Mitochondrial DNA Haplogrouping of the Okhotsk People Based On

ANTHROPOLOGICAL SCIENCE Vol. 117(3), 171–180, 2009

Mitochondrial DNA haplogrouping of the Okhotsk people based on analysis of ancient DNA: an intermediate of gene flow from the continental Sakhalin people to the Ainu Takehiro SATO1, Tetsuya AMANO2, Hiroko ONO2, Hajime ISHIDA3, Haruto KODERA4, Hirofumi MATSUMURA5, Minoru YONEDA6, Ryuichi MASUDA1*

1Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan 2Hokkaido University Museum, Sapporo 060-0810, Japan 3Department of Anatomy, Faculty of Medicine, University of the Ryukyus, Nishihara 903-0215, Japan 4School of Dental Medicine, Tsurumi University, Yokohama 230-8501, Japan 5Department of Anatomy, Sapporo Medical University, Sapporo 060-8556, Japan 6Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8562, Japan

Received 2 December 2008; accepted 28 March 2009

Abstract In order to further understand the genetic status of the Okhotsk people, who were distributed in southern coastal regions of the Okhotsk Sea during the 5th–13th centuries, nucleotide variations in the hypervariable region (HVR) and the coding regions of mitochondrial DNA (mtDNA) were analyzed. Targeting the coding regions provides reliable genetic information even from ancient DNAs that may have suffered post-mortem damage. MtDNA haplogroups of 38 individuals were classified according to mtDNA lineages known in northeastern Asian people. Comparisons of mtDNA haplogroup frequencies between the Okhotsk people and other Asian populations revealed that the genetic structures of the Okhotsk people are very similar to those of populations currently living around lower regions of the Amur River and the Ainu of Hokkaido. The results support our previous study on molecular phylogeny of mtDNA HVR 1 sequences, and strongly suggest that the Okhotsk people originated around the lower regions of the Amur River and became an intermediate of gene flow from the continental Sakhalin people to the Ainu.

Key words: Okhotsk people, genetic origins, ancient DNA, mitochondrial DNA, haplogroup

Introduction clearly different from those of the Epi-Jomon and Satsumon people. Hence, there has been much discussion among ar- The Okhotsk culture developed around the southern chaeologists and anthropologists as to the origins of the coastal regions of the Okhotsk Sea during the 5th–13th cen- Okhotsk people. Morphological studies have revealed that turies (Amano, 2003a). The Okhotsk culture differs in cer- the characteristics of the Okhotsk people are similar to those tain respects from the Epi-Jomon culture (3rd century BC– of the Nivkhi and Ulchi people, currently distributed around 7th century AD) and the Satsumon culture (8th–14th centu- Sakhalin and the lower regions of the Amur River (Ishida, ries: Amano, 2003b), which were contemporary with the 1988, 1996; Kozintsev, 1990, 1992; Komesu et al., 2008). Okhotsk culture and developed in the southern and inner However, the origins of the Okhotsk people have not yet parts of Hokkaido Island. A particular feature of the Okhotsk been clarified. culture is adaptation of their lifestyle to sea fishing and hunt- The closer archaeological relationships between the Ainu ing. Therefore, archaeological sites of the Okhotsk culture and Okhotsk cultures have also been investigated. Utagawa are restricted to coastal regions. Moreover, polygonal large (2002) reported the occurrence of bear-sending ceremonies houses and rituals using animals, including brown bears, based on evidence obtained from archaeological sites of the which are other typical features of the Okhotsk culture, have Okhotsk culture. In the Ainu culture (17th century to the not been observed in the Epi-Jomon and Satsumon cultures. present day), the people consider brown bears to be a moun- In addition, skulls of the Okhotsk people share particular tain god and perform a bear-sending ceremony called “Iom- morphological characteristics, which are anthropologically ante” using juvenile bears nursed in the villages. These facts suggest that the Okhotsk people merged with the Satsumon people (a direct ancestoral lineage of the Ainu people) on * Correspondence to: Ryuichi Masuda, Department of Natural His- Hokkaido, resulting in the establishment of the Ainu people tory Sciences, Graduate School of Science, Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan. (Utagawa, 2002). E-mail: [email protected] Sato et al. (2007) analyzed the mitochondrial DNA (mtD- Publlished online 28 May 2009 NA) hypervariable region (HVR) 1 from bone remains of in J-STAGE (www.jstage.jst.go.jp) DOI: 10.1537/ase081202 the Okhotsk people excavated from archaeological sites, and

© 2009 The Anthropological Society of Nippon 171 172 T. SATO ET AL. ANTHROPOLOGICAL SCIENCE reported that the Okhotsk people were closely related to lacked archaeological information, their dates were deter- populations that are currently distributed around Sakhalin mined by the radiocarbon method (Yoneda et al., 2004). The and the lower regions of the Amur River. In addition, Sato et dates were consequently estimated to be from the 7th to the al. (2007) suggested gene flow from the Okhotsk people to 13th centuries AD, with the marine reservoir effect (400 14C the Ainu people. years; Yoneda et al., 2007), in agreement with the Okhotsk Meanwhile, some mtDNA haplogroups that were consid- culture period. Detailed information for the materials used is ered to be monophyletic in the early literature have turned available on request. out to be paraphyletic, due to insufficient information of coding region sequences. Therefore, the analysis of coding DNA extraction region informations is essential for reliably inferring Total DNA was extracted from femurs, ribs, coxal bones, mtDNA phylogeny (Bandelt et al., 2001). Moreover, it was sacrums, skulls, or teeth. To eliminate the possibility of sur- reported that the distribution patterns of hotspots of post- face contamination of external DNA, each bone piece or mortem damage in HVR 1 correlate with those of the muta- tooth was soaked in sodium hypochlorite solution (8.5– tion sites. On the other hand, the post-mortem damage rate in 13.5% Cl, Nacalai) for 5 min, rinsed with DNase-/RNase- the coding regions is significantly lower than that in HVR 1 free distilled water, and air-dried. The bones and teeth were (Gilbert et al., 2003). Therefore, it is necessary to analyze then powdered with a dental drill. DNA extraction from bone the coding regions in addition to HVR, because post-mortem or tooth powders was carried out according to the method of damage could be involved in the case of ancient DNA Masuda et al. (2001). Approximately 0.2–0.5 g of powders analysis. per specimen was decalcified with 30 ml of 0.5 M ethylene- In the present study, direct sequencing of HVR 1 and diamine tetraacetic acid (EDTA, Nippongene) in a 50 ml HVR 2 as well as amplified product-length polymorphism plastic tube with rotation at room temperature for 24 h. The (APLP) analysis of coding regions of mtDNA were per- decalcified bone powders were suspended in 5 ml of 0.5 M formed to classify mtDNA haplogroups and to clarify the EDTA containing 100 μl of 10 mg/ml proteinase K at 37°C frequencies in the Okhotsk people. Phylogenetic results with rotation for 24 h. The solution was extracted by using from data of mtDNA haplogroups were compared with those the phenol–chloroform extraction method (phenol/chloro- of HVR 1 sequences, and the genetic features of the Okhotsk form/isoamylalcohol, 25:24:1; Sambrook et al., 1989). The people are further discussed. DNA extracts were concentrated into approximately 100 μl of TE buffer with VivaSpin 6 Concentrators (Sartorius) and Materials and Methods subjected to subsequent PCR as templates. Contamination precautions PCR amplification and direct sequencing The following standard contamination precautions were From the HVR 1 sequences (Accession Nos. AB292314– performed: separation of pre- and post-polymerase chain re- AB292350) of 37 Okhotsk people reported by Sato et al. action (PCR) experimental areas, wearing gloves, face (2007), fragments with nucleotide positions (np) 16132– masks and laboratory coats, use of disposable filter-plugged 16402 relative to the revised Cambridge reference sequence pipette tips and disposable tubes, treatment with a DNA- (CRS) (Andrews et al., 1999) were used for the analysis. AWAY (Molecular BioProducts), ultraviolet irradiation of Other fragments (np 16121–16131) of the 37 samples were equipments and bench, negative extraction controls, and newly determined in the present study. The fragments (np negative PCR controls. Moreover, mtDNA HVR sequences 16121–16402) for the additional samples were newly PCR- of members of our laboratory and related archaeologists and amplified and sequenced. In addition, a fragment of HVR 2 anthropologists were determined using DNA extracted from (np 128–267) for each sample was PCR-amplified and se- their hair roots. Extraction of DNA from hair roots was car- quenced. Because ancient DNA was possibly fragmented, ried out by the method of Walsh et al. (1991) and PCR am- the 282 base-pair (bp) fragment of HVR 1 was divided into plification and nucleotide sequencing were the same as de- two overlapping subregions and amplified with primer pairs scribed below. When ancient DNA sequences from the L16120/H16239 (Adachi et al., 2004) and A16208/B16403 Okhotsk remains were found to be identified with any mod- (Horai et al., 1989). Moreover, a segment of the coding re- ern DNA sequences of members of our laboratory and relat- gion (np 10382–10465) that covers part of the NADH dehy- ed archaeologists and anthropologists, those bone samples drogenase 3 and tRNAArg gene was amplified with primer were excluded from the subsequent analysis. pairs L10381/H10466 (Adachi et al., 2004) for direct sequencing, because APLP analysis results of several speci- Sample collection mens for 10398A and 10400T were ambiguous. To determine mtDNA haplogroups of the Okhotsk peo- An aliquot (1 μl) of DNA extract was used as PCR tem- ple, 102 skeletal remains excavated from 10 archaeological plate. Singleplex PCR was carried out with a Multiplex PCR sites on Hokkaido and Sakhalin (Figure 1) were analyzed. Kit (Qiagen) for efficient amplification. The PCR amplifica- The skeletal remains were preserved in the Hokkaido Uni- tions were carried out in a reaction mixture of 20 μl contain- versity Museum and Sapporo Medical University. To avoid ing reagents of the Multiplex PCR Kit (Qiagen), 0.25 μM of duplicate analyses of identical individuals, parts in the same each primer, and 0.4 μg/μl bovine serum albumin (BSA, positions of bones, or bones from different graves within one Boehringer). The BSA was used to eliminate some effects of archaeological site were used. the PCR inhibitors that are often found in ancient bones and Because skulls excavated from the Moyoro site (Figure 1) teeth. The conditions of PCR were incubation at 95°C for Vol. 117, 2009 MITOCHONDRIAL DNA HAPLOGROUPING OF THE OKHOTSK PEOPLE 173

Figure 1. Geographical locations of archaeological sites of the Okhotsk culture where specimens analyzed in the present study were excavated.

15 min to activate the polymerase; 35 cycles of 94°C for Multiplex APLP analysis 30 s, 46°C or 55°C for 90 s, 72°C for 90 s; and final exten- To analyze the haplogroup-diagnostic mitochondrial sin- sion at 72°C for 10 min. gle nucleotide polymorphisms (SNPs) of the Okhotsk peo- After the amplification, PCR products were purified with ple, the multiplex APLP method of Umetsu et al. (2005) was the QIAquick PCR Purification Kit (Qiagen) and used for used. By using this method, 36 diagnostic mutations sequencing; 4 μl of PCR product was used as template. Se- (10398A, intergenic COII/tRNALys 9 bp deletion, 5178A, quence reactions were carried out using the Sequenase Prim- 3010A, 14979C, 8020A, 13104G, 11215T, 11959G, er Cycle sequencing kit (Amersham) and the following se- 10400T, 8793C, 4833G, 8200C, 3394C, 14178C, 3970T, quencing primer pairs: L132/H261 for np 128–267, L10387/ 5417A, 13183G, 3594T, 11969A, 11696A, 6455T, 4386C, H10458 for np 10382–10465, A16126/B16233 for np 12811C, 15487T, 8684T, 1736G, 10873T, 8994A, 4580A, 16121–16238, and A16214/B16398 for np 16209–16402. In 10550G, 12308G, 1719A, 15607G, 7028C, and 12612G) addition, the primer H16167 (Ricaut et al., 2004) was also were analyzed. An aliquot (1 μl) of DNA extract was used as used to resolve sequencing problems related to the polycy- template for the multiplex APLP analysis. The PCR amplifi- tosine region, located in np 16184–16193. The 3′ ends of se- cation was carried out in a reaction mixture of 20 μl contain- quencing primers were labeled with Texas Red. Sequencing ing reagents of the Multiplex PCR Kit (Qiagen), optimum reaction conditions were 95°C for 4.5 min; 30 cycles of concentrations of each primer (Umetsu et al., 2005), and 94°C for 30 s, 55°C for 30 s, 72°C for 1 min; and final ex- 0.4 μg/μl BSA. The PCR conditions were incubation at tension at 72°C for 7 min. The products were analyzed using 95°C for 15 min to activate the enzyme; 35 cycles of 94°C an automated DNA sequencer Hitachi SQ-5500. for 30 s, 52°C for 3 min, 72°C for 90 s; and final extension at 72°C for 10 min. 174 T. SATO ET AL. ANTHROPOLOGICAL SCIENCE

An aliquot (10 μl) of the PCR product was separated by electrophoresis in a 13 cm native polyacrylamide gel (10% T, 5% C) containing 375 mM Tris–NaOH buffer (pH 8.9) with the running buffer (12.5 mM Tris, 96 mM glycine, pH 8.3). The DNA bands were detected with an ultraviolet illuminator after staining with ethidium bromide.

Data analysis To clarify the maternal genetic structures of the Okhotsk people, mtDNA data of the Okhotsk people were assigned to mtDNA haplogroups according to the data and classification tree of East Asian mtDNAs (Kivisild et al., 2002; Yao et al., 2002; Kong et al., 2003, 2006; Maruyama et al., 2003). Be- cause the multiple segments from the identical mtDNA were analyzed independently, the haplogroup assignment was carried out carefully to avoid artificial recombination of the data. If the haplogroup classified by HVR was inconsistent with that indicated by the coding region, that sample was excluded from the subsequent data analysis. To infer the phylogenetic relationships between the Okhotsk people and the other northeastern Asian populations, published mtDNA haplogroup data from 21 popula- Figure 2. Geographical locations of northeastern Asian populations were cited as follows: Mansi (Derbeneva et al., 2002a); tions compared with the Okhotsk people in the present study. OK, Ket, Nganasan (Derbeneva et al., 2002b), Itelmen, Koryak Okhotsk; UL, Ulichi; NV, Nivkhi; NG, Negidal; AI, Ainu; HJ, Hok- (Schurr et al., 1999); Chukuchi, Eskimo (Straikovskaya et kaido Jomon; JP, Mainland Japanese; CN, Chinese; KR, Korean; UD, Udegey; KY, Koryak; IT, Itelmen; ES, Eskimo; CH, Chukuchi; EV, al., 1998); Tubalar, Tuvan, Buryat, Tofalar, Evenki, Evenki; BR, Buryat; TF, Tofalar; TV, Tuvan; TB, Tubalar; NS, Nga- Negidal, Ulchi, Nivkhi, Udegey (Straikovskaya et al., 2005); nasan; KT, Ket; MN, Mansi. Hokkaido Jomon people (Adachi et al., 2006); Ainu (Horai et al., 1996), Mainland Japanese, Chinese, Korean (Umetsu and Yuasa, 2005). Geographic distributions of these popula- that were also observed in MYR-4 that was allocated to hap- tions are shown in Figure 2. Frequencies of mtDNA haplo- logorup G1b. The results showed that the possibility of DNA groups of these populations were compared with that of the contamination or post-mortem damage cannot be excluded F Okhotsk people. The pairwise st and statistical significance for these samples. However, because HVR 1 mutations were of frequencies of haplogroups were computed using Arle- in agreement with results of APLP analyses, we considered quin, version 3.11 (Excoffier et al., 2005). Then, the genetic the haplogroup assignments in Table 1 to be reasonable. The F relationships among the st values of the northeastern Asian nucleotide sequences of HVR 1 and HVR 2 as well as np populations were analyzed using the multidimentional scal- 10382–10465 will appear in the DNA databases (DDBJ/ ing method (Sneath and Sokal, 1973) in Statistica, version EMBL/GenBank) with the following accession numbers: 06J (Statsoft Japan). AB464841–AB464933. The haplogroup frequencies in the Okhotsk specimens Results were as follows: A, 8.1%; B5, 2.7%; C3, 5.4%; G1, 24.3%; M7, 5.4%; N9, 10.8%; Y, 43.2% (Table 2). Thus, in the mi- Nucleotide sequences of HVR 1 and HVR 2 were suc- tochondrial gene pool of the Okhotsk people, haplogroup Y cessfully PCR-amplified from 51 of the 102 specimens. was major. This genetic feature is similar to those of popula- Multiplex APLP analysis was carried out for the successful tions currently living around the lower regions of the Amur 51 specimens. No successful results were obtained from the River, such as the Ulchi, Nivkhi, and Negidal (Table 2). F other 51 specimens, because of possible DNA degradation. Table 3 shows values of pairwise st, estimated by haplo- Since the haplogrouping from APLP analysis for 14 of the group frequencies, among northern Asian populations. The successful 51 specimens did not agree with that from direct exact test demonstrated that differentiations in any pair of sequencing of HVR 1 and HVR 2, mtDNA haplogroups of the populations were statistically significant (P < 0.05). The the 14 specimens could not be correctly determined. The genetic relationships between the Okhotsk people and the cause of such inconsistency is considered to be modern other northeastern Asian populations were shown using the DNA contamination or post-mortem damage. Therefore, the multidimensional scaling method (Figure 3). Based on this, 14 specimens were excluded from the subsequent analysis. the Okhotsk people were closely related to the Ulchi, Ainu, As a result, mtDNA haplogroups of 37 specimens were fi- and Negidal. Although the Nivkhi also neighbored the F nally classified (Table 1). The HVR 2 sequence of OMS-1 Okhotsk people (Figure 3), the st values between the that was allocated to haplogorup G1b possessed 146–263 Nivkhi and Okhotsk people were 0.1556: this high value is mutations that were often observed in haplogroup Y, N9b, probably due to a high frequency of haplogroup Y (66.1%) and M7a. Moreover, the HVR 2 sequence of OMS-3 that in the Nivkhi. The findings show that the Okhotsk people are was allocated to haplogroup Y possessed 207–263 mutations genetically closer to populations currently living around the Vol. 117, 2009 MITOCHONDRIAL DNA HAPLOGROUPING OF THE OKHOTSK PEOPLE 175

Table 1. Nucleotide mutations observed in mtDNA of 37 specimens of the Okhotsk people examined in the present study Nucleotide substitutions in the segmentsa Sample Archeological b Haplogroup 16121–16208 16209–16402 APLP analysis number site 128–267 10382–10465 (+16000) (+16000) MYR-1 Moyoro Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C MYR-2 Moyoro Y1a1 126 189 231 266 146 263 10398 5417A 14178C MYR-3 Moyoro Y1a1 126 183C 189 231 266 200 257 263 10398 5417A 14178C MYR-4 Moyoro G1b 129 223 207 263 10398 10400 4833G 8200C MYR-5 Moyoro N9b 183C 189 223 263 CRS 5417A 13183G UTR-4 Utoro Y1a1 126 189 231 266 146 263 10398 5417A 14178C OMS-1 Omisaki G1b 129 223 146 263 10398 10400 4833G 8200C OMS-2 Omisaki G1b 129 223 263 10398 10400 4833G 8200C OMS-3 Omisaki Y1a1 126 183C 189 231 266 207 263 10398 5417A 14178C OMS-4 Omisaki Y1a1 126 189 231 266 146 263 10398 5417A 14178C OMS-5 Omisaki A CRS 223 242 290 319 185 235 263 CRS 1736G OMS-6 Omisaki G1b 129 223 263 10398 10400 4833G 8200C OMS-7 Omisaki G1b 129 223 263 10398 10400 4833G 8200C OMS-8 Omisaki G1b 129 223 231 263 10398 10400 4833G 8200C OMS-10 Omisaki Y1a1 126 189 231 266 292 146 263 10398 5417A 14178C OMS-11 Omisaki M7a 140 187 209 223 146 263 10398 10400 6455T 4386C HMN-1 Hamanaka Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C HMN-2 Hamanaka C3 183C 189 223 261 288 298 249d 263 10398 10400 15487T HMN-3 Hamanaka G1b 129 223 263 10398 10400 4833G 8200C HMN-4 Hamanaka N9b 183C 189 223 146 263 CRS 5417A 13183G HMN-5 Hamanaka Y1a1 183C 189 231 266 146 263 10398 5417A 14178C HMN-6 Hamanaka Y 189 231 146 263 10398 5417A 14178C HMN-7 Hamanaka G1b 129 223 263 10398 10400 4833G 8200C HMN-8 Hamanaka N9b 183C 189 223 146 263 CRS 5417A 13183G HMN-9 Hamanaka N9b 183C 189 223 212 263 CRS 5417A 13183G HMN-10 Hamanaka Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C HMN-11 Hamanaka G1b 129 223 263 10398 10400 4833G 8200C HMN-12 Hamanaka C3 183C 189 223 261 288 298 249d 263 10398 10400 15487T HMN-13 Hamanaka Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C HMN-14 Hamanaka Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C HMN-15 Hamanaka A CRS 223 242 290 319 185 235 263 CRS 1736G HMN-16 Hamanaka Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C SSY-1 Susuya Y 126 189 231 497 146 263 10398 5417A 14178C PRK-1 Pirikatai M7a 140 187 209 223 146 263 10398 6455T 4386C TMI-1 Tomiiso Y1a1 126 183C 189 231 266 146 263 10398 5417A 14178C FND-1 Funadomari A CRS 223 242 290 319 185 235 263 CRS 1736G FND-2 Funadomari B5b 140 182C 183C 189 234 243 291 131 204 263 10398 9 bp del a Numbers of polymorphic sites are relative to the revised Cambridge reference sequence (CRS) (Andrews et al., 1999), showing that the sequence is consistent with the revised CRS. The suffixes C and d indicate a transversion and d shows deletion, respectively. Diagnostic polymorphisms are emphasized by bold italic characters. b Numbers of polymorphic sites are relative to the revised CRS. The suffixes A, C, G, and T indicate nucleotide substitutions and “del” shows a deletion. lower regions of the Amur River as well as to the Ainu peo- (37.9%, 66.1%, 21.2%, respectively, Table 2). In addition, ple of Hokkaido. Adachi et al. (2006) reported that haplogroup Y was not observed among the Jomon people of Hokkaido, which are ar- Discussion chaeologically considered to be a direct ancestral lineage of the Ainu (Table 2). In the present study, 37 specimens of the Okhotsk people The present study showed that the Okhotsk people are ge- were assigned to mtDNA haplogroups. The most character- netically closely related to the Ulchi, Negidal, and Ainu istic feature of the gene pool of the Okhotsk people is the (Figure 3). In addition to these three populations, the Nivkhi high frequency of haplogroup Y (43.2%; 16 of 37 individu- shared haplogroup Y at high frequencies, suggesting a ge- als, Table 1 and Table 2), followed by haplogroups G1b netic affiliation with the Okhotsk people. Sato et al. (2007) (24.3%), N9b (10.8%), and the others. Recent studies have reported that the Ainu were not clustered with the Okhotsk shown that most people with haplogroup Y are currently dis- people in the neighbor-joining tree using net nucleotide di- tributed around Sakhalin, the lower regions of the Amur versities (dA distances) between populations (Nei and Li,

River, and in the Ainu on Hokkaido Island (Schurr et al., 1979), while the dA distance between the Okhotsk people 1999; Kivisild et al., 2002). The Ulchi, Nivkhi, and Negidal and the Ainu was smaller than those between the Okhotsk shared haplogroup Y at relatively higher frequencies people and other populations except for the Nivkhi and Ul- 176 T. SATO ET AL. ANTHROPOLOGICAL SCIENCE 98) = MN n ( 38) = KT n ( 24) = NS n ( data of Straikovskaya et al. data of Straikovskaya 72) = TB n ( 95) = TV n ( aikovskaya et al. (2005). 46) = TF n ( UD, Udegey; KY, Koryak; IT, Itelmen; ES, Eskimo; Koryak; IT, UD, Udegey; KY, 25) ies are inferred from the = BR n ( 71) = EV n ( hurr et al. (1999), and Str 66) = CH n n Asian populations ( 79) = ES n ( 47) IT = n ( s (%) in northeaster 155) KY = Population ainland Japanese; CN, Chinese; KR, Korean; ), Derbeneva et al. (2002b), Sc n ( 46) = UD n ( 103) KR = n ( 160) Tubalar; NS, Nganasan; KT, Ket; MN, Mansi. Haplogroup frequenc NS, Nganasan; KT, Tubalar; CN = n ( 005), Derbeneva et al. (2002a 874) JP = n ( Table 2. Frequencies of mtDNA haplogroup 44) = HJ n gidal; AI, Ainu; HJ, Hokkaido Jomon; JP, M gidal; AI, Ainu; HJ, Hokkaido Jomon; JP, ( 51) = AI n ( 33) = NG n ( achi et al. (2006), Umetsu (2 56) = NV n ( 87) = UL n ( 37) = OK n ( OK, Okhotsk; UL, Ulchi; NV, Nivkhi; NG, Ne Nivkhi; NG, OK, Okhotsk; UL, Ulchi; NV, 4.3.6.2.1.—.—1.58.79.74.1—————————— — ————4.04.38.54.2——— — ———————1.44.2—— — ——————————— A —B2.7——12.12.0—11.715.714.8— — ————4.0—4.25.68.410.514.3 F 8.1Y43.237.966.121.219.6—0.9— — — — ————4.0—2.1———12.2 — —H — — ———1.4——1.14.2——1.0 —————— — — — ——————————— 0.6— — — ——————————— ——————————— V —————— — —J — — — 1.1 —————— — — ————4.0—————1.0 0.6— T —————— — —U —————— — — — — — — — 0.5— — ——————————7.2 —K — —————— — — — — — — ————12.0—3.226.425.034.225.4 — ——————————— W—————— — — 3.9 — — — — ——————————3.1 —X — —————— — — ——————2.1——2.7— —N — ——————0.4— — — 2.0 — — — ———————1.4——— ——————————— N910.86.9——7.865.94.96.25.930.4———————3.26.9——— R —————— — 6.7 — —C1—1.1———— — — — — ——————————— ———————2.8——— C2 2.5— — 7.1 8.8 —C3 — —1.3—5.11.51.4——3.21.416.7—1.0 —C4——————— — — 7.5 5.4 8.9 — —CZ——————1.58.13.4— — ————4.0—6.3———6.1 6.9D — — 5.7 — — — ——————————— — 6.4 —D1—4.6——5.913.6— —15.210.6———————— —D2——————— — —D3—2.3———— 5.2 — 9.1D4—10.35.46.19.8—36.916.226.5— 1.3— 4.6 6.1D5————7.8—4.010.07.4— 6.4 — 23.2 —D6——————— — 77.2G ——————3.15.03.4— 18.2 — — 68.2G124.310.35.427.215.713.63.21.94.4—41.968.1—9.1———————— — — — — — —G2—1.1——3.9— ————4.0—4.3———— — 5.6 —G3——————— —Z — —————— — — — —M—2.3——2.0—3.53.8—28.3———————1.6———1.0 5.5—5.86.4———4.010.91.61.44.22.9— —M75.4———19.66.815.26.910.3— — — 1.4 —M8—4.6————0.35.01.015.2—————4.015.2————— — — — — 2.1 17.4 11.1 8.7 — — 23.8 — 4.2 12.3 2.1 — 7.9 12.8 1.4 2.5 — 3.1 — — 4.5 23.7 — 6.1 57.8 14.1 1.0 40.0 — 50.0 — — 35.8 10.9 18.3 16.7 7.4 16.0 12.6 2.8 15.8 — 20.8 16.3 9.5 — 12.5 1.0 8.3 2.6 6.2 group Haplo- CH, Chukuchi; EV, Evenki; BR, Burya; TF, Tofalar; TV, Tuvan; TB, Tuvan; TV, Tofalar; Evenki; BR, Burya; TF, CH, Chukuchi; EV, (2005), Horai et al. (1996), Ad Vol. 117, 2009 MITOCHONDRIAL DNA HAPLOGROUPING OF THE OKHOTSK PEOPLE 177 6 UD, Udegey; KY, Koryak; IT, Itelmen; ES, Eskimo; Koryak; IT, UD, Udegey; KY, 114 0.2229 0.1136 0.1768 0.1276 0.0457 0.0836 950 0.1758 0.0652 0.1526 0.0761 0.0285 0.0541 0.051 5 0.3135 0.1801 0.1001 0.1599 0.0905 0.0459 0.05). < P ainland Japanese; CN, Chinese; KR, Korean; values between populations st F values among northeastern Asian populations 9 0.1426 0.1582 0.3039 0.3183 0.2504 0.1541 0.0597 0.1457 0.0669 st F 391 0.1413 0.1328 0.3232 0.3862 0.3108 0.0475 0.0163 0.0544 .2078 0.1787 0.1686 0.3787 0.4570 0.3798 0.0612 0.0613 Pairwise r; NS, Nganasan; KT, Ket; MN, Mansi. r; NS, Nganasan; KT, ions were statistically significant ( 1483 0.1643 0.1587 0.1547 0.3443 0.4111 0.3392 0.0552 0.2377 0.2555 0.2347 0.2000 0.4212 0.4756 0.3963 197 0.1344 0.1537 0.2028 0.2051 0.3472 0.3790 0.3 268 0.2466 0.2627 0.3571 0.3130 0.4247 0.0253 839 0.1134 0.1323 0.1607 0.1669 0.3114 0.3634 0.2 .1843 0.1244 0.1365 0.1812 0.1636 0.3060 0.386 Table 3. Pairwise 2 0.3966 0.3123 0.3303 0.4248 0.4147 0.5331 308 0.3447 0.2800 0.2822 0.3571 0.0867 dal; AI, Ainu; HJ, Hokkaido Jomon; JP, M dal; AI, Ainu; HJ, Hokkaido Jomon; JP, 1 0.3311 0.2321 0.1687 0.1736 0.2080 1419 0.1926 0.2097 0.1389 0.1644 erentiations in any pair of the populat Buryat; TF, Tofalar; TV, Tuvan; TB, Tubala Tuvan; TV, Tofalar; Buryat; TF, OK UL NV NG AI HJ JP CN KR UD KY IT ES CH EV BR TF TV TB NS KT OK, Okhotsk; UL, Ulichi; NV, Nivkhi; NG, Negi Nivkhi; NG, OK, Okhotsk; UL, Ulichi; NV, The exact test demonstrated that diff UL 0.0205 NV 0.1556NG 0.0949 0.0587AI 0.0596HJ 0.1880 0.0422 0.0497JP 0.3271 0.2482 0.2876CN 0.0660 0.2123 0.4886 0.1665 0.1424 0.3031KR 0.3350 0.1215 0.2738 0.1501UD 0.1759 0.2860 0.1238 0.0948 0.1257 0.1824 0.2885 0.3206 0.0534 0.1497 0.1289 0.2500 0.3282 0.0525 0.0544 0.1790 0.2653 0. 0.0238 0.0118 KY 0.1213IT 0.1363ES 0.3393 0.2563 0.0696 0.2724 0.4920 0.121 0.4819 0.4074 0.1663 0.5614 0.2633 0.4004 0.4 0.4597TF 0.549 TV 0.2816 0.2105 0.2006TB 0.4033 0.1533 0.2020 0.1694NS 0.3265 0.2297 0.1436 0.1311 0.1906KT 0.3909 0.2981 0.1444 0.1515 0.2695 0.1244 0.2248 0.3144 0.3170 0.1955 0.1136 0.1932 0.1937 0.1376 0 0.2742 0.3549 0.1209 0.1398 0.1649 0.1835 0.1 0.3208 0.0912 0.1673 0 0.108 0.3465 0.2 CH 0.3610EV 0.3315 0.3356BR 0.4955 0.2488 0.3135 0.2269 0.4212 0.3337 0.1714 0.2116 0.4767 0.3317 0.2874 0.3 0.1405 0.4334 0.1692 0.3099 0.3442 0.2179 0. MN 0.1829 0.1539 0.3112 0.1383 0.1250 0.3087 0.1 CH, Chukuchi; EV, Evenki; BR, CH, Chukuchi; EV, 178 T. SATO ET AL. ANTHROPOLOGICAL SCIENCE

Figure 3. Multidimensional scaling on the basis of Fst values among the 21 northeastern Asian populations. The Okhotsk people are located near the position of the Ulchi, Negidal, and Ainu. chi. These facts indicate that the Ainu are also closely relat- A is shared by many northeastern Asian populations except ed to the Okhotsk people, although the Ainu people have for the Nivkhi, Ulchi, and Negidal. The HVR 1 sequence of been considered to be one of descendants of the Jomon peo- haplogroup A observed among the Okhotsk people is shared ple on the Japanese islands. The results obtained in the by the Koryak living around the Kamchatka peninsula. This present study suggest that the Okhostk people genetically sequence of HVR 1 corresponds with Type 3 reported by originated from people living around the lower regions of Sato et al. (2007). In addition, the Koryak are closely related the Amur River and that gene flow from the Okhotsk people to the Okhotsk people (Figure 3). These suggest that gene to the Ainu occurred (Figure 4), in agreement with the report flows between the ancient Koryak and Okhotsk people also of Sato et al. (2007). Tajima et al. (2004) reported occur- occurred (Figure 4). rence of gene flow from the Nivkhi to the Ainu. In addition, Haplogroups N9 and G1 were also major among the Hanihara et al. (2008) reported some morphological associ- Okhotsk people (Table 1, Table 2). The two haplogroups ation of the Ainu with continental populations of northeast- are also major among northeastern Asian populations. Hap- ern Asia. The present study and the previous study (Sato et logroup N9 is shared by both of the Hokkaido Jomon people al., 2007) strongly show that the Okhotsk people played a (65.9%) and the Udegey (30.4%) at high frequencies role as an intermediate in gene flow from the populations (Table 2). Meanwhile, haplogroup G1 is observed among living in the lower regions of the Amur River to the Ainu. native populations of the Kamchatka peninsula (68.1% in On the other hand, some of the Okhotsk people shared the Itelmen and 41.9% in the Koryak) at high frequencies haplogroup A, which is not seen in populations currently liv- (Table 2), and is also shared by the populations around the ing around the lower regions of the Amur River and in the lower regions of the Amur River (10.3% in the Ulchi; 5.4% Jomon people of Hokkaido (Table 1, Table 2). Haplogroup in the Nivkhi; and 27.2% in the Negidal) and the Hokkaido Vol. 117, 2009 MITOCHONDRIAL DNA HAPLOGROUPING OF THE OKHOTSK PEOPLE 179

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