Brief communication Mitochondrial DNA variation suggests extensive gene flow from Polynesian ancestors to indigenous Melanesians in the northwestern Bismarck Archipelago.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 130:551–556 (2006) Brief Communication: Mitochondrial DNA Variation Suggests Extensive Gene Flow from Polynesian Ancestors to Indigenous Melanesians in the Northwestern Bismarck Archipelago Jun Ohashi,1* Izumi Naka,1 Katsushi Tokunaga,1 Tsukasa Inaoka,2 Yuji Ataka,3 Minato Nakazawa,4 Yasuhiro Matsumura,5 and Ryutaro Ohtsuka6 1 Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033 Japan Department of Environmental Sociology, Faculty of Agriculture, Saga University, Saga, 840-8502 Japan 3 Research Center for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki, 852-8523 Japan 4 Socio-Environmental Health Sciences, Graduate School of Medicine, Gunma University, Gunma, 371-8511 Japan 5 Division of Health Informatics and Education, National Institute of Health and Nutrition, Tokyo, 162-8636 Japan 6 Department of Human Ecology, Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033 Japan 2 KEY WORDS Austronesian; D-loop; Melanesian; mtDNA; 9-bp deletion; Polynesian ABSTRACT Archaeological, linguistic, and genetic studies show that Austronesian (AN)-speaking Polynesian ancestors came from Asia/Taiwan to the Bismarck Archipelago in Near Oceania more than 3,600 years ago, and then expanded into Remote Oceania. However, it remains unclear whether they extensively mixed with indigenous Melanesians who had populated the Bismarck Archipelago before their arrival. To examine the extent of admixture between Polynesian ancestors and indigenous Melanesians, mitochondrial DNA (mtDNA) variations in the D-loop region and the cytochrome oxidase and lysine transfer RNA (COII/tRNALys) intergenic 9-bp deletion were analyzed in the following three Oceanian populations: 1) Balopa Islanders as AN-speaking Melanesians living in the northwestern end of the Bismarck Archipelago, 2) Tongans as AN-speaking Polynesians, and 3) Gidra as non-Austronesian-speaking Melanesians in the southwestern lowlands of Papua New Guinea. Phylogenetic analysis of mtDNA sequences revealed that more than 60% of mtDNA sequences in the Balopa Islanders were very similar to those in Tongans, suggesting an extensive gene ﬂow from Polynesian ancestors to indigenous Melanesians. Furthermore, analysis of pairwise difference distributions for the D-loop sequences with the 9-bp deletion and the Polynesian motif (i.e., T16217C, A16247G, and C16261T) suggested that the expansion of Polynesian ancestors possessing these variations occurred approximately 7,000 years ago. Am J Phys Anthropol 130:551–556, 2006. V 2006 Wiley-Liss, Inc. Recent genetic studies showed that Austronesian (AN)-speaking Polynesian ancestors came from Asia/Taiwan to the Bismarck Archipelago in Near Oceania, and then expanded into Remote Oceania. The Bismarck Archipelago had been colonized by Melanesians before the arrival of the Polynesians ancestors. However, there are two contradictory models in terms of admixture between these groups: ‘‘express train’’ (Diamond, 1988; Bellwood, 1989) and ‘‘slow boat’’ (Kayser et al., 2000). The former model supposes a rapid expansion of Polynesian ancestors along the coast of New Guinea to Polynesia, with negligible admixture with indigenous Melanesians. The latter model proposes slow movement of the Polynesian ancestors, with extensive admixture. Although both geneticists and archaeologists now generally accept some degree of admixture between the Polynesian newcomers and indigenous Melanesians, the extent of admixture and direction of gene ﬂow remain under debate. Early studies of maternally inherited mitochondrial DNA (mtDNA) supported the express-train model, because a 9-base pair (bp) deletion between the genes for cytochrome oxidase (COII) and lysine transfer RNA (tRNALys), here termed the 9-bp deletion, was found in Southeast Asians and Polynesians, but rarely in Melanesians, and especially not in Papua New Guinea highlanders (Hertzberg et al., 1989; Stoneking et al., 1990). Recent studies, however, revealed that the 9-bp deletion occurs frequently in AN-speaking Melanesians in Island Melanesia, suggesting admixture between Polynesian ancestors and indigenous Melanesians (Merriwether et al., 1999). C 2006 V WILEY-LISS, INC. C Grant sponsor: Ministry of Education, Culture, Sports, Science, and Technology of Japan, Grant-in-Aid for Scientiﬁc Research. *Correspondence to: Jun Ohashi, Ph.D., Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: firstname.lastname@example.org Received 25 March 2005; accepted 11 August 2005. DOI 10.1002/ajpa.20383 Published online 19 January 2006 in Wiley InterScience (www.interscience.wiley.com). 552 J. OHASHI ET AL. Fig. 1. Map of locations of populations studied. The slow-boat model was based on analyses of Y-chromosome polymorphisms (Kayser et al., 2000). Unlike mtDNA, major Y-haplotypes of Polynesians were also found in Melanesians but not in Asians, suggesting that Polynesian ancestors extensively mixed with indigenous Melanesians, and that many Melanesian genes were incorporated into Polynesians. More recently, we reported that the ABO*A102 allele of the ABO blood-group gene could serve as a genetic marker reﬂecting gene ﬂow from Polynesian ancestors to Melanesians (Ohashi et al., 2004). We compared ABO allele frequencies among three groups: 1) AN-speaking Melanesians in the Balopa Islands of the Bismarck Archipelago, 2) AN-speaking Polynesians in Tonga, and 3) the Gidra, a non-Austronesian (NAN)-speaking Melanesian people of the southwestern lowlands of Papua New Guinea (PNG) (Ohashi et al., 2004). The Balopa Islands of the Manus province of PNG are located at the northwestern end of the Bismarck Archipelago (i.e., the northwestern end of Near Oceania). Because a Pleistocene site, the Pamwak rock shelter, was found in Manus (Fredericksen et al., 1993), and the oldest Lapita potteries, dating to 3,600 years ago, were discovered in Mussau Island located close to Balopa Island, the Balopa Islanders are considered to be descendants of the indigenous Melanesians who were initially contacted by the Polynesian ancestors in Near Oceania. Thus, the genetic trail of Polynesian newcomers to Near Oceania, if it still remains, should be detectable in Balopa Islanders. From the ABO allele-frequency data, we concluded that Balopa Islanders were genetically more similar to the Polynesian Tongans than to the Melanesian Gidra (Ohashi et al., 2004). Because mtDNA and Y-chromosome studies show that the extent of admixture and direction of gene ﬂow might differ among genetic markers, our perspective, based on the ABO allele frequency, that Polynesian ancestors mixed with the ancestors of Balopa Islanders should be reevaluated with other genetic markers. Thus, in this study, to obtain more conclusive evidence on the genetic proximity of Balopa Islanders to Polynesians, mtDNA polymorphisms in the Dloop region and the intergenic COII/tRNALys 9-bp deletion were analyzed in the same Oceanian populations that we previously studied: Balopa Islanders, Tongans, and the Gidra. In addition, pairwise difference distributions for the D-loop sequences with the 9-bp deletion and the so-called ‘‘Polynesian motif’’ have implications for the evolutionary history of Polynesian ancestors. MATERIALS AND METHODS Study populations Balopa. The Balopa Islands, which consist of three inhabited islands (Lou, Pam, and Baluan) in the Manus province of PNG, are located at 1478150 –1478250 East longitude and 28200 –28350 South latitude. In total, 59 Balopa Islanders were analyzed in this study, and each individual was not consanguineously related to others until three generations back, based on villagers’ genealogical records as reconstructed by the authors (Ataka, 1997; Ataka and Ohtsuka, 2000). Tonga. Twenty Tongans living in Ha’ano and Fakakakai villages of Ha’apai Island and 23 Tongans in Nukualofa, the capital city of the Kingdom of Tonga, were recruited in this study. Gidra. The Gidra-speaking population, included in the NAN-speaking group, is located in the southwestern lowlands of PNG. Their population structure and subsistence were described previously (Ohtsuka et al., 1985; Ohtsuka, 1986). For the analysis of mtDNA variation, 59 Gidra were selected from 192 unrelated individuals living in 12 villages. The geographic locations of the populations studied are shown in Figure 1. This study was approved by the Ethics Committee of the Graduate School of Medicine, University of Tokyo. The D-loop sequences from nine aboriginal Taiwanese groups were drawn from Tajima et al. (2003). Molecular typing of mtDNA Typing of the COII/tRNALys intergenic deletion. The mtDNA 9-bp deletion in the COII/tRNALys intergenic region was detected by the polymerase chain reaction (PCR) method established previously (Horai et al., 1996), with a slight modiﬁcation of ampliﬁcation conditions. The ampliﬁed fragments (100 or 91 bp in length) encompassing the intergenic region were separated by electrophoresis on 10% acrylamide gels, followed by staining with ethidium bromide. mtDNA VARIATION IN OCEANIAN POPULATIONS TABLE 1. Frequencies of 9-bp deletion and Polynesian motifs (T16217c, A16247G, and C16261T) in Oceanian populations1 Population Balopa Tonga Gidra Deletion frequency (%) Polynesian motif frequency (%) 37/59 (62.7) 39/43 (90.7) 0/59 (0.0) 29/59 (49.2) 34/43 (79.1) 0/59 (0.0) 1 All mtDNA sequences with Polynesian motifs have 9-bp deletion. Direct sequencing of the D-loop region. A fragment of the D-loop region of mtDNA was ampliﬁed with the primer pair B (15985-50 -AGCACCCAAAGCTAAGATTC30 -16004) and E (100-50 -CAGCGTCTCGCAATGCTATCGCGTG-30 -76), as described elsewhere (Fucharoen et al., 2001; Qian et al., 2001). The notation of Anderson et al. (1981) was used for numbering of bases. Ampliﬁcation conditions consisted of an initial denaturation at 968C for 10 min, followed by 30 cycles of denaturation at 968C for 15 sec, annealing at 528C for 15 sec, and extension at 728C for 35 sec, using a thermal cycler (GeneAmp PCR System 9700; Perkin-Elmer Applied Biosystems). Nucleotide sequences of both strands were determined with an automated sequencer (ABI Prism 3100). Data analysis Phylogenetic tree and nucleotide diversity. After eliminating insertion and deletion polymorphisms from the D-loop sequences, the number of nucleotide substitutions per site between individual sequences was estimated, based on the two-parameter method of Kimura (1980). In total, 563 nucleotide sites (positions 16048–16569 followed by positions 1–41: position numbers which followed the standard numbering system of Anderson et al., 1981) were compared for phylogenetic analysis among the Balopa Islanders, Tongans, Gidra, and nine aboriginal Taiwanese populations previously reported (Tajima et al., 2003). Based on the estimated number of substitutions, a phylogenetic tree was constructed by using the unweighted pair group method with arithmetic mean (UPGMA) method. In addition, nucleotide diversity within and between populations (dX and dXY, respectively), and net nucleotide diversity between populations (dA ¼ dXY (dX þ dY)/2; Nei and Li, 1979), were calculated. These analyses were conducted using MEGA version 2.1 (Kumar et al., 2001). Pairwise difference distributions of D-loop sequences. After eliminating insertion and deletion polymorphisms, pairwise differences of mtDNA D-loop sequences were calculated for a total of 623 nucleotide sites (positions 16007–16569, followed by positions 1–60) for the Balopa Islanders, Tongans, and Gidra. The time of population expansion of Polynesian ancestors was inferred based on the pairwise difference distributions of 63 D-loop sequences with the Polynesian motif, assuming the sudden-expansion model (Rogers and Harpending, 1992; Schneider and Excoffier, 1999). These analyses were conducted using Arlequin version 2.000 (Schneider et al., 2000). RESULTS The frequencies of the 9-bp deletion among Balopa Islanders, Gidra, and Tongans are presented in Table 1. The frequency was high among Tongans, as previously reported (Hertzberg et al., 1989; Sykes et al., 1995), while no mtDNA with the 9-bp deletion was found 553 among the Gidra. Because the Gidra are NAN-speaking Melanesians living in the southwestern lowlands of PNG, they as well as PNG highlanders (Hertzberg et al., 1989) are believed to be free from gene ﬂow from AN-speaking groups such as Polynesian ancestors. Thus, the present result is consistent with this perspective. Among Polynesians, the 9-bp deletion is associated with the following three transitions: T16217C, A16247G, and C16261T (Hagelberg and Clegg, 1993; Lum et al., 1994; Melton et al., 1995; Redd et al., 1995). This haplotype was called the ‘‘Polynesian motif’’ (Melton et al., 1995; Redd et al., 1995). The frequencies of the Polynesian motif in the populations studied are shown in Table 1. It should be noted here that all mtDNA sequences with the Polynesian motif had the 9-bp deletion. Figure 2 shows a phylogenetic tree that combines Dloop polymorphisms and the 9-bp deletion for three Oceanian populations and nine aboriginal Taiwanese groups. The Balopa Islanders’ sequences fell into four clusters. Of particular interest, the Tongans and Gidra shared scarcely any sequence features, while the Balopa Islanders in cluster I shared similar mtDNA sequences with Tongans, and the Balopa Islanders in clusters II–IV had sequences similar to the Gidra. Figure 3 shows the distributions of pairwise nucleotide differences within populations. As expected from the results of phylogenetic analysis, a bimodal distribution was observed in the Balopa Islanders. The peak at one difference reﬂects the sequence similarity in cluster I of Figure 2, and the peak at 14 differences result from the comparison between sequences in clusters I and IV. To estimate the time of expansion of Polynesian ancestors with the Polynesian motif, we calculated three demographic parameters that could ﬁt the observed pairwise difference distributions of 63 D-loop sequences with the Polynesian motif, assuming an instantaneous stepwise demographic expansion model (Rogers and Harpending, 1992; Schneider and Excofﬁer, 1999). This approach enables us to estimate a demographic parameter s ¼ 2 ut), where u is the total mutation rate per generation, and t measures time in generations since the sudden expansion. The estimated s was 1.005, with a 95% conﬁdence interval (CI) of 0.034–1.525 obtained from the bootstrap method with 10,000 replications. The substitution rate of the mtDNA control region was estimated to be 1.142 3 107 per site per year, based on the divergence of mtDNA sequences in PNG groups (Stoneking et al., 1992); 623 sites, excluding insertions and deletions, were examined here. The corresponding expansion time t is therefore 7,060 (95% CI, 240–10,720) years ago. DISCUSSION The frequency of the 9-bp deletion was found to exceed 60% among the Balopa Islanders. Thus, as expected from our previous study on the ABO blood-group gene (Ohashi et al., 2004), Balopa Islanders showed genetic proximity to Tongans. Although the 9-bp deletion had been regarded as a useful marker for tracing the expansion of Polynesian ancestors, there is increasing evidence that the same deletion occurred independently in other populations (Ballinger et al., 1992; Soodyall et al., 1996). Thus, analysis of only the 9-bp deletion does not provide conclusive evidence, and so we analyzed mtDNA variations in the D-loop region. A phylogenetic tree for the Dloop region in Figure 2 revealed that Balopa Islanders shared similar mtDNA sequences with Tongans and also with the Gidra. Because all mtDNA sequences in cluster I 554 J. OHASHI ET AL. Fig. 2. UPGMA tree for 341 mtDNA sequences from Balopa Islanders, Tongans, Gidra, and nine aboriginal Taiwanese groups. The mtDNA sequences from Oceanian populations are distinguished as four clusters: cluster I (Polynesian origin), cluster II (Melanesian origin), cluster III (Melanesian origin), and cluster IV (Melanesian origin). Individual sequences at tips of branches are represented by population names followed by sample ID numbers. M, Balopa Islanders; T, Tongans in Ha’ano and Fakakakai villages of Ha’apai island; TG, Tongans in Nukualifa; G, Gidra. Aboriginal Taiwanese groups are designated as in Tajima et al. (2003): ATA, Atayal; SAI, Saisiat; BUN, Bunun; TSO, Tsou; RUK, Rukai; PAI, Paiwan; AMI, Ami; PUY, Puyuma; YAM, Yami (Tajima et al., 2003). The 9-bp deletion appears to have occurred at position indicated by arrow. of Figure 2 had the 9-bp deletion and the sequences of aboriginal Taiwanese diverged prior to those of Balopa and Tonga, the deletion seems to have arisen only once in these populations. Thus, the mtDNA sequences of Balopa in cluster I appear to be derived from Polynesian ancestors who came from Asia/Taiwan. In contrast, the mtDNA sequences of Balopa in clusters II–IV seem to have derived from NAN-speaking Melanesians or indigenous Melanesians who had populated the Bismarck Archipelago before Polynesian ancestors arrived. This result strongly suggests that extensive gene ﬂow from Polynesian ancestors to ancestors of the Balopa Islanders occurred when Polynesian ancestors arrived at the Bismarck Archipelago. Thus, our data do not support the ‘‘express-train’’ model hypothesizing that admixture between Polynesian ances- tors and indigenous Melanesians is negligible. In the ‘‘slowboat’’ model proposed by Kayser et al. (2000), based on Y-chromosome polymorphisms, Polynesian ancestors moved gradually across Melanesia and left behind their genes, but also incorporated many Melanesian genes. The present data from mtDNA sequences are not compatible with the incorporation of many Melanesian genes into Polynesian ancestors. The main direction of gene ﬂow appears to have been from Polynesian ancestors to indigenous Melanesians. Of particular interest, this perspective is supported by mtDNA following maternal inheritance. Thus, not only males but also many females may have gotten off the ‘‘slow boat.’’ Although the phylogenetic tree in Figure 2 does not allow us to infer from which aboriginal Taiwanese groups mtDNA VARIATION IN OCEANIAN POPULATIONS Fig. 3. Distributions of pairwise nucleotide differences of Dloop region in Balopa, Tonga, and Gidra. the Polynesian ancestors are derived, sequences in the Ami were found to be most similar to those in Tongans. Furthermore, the net nucleotide diversity dA between the Ami and Tongans (0.0039) was smaller than that between the other aboriginal Taiwanese groups and Tongans (data not shown). Also, dA between the Ami and Balopa Islanders (0.0020) was smaller than that between the other aboriginal Taiwanese groups and Balopa (data not shown). The Ami inhabit the eastern coast of the main island of Taiwan, geographically close to Near Oceania. Taking these results together, we speculate that Polynesian ancestors are partly derived from ancestors of the Ami. This view should be clariﬁed by future studies. The distributions of pairwise nucleotide differences within populations were different among three populations (Fig. 3). A unimodal distribution with a strong peak at one difference was observed in Tongans, suggesting a recent population expansion of Polynesians (Rogers and Harpending 1992). In contrast, the Gidra showed multiple peaks, resulting from three mtDNA lineages indicated by clusters II–IV in Figure 2. The multiple peaks imply that the Gidra did not expand recently, unless they mixed with other populations. The Gidra are thought to be descendants of early migrants into PNG, and have been well-isolated from other Papuan groups due to the linguistic boundary. It is therefore surprising that the three allelic lineages that diverged a long time ago have been maintained in the small population of Gidra (approximately 2,000 in 1989), regardless of random genetic drift. Overall, our results seem to be consistent with demographic histories of the studied populations. The Polynesian motif was frequently found in Balopa Islanders as well as in Tongans (Table 1), suggesting that the appearance of the Polynesian motif had occurred before Polynesian ancestors arrived at the Bismarck Archipelago. As shown in Figure 2, a number of sequence features are shared by Balopa Islanders and Tongans in cluster I. This observation implies a rapid expansion of Polynesian ancestors before their signiﬁcant admixture with indigenous Melanesians. Our estimated time of the expansion, 7,060 years ago, is similar to the 5,500 years ago inferred by Redd et al. (1995), based on mtDNA sequences from Indonesia, coastal PNG, and Samoa. Haplotypes of the 9-bp deletion with the Polynesian motif have never been found in aboriginal Taiwanese groups (Melton et al., 1995, 1998; Sykes et al., 1995; Tajima 555 et al., 2003). Thus, the Polynesian motif appears to have arisen in Polynesian ancestors after they left Asia/Taiwan. Interestingly, the estimated expansion time of 7,000 years ago is consistent with the estimated date, 6,000–6,500 years ago, of the ﬁrst split with AN languages into Formosan (one or more primary subgroups in Taiwan) and Malayo-Polynesian (all AN languages outside Taiwan) (Blust, 1988; Bellwood, 1991). Thus, the population expansion of Polynesian ancestors with the Polynesian motif may have occurred soon after they left Asia/Taiwan. Haplogroups P and Q, as deﬁned by Forster et al. (2001), are commonly found in Papua New Guinea. We examined if the present D-loop sequences belong to P or Q by using the same mtDNA sequences in Figures 2 and 4 of Friedlaender et al. (2005), although such conventional haplogroups can be distinguished correctly based on whole-genome sequencing. We found that approximately 20% and 60% of Gidra mtDNA sequences were similar to haplogroups P and Q, respectively, and 5% and 27% of Balopa Islanders were similar to P and Q, respectively. Interestingly, 20% of Gidra mtDNA sequences seemed to be placed between haplogroups P and Q (data not shown). In Australian Aborigines, mtDNA sequences belonging to haplogroup P are found, while those belonging to haplogroup Q are not (Huoponen et al., 2001; Ingman and Gyllensten, 2003; Friedlaender et al., 2005). Thus the Gidra, NAN-speaking Melanesians, are considered to be genetically close to Australian Aborigines compared to Balopa Islanders, AN-speaking Melanesians. CONCLUSIONS The analysis of mtDNA variations revealed that Balopa Islanders were genetically similar to Tongans rather than the Gidra. The present results suggest that extensive gene ﬂow occurred from Polynesian ancestors to indigenous Melanesians. Based on pairwise difference distributions of D-loop sequences with the Polynesian motif (i.e., T16217C, A16247G, and C16261T) in Balopa Islanders and Tongans, the expansion of Polynesian ancestors possessing the motif was presumed to have occurred approximately 7,000 years ago. SEQUENCE AVAILABILITY The sequences reported here were deposited in DDBJ/ EMBL/GenBank under accession numbers AB119285– AB119445. ACKNOWLEDGMENTS The authors are indebted to the people of Papua New Guinea and Tonga for their cooperation in providing blood samples for testing. We also thank Drs. Taniela Palu (Ministry of Health, Kingdom of Tonga), Viliami Tangi (Diabetes Clinic, Kingdom of Tonga), and Kazumichi Katayama (Primate Research Institute, Kyoto University) for research on the Tongan population. We thank three anonymous reviewers for valuable comments and suggestions about a previous version of the manuscript. 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