Bull. Natl. Mus. Nat. Sci., Ser. D, 39, pp. 1–8, November 22, 2013

Ancient DNA Analyses of Human Skeletal Remains from the Period in the ,

Ken-ichi Shinoda1, Tsuneo Kakuda1 and Naomi Doi2

1 Department of Anthropology, National Museum of Nature and Science, 4–1–1, Amakubo, Tsukua City, Ibaraki Prefecture 305–0005, Japan 2 Department of Anatomy, Faculty of Medicine, University of the Ryukyus, Uehara 207, Nishihara, 203–0216, Japan

Abstract Identifying genetic characteristics of the Gusuku period is indispensable to clarifying the colonizing process of the Ryukyu Islands and formation of the modern Ryukyu population. However, no genetic information on this period has hitherto been reported. In the present study, we analyzed ancient DNA recovered from Gusuku skeletons excavated from (Nakan- dariyama site) and Island (Moubaru site). Sequence polymorphisms in hypervariable region 1 (HVR1) and 24 single nucleotide polymorphisms in the coding region of mitochondrial DNA (mtDNA) were analyzed in eight tooth samples from Nakandariyama and seven samples from Moubaru. We assigned 12 mtDNAs (six from both sites) to relevant haplogroups. Hap- logroups M7a, M7b, D4, A, and B were observed in these individuals, with M7a and D4 predomi- nant in both archaeological sites, indicating characteristics of the contemporary Okinawan popula- tion. The geographic distribution of haplogroups M7a and D4 at both sites seems to support the claim inferred from the archaeological evidence for a relationship between the Okinawa and during the Gusuku period. The fact that all these haplogroups were observed with relatively high frequencies in the late Kaizuka, Gusuku, and contemporary Ryukyu popula- tions implies that formation of the extinct population from the Ryukyu Islands might result from a population expansion after the late Kaizuka period. Key words : Ancient DNA, Mitochondria, Gusuku period, Okinawa, Population genetics

et al., 2012; Matsukusa et al., 2010) and research Introduction into historical, cultural, and classic anthropologi- Due to its geographical position and distinc- cal sources from the Ryukyu Islands (e.g., Fuku- tion as the largest of the Ryukyu Islands, Oki- mine et al., 2006; Takamiya, 2005; Asato, 1996; nawa may have been an important place along Tagaya and Ikeda, 1976). However, the consis- the migration route from southern Asia to the tency in the information from these myriad Japanese archipelago. The first human skeletal sources is still debated. evidence on Okinawa dates to the Paleolithic Because of the Ryukyu Islands’ complex his- period (Matsu’ura, 1982). Human genetic diver- tory and prehistory, it is not surprising that they sity on this island has largely been shaped by have often attracted the interest of population migration processes occurring after the initial geneticists (e.g., Jinam et al., 2012). Neverthe- Paleolithic occupations. This was followed by less, we still know very little about the genetic the spread of farming, which probably entailed structure of their human populations. To answer population movements from Kyushu Island. questions regarding the genotypes of ancient Much is already known about the main demo- populations, genetic analysis of skeletons exca- graphic transitions revealed by studies of genetic vated from archaeological sites is the most diversity in extant populations (Koganebuchi straightforward approach. Ancient DNA analyti- 2 Ken-ichi Shinoda, Tsuneo Kakuda and Naomi Doi

Fig. 1. Geographic distribution of the Ryukyu archipelago. Map showing location of the Nakandariyama old tomb and the Moubaru site. cal methods developed significantly in the last to clarify the human genetic characteristics of two decades directly compare ancestral popula- this period. The present study aims to explain the tions to their present-day counterparts. They per- genetic composition of the population from the mit direct reconstruction of the genetic history of Gusuku period, and address their genetic rela- populations through time, providing a temporal tionship (at a molecular level) with other Ryukyu dimension of the study of past human popula- groups from different cultural periods. tions. Our previous investigation of the genetic Materials and Methods structure of humans from the Shell Midden (Kai- zuka) period on Okinawa provides proof of Archaeological sites and specimens genetic continuity between the ancient and con- In this analysis, we used human skeletal temporary populations of this island from at least remains excavated from two archaeological sites the late Kaizuka period (Shinoda et al., 2012). on mainland Okinawa (Nakandariyama site) and However, no genetic evidence has been reported Hateruma Island (Moubaru site), the two south- from the intermediate Gusuku period (thirteenth– ernmost and inhabited islands of Japan. The sixteenth centuries) mainly because sufficient approximate locations of these sites are shown in skeletal remains for ancient DNA analysis are Figure 1. lacking. Nakandariyama is a cliff burial site located in During this period, agricultural production of the city of . It was excavated in 2003 by the rice, wheat, and millet was developed and over- Center for Buried Cultural Properties (Okinawa seas trading of these goods began. All areas of Prefecture) and comprises at least 47 individuals. the Ryukyu Islands formed a common culture These consisted of 15 males, 10 females, 15 during this time (Asato, 1996). In order to under- juveniles, and 7 individuals of indeterminate stand the history of these islands, it is important gender, ranging from infants to mature adults. MtDNA Polymorphisms in Gusuku Period 3

These skeletons generally display a prolonged In order to prevent contamination from post- upper jaw, which is characteristic of medieval excavation handling, the tooth samples were humans but different from former Kaizuka peo- rinsed with DNA-decontamination agents and ple (Doi, 2005). then washed thoroughly with distilled water Since no cemetery artifacts remain, the chro- before drying. Next, the samples were crushed nology of this site was determined based on the into powder using a Multi-beads Shocker (Yasui design of burial goods. All tombs except No. 7 Kikai Corporation). DNA was extracted from belong to the early modern period. In contrast, 0.5 g of powder from each sample using a com- the age of tomb No. 7 is estimated to be in the mercial DNA extraction kit (Qiagen, DNA Inves- Gusuku period. In order to determine the genetic tigator kit, Germany) after Protainase K diges- characteristics of the Gusuku population from tion. In the case of the Moubaru sample, the mainland Okinawa, eight well-preserved samples tooth root was cut off and used for 14C dating and excavated from tomb No. 7 were selected for stable isotope analysis. DNA analysis. A sediment production project in the 1980s Amplification and sequencing of HVR1 identified the Moubaru site, located in the Segments of HVRs (nucleotide positions Misyuku village in the northwest part of Hat- 16121-16238, HVR1-1; 16209-16291, HVR1-2; eruma Island. By counting skulls and limb bones, and 160289-16366; HVR1-3) of mtDNAs, as per the minimum number of individuals was esti- the revised Cambridge reference sequence mated at 29 (13 adult males, 10 adult females, 6 (Andrews et al., 1999), were sequenced in all immature individuals) (Doi, 1998). According to samples. 14C dating of bone samples, the remains belong Aliquots (2 μl) of the extracts were used as to the Gusuku period (Dr. Yoneda, personal com- templates for PCR. Amplifications were carried munication). Seven well-preserved samples were out in a reaction mixture (total volume, 15 μl) selected for DNA analysis. A list of all samples containing 1 unit of Taq DNA polymerase (Hot- used in this study is presented in Table 1. StarTaqTM DNA polymerase; Qiagen, Germany), 0.1 M of each primer and 100 mM of deoxyribo- Authentication methods and extraction of DNA nucloside triphosphates in 1×PCR buffer pro- DNA analyses were performed at the National vided by the manufacturer. The PCR conditions Museum of Nature and Science, which is dedi- were as follows: incubation at 95°C for 15 min; cated to ancient DNA analysis. In the present followed by 40 cycles of heat treatment at 94°C study, we employed standard precautions to for 20 sec; 50°C–56°C for 20 sec, and 72°C for 15 avoid contamination, e.g., separation of pre- and sec; and final extension at 72°C for 1 min. post-PCR experimental areas, use of disposable The following primers were used to amplify laboratory wares and filter-plugged pipette tips, HVR1-1, HVR1-2, HVR1-3: treatment with DNA contamination removal L16120 5′-TTACTGCCAGCCACCATGAA-3′ solution (DNA Away; Molecular Bio Products, H16239 5′-TGGCTTTGGAGTTGCAGTTG-3′ San Diego, CA, USA), UV irradiation of equip- L16208 5′-CCCCATGCTTACAAGCAAG-3′ ment and benches, and negative extraction and H16312 5′-ACTATGTACTGTTAAGGGTG-3′ PCR controls (Shinoda et al., 2006). L16269 5′-CTAGGATACCAACAAACCTA-3′ Extraction and purification of DNA and H16367 5′-ATCTGAGGGGGGTCATCCAT-3′ sequence analysis were mainly performed by The PCR products were subjected to agarose T.K. and K.S. The multiplex amplified product- gel electrophoresis on a 1.5% gel and were length polymorphism (APLP) analysis (Umetsu recovered by using a QIAEX II agarose gel et al., 2005) was conducted in different laborato- extraction kit (Qiagen, Germany). Aliquots of the ries as described in the genotyping section. samples were prepared for sequencing on a Big- 4 Ken-ichi Shinoda, Tsuneo Kakuda and Naomi Doi A M M B4 D4 D4 D4 D4 (B) D4a M7a M7a M7a M7a M7b N.D. N.D. 8272 delate not delete not delete not delete not delete not delete not delete not delete not delete not delete not delete not delete not delete not delete Haplogroup B M M D4 D4 D4 D4 D4a SNP SNP M7a M7a M7a M7a M7b N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 6689 (D4b1b) PCR-Luminex T T T T T T T T T T T A A A A A A A A A A A A A N.D. N.D. N.D. N.D. N.D. N.D. 5964 15874 (D4e1) (D4e2) A A A A A A A A A A A A A A A G G G G G G G G G G G G N.D. N.D. N.D. 5231 (N9a) 15524 (D4b2a) A A A C C C C C C C C C C C G G G G G G G G G G (D) (G1) N.D. N.D. N.D. N.D. N.D. N.D. 5178 15497 G G G G G G G G G G G G G G G G G G G G G G G G G G N.D. N.D. N.D. N.D. 5147 (B4c) (N9b) 15346 , 1999). T T T T T T T T T T T T T T C G G G G G G G G N.D. N.D. N.D. N.D. N.D. N.D. N.D. 4820 (B4b) (D4a) 14979 et al. T T T T T T T T T C C C C (G2) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 4386 (M7a) 13563 T T T T T T T T A C G G G G G G G G G G G G G G (M) N.D. N.D. N.D. N.D. N.D. N.D. 4048 10400 (M7b) T T T T T T T T T T T T T T T G G G (D5) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 3644 (D4h) 10397 A A A A A G G G G G G G G G G G G G G G G G G G G G G G G G (F) (D4) 3010 10310 T T T T T T T T T T T T T A A 856 (B5) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 9950 (D4d1b) C C C C C C C C C 362 CRS CRS N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 9293 HV1-3 16366) (16289- (D4b2b) (16,000+) 290,311,319 T T T T T 223 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 8793 (M10) HV1-2 16291) (16209- 217,249 (16,000+) ” . A G G G G G G N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 8701 HV1-1 16238) (16121- (N,D4g) (16,000+) Sample Sample (Okinawa Island) (Okinawa Island) Maxilla, Left, M3 Mandible, Left, M2 Maxilla, Right, M2 Maxilla, Left, M2 Maxilla, Right, M2 Mandible, Right, M3 Maxilla, Right, M2 Mandible, Right, M1 Maxilla, Left, M2 Mandible, Left, M2 Maxilla, Left, M3 Maxilla, Right, M3 Maxilla, Right, M2 Maxilla, Right, M3 Mandible, Right, M1 Maxilla, Right, M3 Maxilla, Left, M3 Maxilla, Right, M3 Maxilla, Right, M3 Mandible, Left, M3 Maxilla, Right, M3 Maxilla, Right, M3 Maxilla, Left, M1 Mandible, Left, M3 Maxilla, Right, M3 Maxilla, Left, M1 Maxilla, Right, M3 Maxilla, Right, M3 Maxilla, Left, M3 Mandible, Right, M3 1 4 1 4 41 32 35 35 32 41 10 10 7-6 2-2 9-1 2-2 9-1 7-6 11-1 11-2 11-1 11-2 56-1 56-1 Code Code Name Name 7-21(2) 7-13(2) 7-21(2) 7-13(2) no name no name All polymorphic sites are numbered according to the revised Cambridge reference sequence (Andrews CRS indicates that the sequence of segment is identical to revised Cambridge reference sequence. Diagnostic polymorphisms of the PCR-Luminex analysis are emphasized by bold italic type. N.D. denotes “ not determined Sample used for DNA exreaction and the result of analysis. 1. Sample used for DNA Table 1 3 6 2 4 5 7 5 8 3 4 6 2 1 7 3 8 1 4 2 3 6 4 5 7 5 6 7 1 2 No. Nakandariyama site No. Moubaru site (Hateruma Island) Moubaru site (Hateruma Island) Nakandariyama site MtDNA Polymorphisms in Gusuku Period 5

Dye cycle sequencing kit Ver.3.1 (Applied Bio- other questionable data were omitted from this systems, Foster City, CA, USA), which was per- study. In these samples, 6 out of 45 PCR amplifi- formed using forward and reverse primers. The cations were successfully analyzed. In contrast, primers used in the PCR amplification were also 12 of the 15 samples were successfully typed in used in the sequencing reaction. Sequencing was each haplogroup by multiplex SNP typing assays performed in both directions to enable identifica- to simultaneously analyze important SNPs within tion of polymorphisms or ambiguous bases by the mtDNA coding region. Two samples that using a single primer. The sequencing reactions defined macrohaplogroup M could not be identi- were performed on a DNA Sequencer (ABI fied further to haplogroup. model 3130) equipped with SeqEd software. In the case of multiplex analysis, to minimize the risk of contamination and to raise the effi- Genotyping of Polymorphisms ciency of PCR, very short DNA fragments (aver- To confidently assign mtDNAs to the relevant age 60–80 bp) were amplified. However, the haplogroups, 24 haplogroup-diagnostic single length of amplified HVR1 portion exceeded 100 nucleotide polymorphisms (SNPs) including a bp in this experiment. Therefore, it is presumable nine b.p. repeat variation in the non-coding cyto- that DNA extraction products of both archaeo- chrome oxidase II/tRNALys intergenic region logical sites were degenerated and most of their that define major haplogroups found in Japanese lengths were under 80 bp. and East Asian population were analyzed by The haplogroup composition of the samples is multiplex APLP (Umetsu et al., 2005). SNPs that M7a (33.3%); M7b (8.3%); D4 (41.7%); A define major haplogroups were detected by use (8.3%); and B (8.3%) in total (Table 1). Owing to of suspension-array technology (Luminex 100; the small sample size, it is difficult to verify the Luminex) at the laboratory of G&G Science, genetic characteristics by statistical methods, Fukushima. The methodology used for genotyp- although it is noteworthy that the M7a and D4 ing and primer sequences have been described in haplogroups from both archaeological sites were detail elsewhere (Itoh et al., 2005, Shinoda et al., well-represented. The distribution of mtDNA 2012). haplogroups in this period will provide some insight on the human population history of the Ryukyu Islands. Results and Discussion Figure 2 shows the haplogroup frequencies of There are many advantages to using mtDNA. the Gusuku period (present study), modern Because it is present in high copy numbers in Ryukyu, and Taiwanese aboriginal populations. mammalian cells, mtDNA can most likely be Regardless of their geographical location, the detected even in highly degraded samples, such Ryukyu and Taiwanese aboriginal populations as the ancient ones studied here. However, hot differed when compared according to their share and humid conditions are generally unfavorable of haplogroups. Taiwanese aborigines do not to the preservation of DNA in human skeletal have haplogroup M7a and D4 that dominate the remains, limiting the possibilities for finding extant Ryukyu and Gusuku populations. There- well-preserved DNA in subtropical regions such fore, we assume there was no genetic connection as the Ryukyu Islands (Shinoda et al., 2012). between the Taiwanese and Gusuku populations, Due to the poor quality of mtDNA extracted who had different origins and population histo- from the ancient material, it was not possible to ries overall. amplify all samples. The biased distribution pattern of haplogroup Table 1 shows the results of PCR amplifica- M7a in Japan may have diversified from the tion. Suspected false positive results stemming ancestral M7 haplogroup, which was introduced from contamination with contemporary DNA and to Japan around the Last Glacial Maximum 6 Ken-ichi Shinoda, Tsuneo Kakuda and Naomi Doi

Fig. 2. Estimated frequencies of mtDNA haplogroups in regional populations. References: Ryukyu: Umetsu et al., (2005) aborigines: Trejaut et al., (2005) within the Japanese archipelago (Adachi et al., been present in the late Kaizuka population (Shi- 2011). The Ryukyu Islands are candidates for the noda et al., 2012) and that haplogroup D4 most origin of this haplogroup (Shinoda, 2007). On probably reached the from the other hand, haplogroup D4 is observed in mainland Japan at least by the end of the late northeastern and Korea as well as main- Kaizuka period. land Japan (Tanaka et al., 2004) and there is no High frequencies of haplogroups M7a and D4 biased distribution among the mainland Japan. are characteristic of the contemporary Okinawan Moreover, this haplogroup apparently occurred at population (Tanaka et al. 2004). The distribution a low frequency in the Jomon population (Shi- of these haplogroups among the late Kaizuka, noda and Kanai, 1999; Adachi et al., 2011). A Gusuku, and modern Ryukyu populations sug- reasonable theory is that the Yayoi immigrants gests that the formation of the extinct Ryukyu were predominantly from haplogroup D4. Islands population was the result of population Archaeological findings indicate that travel expansion. and trade between mainland Japan and the The number of samples for which DNA hap- Ryukyu archipelago began around the twelfth logroups could be determined was small in the century. There are also some indications that present analysis, so unfortunately, the results pro- waves of immigrants moved to Okinawa from vided minimal insight. However, the establish- mainland Japan and reached the Sakishima area ment of genetic characteristics among numerous (Asato, 1996). The geographic distribution of burial sites in the Gusuku period may provide us haplogroups M7a and D4 in both sites seems to with extremely valuable information regarding support the claim inferred from the archaeologi- human migration and population dynamics in the cal evidence that a relationship existed between Ryukyu Islands. The preliminary experiment pre- the Okinawa, Sakishima, and Hateruma Islands sented here proved that sufficient amounts of in the Gusuku period. DNA are retained in some human skeleton sam- This study identified four distinct haplogroups ples, even though the analytical efficiency may (M7a, M7b, D4, A, B) in the archaeological be poor. Thus, we believe it is worthwhile to con- specimens sampled from the Okinawa and Hat- tinue the experiments to obtain more detailed eruma Islands, which agree well with the genetic data on the human skeletal remains from the variation of extant Okinawan and mainland Japa- Gusuku period site. Moreover, insight on the nese populations (Figure 2). It follows that the population history of the Ryukyu Islands could these mtDNA haplogroups except M7b have be obtained by comparing ancient mtDNA MtDNA Polymorphisms in Gusuku Period 7 sequences collected from the Kaizuka sites that Saitou. 2012. The history of human populations in the Japanese Archipelago inferred from Genomewide SNP are contemporaneous with the Jomon period in data with a special reference to the Ainu and the mainland Japan with sequences from the late- Ryukyuan Populations. Journal of Human Genetics, Kaizuka, Gusuku, and present-day populations. 57: 787–795. Koganebuchi, K., T. Katsumura, S. Nakagome, H. Ishida, S. Kawamura, and H. Oota, The Asian Archival DNA Acknowledgments Repository Consortium, 2012. Autosomal and Y-chro- mosomal STR markers reveal a close relationship We want to thank Mr. Katagiri of the Okinawa between Hokkaido Ainu and Ryukyu islanders. 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