allotypes in 74 Chinese populations: a hypothesis of the origin of the Chinese nation. Hum. Genet. 83, 101–110 (1989). 7. Du, R. F., Xiao, C. J. & Cavalli-Sforza, L. L. Genetic distances calculated on gene frequencies of 38 loci. Sci. China 40, 613 (1997). 8. Chu, J. Y. et al. Genetic relationship of populations in China. Proc. Natl Acad. Sci. USA 95, 11763–11768 (1998). 9. Xiao, C. J. et al. Principal component analysis of gene frequencies of Chinese populations. Sci. China 43, 472–481 (2000). 10. Xu, Y. T. A brief study on the origin of Han nationality. J. Centr. Univ. Natl 30, 59–64 (2003). 11. Su, B. et al. Y chromosome haplotypes reveal prehistorical migrations to the Himalayas. Hum. Genet. 107, 582–590 (2000). 12. Yao, Y. G. et al. Phylogeographic differentiation of mitochondrial DNA in Han Chinese. Am. J. Hum. Genet. 70, 635–651 (2002). 13. Cavalli-Sforza, L. L. & Feldman, M. W. The application of molecular genetic approaches to the study of human evolution. Nature Genet. 33, 266–275 (2003). 14. Wallace, D. C., Brown, M. D. & Lott, M. T. Nucleotide mitochondrial DNA variation in human evolution and disease. Gene 238, 211–230 (1999). 15. Underhill, P. A.et al. Y chromosome sequence variation and the history of human populations. Nature Genet. 26, 358–361 (2000). 16. Jobling, M. A. & Tyler-Smith, C. The human Y chromosome: an evolutionary marker comes of age. Figure 2 Principal component plot. a, b, Plots are of Y-chromosome (a) and mtDNA (b) haplogroup frequency. Population groups: H-M, Hmong-Mien; DAC, Daic; A-A, AustroAsiatic; SH, southern Han; NH, northern Han. letters to nature 304 NATURE | VOL 431 | 16 SEPTEMBER 2004 | www.nature.com/nature
letters to nature Narture Rev. gene.4,598-612(2003) parental care is provided. 'Pirate males search for freshly laid .Ham. Genet.65,178-1724(1999) clutches, clasp them as they would do a female and fertilize the 8. Kivisild,Tet al. The emerging limbs and twigs of the East Asian mtDNA tree. Mol Biol Evol 19, eggs that were left unfertilized by the parental male. This 1737-1751(2002 behaviour does not seem to be size-dependent, and some males 19.Roberts, DE&Hioms,RW Methods of analysis of the genetic compos ition ofa hybrid population mate with a female and perform clutch piracy in the same season. 20. Bertorelle, G. Excoffier, L Inferring admixture proportions from molecular data. Mol BioL. EvoL. Piracy affected 84% of the clutches and in some cases increased 1298-1311(1998) the proportion of eggs fertilized, providing direct fitness benefits um-likelithood estimation of admixture proportions from genetic data. Genetics 164. both for the pirate males and the females 7. Sexual selection- 22. Wen, B ct aL Analyses of genetic structure of Tibeto-Burman populations revealed a gender-biased probably caused by a strong male-biased sex ratio--occurs in this admixture in southern Tibeto-Burmans. Am. Hum. Genet 74, 856-865(200 population, as indicated by size-assortative mating; however, 23. Jin, L& Su, B Natives or immigrants modern human origin in East Asia, Nature Rev Genet. 1 clutch piracy may reduce its impact. This provides a good L. The Y Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal model to explore how alternative mating strategies can affect binary haplogroups. Genome Res. 12, 339-348(2002 the intensity of sexual selection. 25. Excoffier, L Smouse, P. E. Quattro I. M. Analysis of molecular variance inferred from metri Anuran amphibians have a wide diversity of reproductive mod mong DNA haplotypes application to human chondrial DNA restriction data. but external aquatic fertilization without parental care is the Genetics131,479-491(1992 for population genetic analysis. (Genetics and ancestral and most widespread strategy. Only a few instances of Biometry Laboratory, Univ of Geneva, Geneva, 2000) multiple paternity have been demonstrated in frogs and those were 27. Dupanloup. L &t Bertorelle, G Inferring admistureproportions from molecular data extenson to any considered to be the result of polyandrous matings, in which several ort, R. Geme admixture in human populations Models and predictions. Yh Phya Anthropol. males mate simultaneously with a female In the common frog 29,1-43(1986) R temporaria, one of the most widespread Palaearctic amphibians", 29. Sans, M. et aL Unequal contributions of male and female gene pools from parental populations in the multiple paternity has been detected through allozyme analyses of African descendants of the city of Melo, Uruguay. Arm /. Plys. Anthropol. 118, 33-44(2002). tadpole kin groups, and was interpreted as being the consequence of SupplementaryInformationaccompaniesthepaperonwww.nature.com/nature. high concentrations of spermatozoa in the water during simul taneous making this work possible. The data collection R temporaria is an explosive pond breeder that often reproduces and a NSF grant to.J L-RD and R C are immediately after the melting of the ice cover. Breeding is usually supported by NIH nocturnal and males form large breeding aggregations. We Competing interests statement The authors declare that they have no competing financial monitored a high altitude population of common frogs in a als should be addressed to LL(ljinefudaneduce or periods(2001-2003)in the central Pyrenean mountains, Spain li jin@uc. edu). The mtDNA I sequences of 711 individuals from 15 Han populations were (42 49N, 017'w, about 2200 m above sea level). Breeding was submitted to Gen Bank with accession numbers Ay594701-AY595411 exclusively diurnal due to low temperatures at night( Supplementary Information A), which permitted us to conduct detailed beha- vioural observations in the field and to measure and mark most individuals in this population. Males aggregated in a specific area of the pond, where clutches were also laid. Male density at the Post-mating clutch piracy in an amphibian Amplexus Clutch deposition David R. Vieites', Sandra Nieto-Roman", Marta Barlueng Antonio Palanca, Miguel Vences& Axel Meyer Clutch Laboratorio de Anatomia Animal, Departamento de ecoloxia e Bioloxia Animal, Facultade de Ciencias Bioloxicas, Universidade de vigo, Buzon 137, 36201 vigo Lehrstuhl fiir Zoologie und Evoluetionsbiologie, Department of Biology University Parental male of Konstanz, 78457 Konstanz, German parental on by INstitute for Biodiversity and Ecosystem Dynamics and Zoological Museun University of Amsterdam, Mauritskade 61, 1092 AD Amsterdam, The Netherlands b Female Present address Museum of vertebrate Zoology and Department of Integrative Biology, 3101 Valley Life the clutch Sciences Building, Universty of California, Berkeley, California 94720-3160, USA Female multiple mating and alternative mating systems can at→ decrease the opportunity for sexual selectionl-. Spero je males tition is often the outcome of females mating with multipl d has been observed in many animals, and alternative Fertilization by reproductive systems are spread am ong species with exte Parental male nal fertilization and parental careB-lo. Multiple paternity with- out associated complex behaviour related to mating or parental Figure 1 Schematic representation of mating mporaria. a, Females arrive care is also seen in simultaneously spawning amphibians -and at the breeding ponds and are clasped in the ion (amplexus ) by a male (the fishes that release gametes into water. Here we report clutch parental male). The female deposits a single, utah of eggs. The parental male piracy' in a montane population of the common frog Rana simultaneously releases his sperm and thereby fertilizes the eggs extemally temporaria, a reproductive behaviour previously unknown in ntly both parents leave the clutch. b, Pirate males search for freshly vertebrates with external fertilization. Males of this species clasp clasp them and release their sperm, sometimes crawling into the clutch to gain the females and the pair deposits one spherical clutch of eggs. No the internal eggs NatuRejVol43116SepTemBer2004www.nature.com/nature02004NaturePublishingGroup
© 2004 NaturePublishingGroup Nature Rev. Genet. 4, 598–612 (2003). 17. Su, B. et al. Y-chromosome evidence for a northward migration of modern humans into eastern Asia during the last ice age. Am. J. Hum. Genet. 65, 1718–1724 (1999). 18. Kivisild, T. et al. The emerging limbs and twigs of the East Asian mtDNA tree. Mol. Biol. Evol. 19, 1737–1751 (2002). 19. Roberts, D. F. & Hiorns, R. W. Methods of analysis of the genetic composition of a hybrid population. Hum. Biol. 37, 38–43 (1965). 20. Bertorelle, G. & Excoffier, L. Inferring admixture proportions from molecular data. Mol. Biol. Evol. 15, 1298–1311 (1998). 21. Wang, J. Maximum-likelihood estimation of admixture proportions from genetic data. Genetics 164, 747–765 (2003). 22. Wen, B. et al. Analyses of genetic structure of Tibeto-Burman populations revealed a gender-biased admixture in southern Tibeto-Burmans. Am. J. Hum. Genet. 74, 856–865 (2004). 23. Jin, L. & Su, B. Natives or immigrants: modern human origin in East Asia. Nature Rev. Genet. 1, 126–133 (2000). 24. The Y Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups. Genome Res. 12, 339–348 (2002). 25. Excoffier, L., Smouse, P. E. & Quattro, J. M. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491 (1992). 26. Schneider, S., et al. Arlequin: Ver. 2.000. A software for population genetic analysis. (Genetics and Biometry Laboratory, Univ. of Geneva, Geneva, 2000). 27. Dupanloup, I. & Bertorelle, G. Inferring admixture proportions from molecular data: extension to any number of parental populations. Mol. Biol. Evol. 18, 672–675 (2001). 28. Chakraborty, R. Gene admixture in human populations: Models and predictions. Yb. Phys. Anthropol. 29, 1–43 (1986). 29. Sans, M. et al. Unequal contributions of male and female gene pools from parental populations in the African descendants of the city of Melo, Uruguay. Am. J. Phys. Anthropol. 118, 33–44 (2002). Supplementary Information accompanies the paper on www.nature.com/nature. Acknowledgements We thank all of the donors for making this work possible. The data collection was supported by NSFC and STCSM to Fudan and a NSF grant to L.J. L.J., R.D. and R.C. are supported by NIH. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to L.J. (lijin@fudan.edu.cn or li.jin@uc.edu). The mtDNA HVS-1 sequences of 711 individuals from 15 Han populations were submitted to GenBank with accession numbers AY594701–AY595411. .............................................................. Post-mating clutch piracy in an amphibian David R. Vieites1,2*, Sandra Nieto-Roma´n1,3, Marta Barluenga2 , Antonio Palanca1 , Miguel Vences3 & Axel Meyer2 1 Laboratorio de Anatomı´a Animal, Departamento de Ecoloxı´a e Bioloxı´a Animal, Facultade de Ciencias Biolo´xicas, Universidade de Vigo, Buzo´n 137, 36201 Vigo, Spain 2 Lehrstuhl fu¨r Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, 78457 Konstanz, Germany 3 Institute for Biodiversity and Ecosystem Dynamics and Zoological Museum, University of Amsterdam, Mauritskade 61, 1092 AD Amsterdam, The Netherlands * Present address: Museum of Vertebrate Zoology and Department of Integrative Biology, 3101 Valley Life Sciences Building, University of California, Berkeley, California 94720-3160, USA ............................................................................................................................................................................. Female multiple mating and alternative mating systems can decrease the opportunity for sexual selection1–3. Sperm competition is often the outcome of females mating with multiple males and has been observed in many animals1,4–7, and alternative reproductive systems are widespread among species with external fertilization and parental care3,8–10. Multiple paternity without associated complex behaviour related to mating or parental care is also seen in simultaneously spawning amphibians11–15 and fishes16 that release gametes into water. Here we report ‘clutch piracy’ in a montane population of the common frog Rana temporaria, a reproductive behaviour previously unknown in vertebrates with external fertilization. Males of this species clasp the females and the pair deposits one spherical clutch of eggs. No parental care is provided. ‘Pirate’ males search for freshly laid clutches, clasp them as they would do a female and fertilize the eggs that were left unfertilized by the ‘parental’ male. This behaviour does not seem to be size-dependent, and some males mate with a female and perform clutch piracy in the same season. Piracy affected 84% of the clutches and in some cases increased the proportion of eggs fertilized, providing direct fitness benefits both for the pirate males and the females17. Sexual selection— probably caused by a strong male-biased sex ratio—occurs in this population, as indicated by size-assortative mating; however, clutch piracy may reduce its impact. This provides a good model to explore how alternative mating strategies can affect the intensity of sexual selection. Anuran amphibians have a wide diversity of reproductive modes, but external aquatic fertilization without parental care is the ancestral and most widespread strategy18. Only a few instances of multiple paternity have been demonstrated in frogs and those were considered to be the result of polyandrous matings, in which several males mate simultaneously with a female11,13,14. In the common frog R. temporaria, one of the most widespread Palaearctic amphibians19, multiple paternity has been detected through allozyme analyses of tadpole kin groups, and was interpreted as being the consequence of high concentrations of spermatozoa in the water during simultaneous spawning12. R. temporaria is an explosive pond breeder that often reproduces immediately after the melting of the ice cover. Breeding is usually nocturnal12,20 and males form large breeding aggregations. We monitored a high altitude population of common frogs in a medium-sized pond (540 m2 ) during three consecutive breeding periods (2001–2003) in the central Pyrenean mountains, Spain (428490 N, 08170 W, about 2200 m above sea level). Breeding was exclusively diurnal due to low temperatures at night (Supplementary Information A), which permitted us to conduct detailed behavioural observations in the field and to measure and mark most individuals in this population. Males aggregated in a specific area of the pond, where clutches were also laid. Male density at the Figure 1 Schematic representation of mating systems in R. temporaria. a, Females arrive at the breeding ponds and are clasped in the axillary region (‘amplexus’) by a male (the ‘parental’ male). The female deposits a single, spherical clutch of eggs. The parental male simultaneously releases his sperm and thereby fertilizes the eggs externally. Subsequently both parents leave the clutch. b, ‘Pirate’ males search for freshly laid clutches, clasp them and release their sperm, sometimes crawling into the clutch to gain access to the internal eggs. letters to nature NATURE | VOL 431 | 16 SEPTEMBER 2004 | www.nature.com/nature 305
