Mitochondrial DNA Analysis of Human Skeletal Remains Obtained from the Old Tomb of Suubaru: Genetic Characteristics of the Westernmost Island Japan
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Bull. Natl. Mus. Nat. Sci., Ser. D, 34 pp. 11–18, December 22, 2008 Mitochondrial DNA Analysis of Human Skeletal Remains Obtained from the Old Tomb of Suubaru: Genetic Characteristics of the Westernmost Island Japan Ken-ichi Shinoda1 and Naomi Doi2 1 Department of Anthropology, National Museum of Nature and Science, 3–23–1 Hyakunincho, Shinkyuku-ku, Tokyo, 169–0073 Japan 2 Department of Anatomy, Faculty of Medicine, University of the Ryukyus, Uehara 207, Nishihara, Okinawa, 203–0216 Japan Abstract Yonaguni-jima is the westernmost island in Japan. Because of the geographical posi- tion of this island, it is considered to have played an important role as a route of migration from the southern part of Asia to the Japanese archipelagos. In order to investigate the genetic structure of the ancient Yonaguni people and to assess their genetic relationship with other East Asian popula- tions at a molecular level, we analyzed the hypervariable regions (HVR) 1 and 2 of the mitochon- drial DNA (mtDNA) from 16 tooth samples excavated from the old tomb of Suubaru, located on the north coast of the island. This tomb, which belongs to the early modern to modern periods, contained 32 graves. The distribution of mtDNA haplotypes among the skeletal remains obtained from the cemetery indicated the existence of several different maternal lineages. The mtDNA se- quences can be tentatively classified under specific haplogroups on the basis of mutations in the HVR1 and HVR2 regions. The frequencies of these haplogroups were compared with those of haplogroups present in Asian populations. The fact that the haplogroup M7a was found at a high frequency and that dominant haplogroups of Northeast Asian populations were found among the Suubaru people indicates that the Okinawa mainland had an enormous influence on the formation of the modern people in Sakishima Islands. Key words : Ancient DNA, Mitochondria, Yonaguni-jima, Old tomb of Suubaru, Population ge- netics 520 km from the main island of Okinawa (Figure Introduction 1). Because of the geographical position of Yona- The origin of the genetic diversity in the guni-jima, this island is considered to have Japanese population remains controversial, al- played an important role as a route of migration though multidisciplinary approach is being used from Taiwan and Southeast Asia to the Ryukyu to address this issue. The DNA analysis of an- and Japanese archipelagos. Archaeological exca- cient human remains is useful in this regard be- vations have been conducted at more than 20 cause it provides information on the genetic char- sites on the island; the skeletal remains excavated acteristics of a society that existed at a specific were from sites ranged from the Neolithic period time in the past (Alzualde et al. 2006; Wang et al. to the modern period, and many of these were 2007). DNA analysis can shed light on the origin built after the 14th century. of the genetic composition of the present popula- The archaeological evidence suggests that tion (Maca-Mayer et al. 2005; Casas et al. 2006). until the 12th century, the Sakishima area, which Yonaguni-jima is the westernmost island in comprised the Miyako and Yaeyama island Japan located at the end of the Ryukyu Archipel- groups, formed a cultural sphere different from ago, 125 km from the east coast of Taiwan and the main island of Okinawa. This culture was not 12 Ken-ichi Shinoda and Naomi Doi Fig. 1. Map of Ryukyu Archipelago and Taiwan. Map showing the location of Yonaguni-jima and the old tomb of Suubaru. influenced by the Japanese mainland (Jomon and Japan, we are currently analyzing DNA extracted Yayoi cultures), and the evidence obtained from from human remains that were excavated from existent remains indicated that the Sakishima cemeteries belonging to the early modern and area had more in common with the southern re- modern periods. gions of Asia. Perhaps the seas lying between the main islands of Okinawa and Miyako formed the Materials and Methods boundaries for the extension of the Japanese cul- ture toward the south (Takamiya, 2005). Archaeological site and specimens In the 12th century, the hunter-gatherer The human skeletal remains used for the lifestyle began to change with the advent of an analysis were obtained from the old tomb of Su- agricultural society in all parts of Ryukyu Is- ubaru, which is located toward the north coast of lands; thus, the cultural differentiation was elimi- Yonaguni-jima (Figure 1). As a part of the airport nated. The relationship between the hunter-gath- expansion project that was undertaken in Oki- erers who inhabited this region in ancient times nawa Prefecture in 2004–2005, the site was fully and between the farmers who arrived later on has excavated by the government to obtain any items been particularly interesting; however, little is of cultural significance that may have been known about their biological relationships. This buried. At least 77 skeletons were excavated is because none of the human remains that have from 32 graves. On the basis of the funerary ob- been excavated from the Yaeyama islands belong jects procured, it was concluded that the tomb be- to the Neolithic period (Asato and Doi, 1999). longed to the early modern to modern period. Genetic analysis of ancient human remains is The skeletons excavated generally had a lower the most effective biological approach for deter- face and long head—a characteristic similar to mining the relationships between these people. In that observed in the modern Okinawa population order to obtain more information on the genetic (Figure 2). The average height of the skeletons characteristics of this westernmost island of was estimated at 157.5 cm for males and 144.6 DNA Analysis of the Old Tomb of Suubaru 13 DNA-OFF (TaKaRa Co.) and ultraviolet (UV) light. All metallic materials used were sterilized in an oven at 210°C for at least 6 h. Other rigor- ous authentication methods that have been de- scribed previously were employed throughout the DNA-based analyses (Shinoda et al., 2006). Tooth sample preparation, DNA extraction, and PCR amplification were performed in a laborato- ry room that was reserved for analysis of the an- cient DNA. DNA Extraction and purification The tooth samples were dipped in a DNA-OFF solution for 5 min to eliminate contamination, rinsed several times with DNase-/RNase-free dis- Fig. 2. Frontal and lateral views of the adult male tilled water, and air dried. When the samples and female skulls excavated from the old tomb were completely dry, they were pulverized in a of Suubaru. mill (Multi-beads shocker MB400U; Yasui Kikai, Osaka, Japan). cm for females, indicating that the ancient hu- DNA was extracted in 2 steps by using a DNA mans were fairly short in stature. Further, perios- extraction kit (Mo Bio Co.). The pulverized tooth titis was observed in the lower limb of many of powder (0.3 g) was placed in a 15-ml conical the skeletons; this indicated that the people had tube and demineralized in 5 ml of 0.5 M ethyl- been exposed to severe environmental conditions enediamminetetraacetic acid (EDTA). The sam- (Doi, 2007). ples were rotated and incubated at 37°C for A total of 16 remains obtained from 12 graves 12–15 h. After digestion with proteinase K (0.5 were selected for the DNA analysis. Of these mg/ml), the resultant pellet was used for DNA samples, 3 were collected from a single skeleton extraction. The eluted DNA (approximately 50 in order to verify the authenticity of the retrieved ml) was amplified by PCR, without prior process- DNA. Almost all the samples were collected ing. from the surface of the tomb chamber. Amplification and sequencing of HVR1 and Authentication and prevention of contamina- HVR2 tion In all the samples, segments of hypervariable During analysis of ancient DNA samples, it is region (HVR) 1 (nucleotide positions 16121– necessary to exclude false positive results that 16238 and 16209–16402, as per the revised can arise because of postmortem damage and Cambridge reference sequence; Andrews et al., contamination with more recent DNA samples 1999) and HVR 2 (nucleotide positions 128–267) (Cooper and Poinar, 2000; Bandelt, 2005). Dur- were sequenced. ing each step of sample preparation, all possible Aliquots (2 ml) of the extracts were used as precautions were taken to prevent contamination templates for PCR. Amplifications were carried of the DNA samples with more recent samples. out in a reaction mixture (total volume, 25 ml) Separate laboratory rooms were used for extrac- containing 1 unit of Taq DNA polymerase (Hot- tion, amplification, and post-polymerase chain StarTaqTM DNA polymerase; Qiagen), 0.1 mM of reaction (PCR) analysis of the ancient DNA. The each primer, and 100 mM of deoxyribonucloside samples were kept pure by regular treatment with triphosphates (dNTPs) in 1ϫPCR buffer provid- 14 Ken-ichi Shinoda and Naomi Doi ed by the manufacturer. The PCR conditions motif specific to a haplogroup and by matching were as follows: incubation at 95°C for 15 min; or almost matching the values with the mitochon- followed by 40 cycles of heat treatment at 94°C drial mtDNA haplotypes in the global database. for 20 s; 50–56°C for 20 s; and 72°C for 15 s; and The haplogroup status was further characterized final extension at 72°C for 1 min. on the basis of other specific mutations in the The following primers were used to amplify HVR2 motif. HVR1 and HVR2. L16120 5Ј-TTACTGCCAGCCACCATGAA-3Ј Results and discussion H16239 5Ј-TGGCTTTGGAGTTGCAGTTG-3Ј L16208 5Ј- CCCCATGCTTACAAGCAAG-3Ј In this study, we assessed the mtDNA varia- H16403 5Ј-TTGATTTCACGGAGGATGGTG-3Ј tions in samples obtained from the old tomb of L127 5Ј-AGCACCCTATGTCGCAGTAT-3Ј Suubaru.