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 distrib- uted 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 an- alyzed. 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 haplo- group 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 mo- lecular phylogeny of mtDNA HVR 1 sequences, and strongly suggest that the Okhotsk people origi- nated 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 se- quencing, 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.