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 ofthe 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 Japan2
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 position of this island, it is considered to have played an important role as a route of migration from thesouthern 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 populations at a molecular level, we analyzed the hypervariable regions (HVR) 1 and 2 of the mitochondrial 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 sequences 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 highfrequency 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 formationof the modern people in Sakishima Islands.
Key words : Ancient DNA, Mitochondria, Yonaguni-jima, Old tomb of Suubaru, Population genetics

Introduction
The origin of the genetic diversity in theJapanese population remains controversial, although multidisciplinary approach is being used
to address this issue. The DNA analysis of ancient human remains is useful in this regard because it provides information on the genetic characteristics of a society that existed at a specifictime in the past (Alzualde et al. 2006; Wang et al.
2007). DNA analysis can shed light on the originof the genetic composition of the present population (Maca-Mayer et al. 2005; Casas et al. 2006).
Yonaguni-jima is the westernmost island inJapan located at the end of the Ryukyu Archipelago, 125 km from the east coast of Taiwan and520 km from the main island of Okinawa (Figure1). Because of the geographical position of Yonaguni-jima, this island is considered to haveplayed an important role as a route of migrationfrom Taiwan and Southeast Asia to the Ryukyu
and Japanese archipelagos. Archaeological excavations have been conducted at more than 20
sites on the island; the skeletal remains excavatedwere from sites ranged from the Neolithic period
to the modern period, and many of these werebuilt after the 14th century.
The archaeological evidence suggests that
until the 12th century, the Sakishima area, which
comprised the Miyako and Yaeyama islandgroups, formed a cultural sphere different fromthe main island of Okinawa. This culture was not

12Ken-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 andYayoi cultures), and the evidence obtained fromexistent remains indicated that the Sakishima
area had more in common with the southern regions of Asia. Perhaps the seas lying between themain islands of Okinawa and Miyako formed theboundaries for the extension of the Japanese culture toward the south (Takamiya, 2005).
In the 12th century, the hunter-gatherer
lifestyle began to change with the advent of an
agricultural society in all parts of Ryukyu Islands; thus, the cultural differentiation was eliminated. The relationship between the hunter-gatherers who inhabited this region in ancient times
and between the farmers who arrived later on has
been particularly interesting; however, little is
known about their biological relationships. Thisis because none of the human remains that havebeen excavated from the Yaeyama islands belong
to the Neolithic period (Asato and Doi, 1999).
Genetic analysis of ancient human remains is
the most effective biological approach for determining the relationships between these people. In
order to obtain more information on the genetic
characteristics of this westernmost island ofJapan, we are currently analyzing DNA extracted
from human remains that were excavated fromcemeteries belonging to the early modern andmodern periods.
Materials and Methods
Archaeological site and specimens
The human skeletal remains used for the
analysis were obtained from the old tomb of Suubaru, which is located toward the north coast of
Yonaguni-jima (Figure 1). As a part of the airport
expansion project that was undertaken in Okinawa Prefecture in 2004–2005, the site was fully
excavated by the government to obtain any itemsof cultural significance that may have beenburied. At least 77 skeletons were excavatedfrom 32 graves. On the basis of the funerary objects procured, it was concluded that the tomb belonged to the early modern to modern period.
The skeletons excavated generally had a lower
face and long head—a characteristic similar tothat observed in the modern Okinawa population(Figure 2). The average height of the skeletons
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 rigorous authentication methods that have been described previously were employed throughout the
DNA-based analyses (Shinoda et al., 2006).
Tooth sample preparation, DNA extraction, and
PCR amplification were performed in a laboratory room that was reserved for analysis of the ancient DNA.

Fig. 2. Frontal and lateral views of the adult maleand female skulls excavated from the old tombof Suubaru.

cm for females, indicating that the ancient humans were fairly short in stature. Further, periostitis was observed in the lower limb of many of
the skeletons; this indicated that the people had
been exposed to severe environmental conditions(Doi, 2007).
A total of 16 remains obtained from 12 graves
were selected for the DNA analysis. Of thesesamples, 3 were collected from a single skeleton
in order to verify the authenticity of the retrieved
DNA. Almost all the samples were collectedfrom the surface of the tomb chamber.
Authentication and prevention of contamination
During analysis of ancient DNA samples, it isnecessary to exclude false positive results that
can arise because of postmortem damage andcontamination with more recent DNA samples(Cooper and Poinar, 2000; Bandelt, 2005). During each step of sample preparation, all possible
precautions were taken to prevent contaminationof the DNA samples with more recent samples.
Separate laboratory rooms were used for extraction, amplification, and post-polymerase chain
reaction (PCR) analysis of the ancient DNA. The
samples were kept pure by regular treatment withDNA 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 distilled water, and air dried. When the samples
were completely dry, they were pulverized in amill (Multi-beads shocker MB400U; Yasui
Kikai, Osaka, Japan).
DNA was extracted in 2 steps by using a DNA
extraction kit (Mo Bio Co.). The pulverized tooth
powder (0.3 g) was placed in a 15-ml conicaltube and demineralized in 5 ml of 0.5 M ethylenediamminetetraacetic acid (EDTA). The samples were rotated and incubated at 37°C for
12–15 h. After digestion with proteinase K (0.5mg/ml), the resultant pellet was used for DNA
extraction. The eluted DNA (approximately 50m l) was amplified by PCR, without prior processing.
Amplification and sequencing of HVR1 andHVR2In all the samples, segments of hypervariable
region (HVR) 1 (nucleotide positions 16121–
16238 and 16209–16402, as per the revised
Cambridge reference sequence; Andrews et al.,1999) and HVR 2 (nucleotide positions 128–267)
were sequenced.
Aliquots (2 m l) of the extracts were used as
templates for PCR. Amplifications were carriedout in a reaction mixture (total volume, 25 m l)containing 1 unit of Taq DNA polymerase (HotStarTaqTM DNA polymerase; Qiagen), 0.1 m M ofeach primer, and 100 m M of deoxyribonucloside
triphosphates (dNTPs) in 1PCR buffer provid-

14

Ken-ichi Shinoda and Naomi Doi

ed by the manufacturer. The PCR conditions
were as follows: incubation at 95°C for 15 min;followed by 40 cycles of heat treatment at 94°C
for 20 s; 50–56°C for 20 s; and 72°C for 15 s; andfinal extension at 72°C for 1 min.
The following primers were used to amplify
HVR1 and HVR2.
L16120 5-TTACTGCCAGCCACCATGAA-3
H16239 5-TGGCTTTGGAGTTGCAGTTG-3
L16208 5- CCCCATGCTTACAAGCAAG-3
H16403 5-TTGATTTCACGGAGGATGGTG-3
L127 5-AGCACCCTATGTCGCAGTAT-3
H268 5-GTTATGATGTCTGTGTGG-3
The PCR products were subjected to agarose
gel electrophoresis on a 1.5% gel and were recovered by using a QIAEX II agarose gel extraction kit (Qiagen, Germany). Aliquots of the samples were prepared for sequencing, on a BigDyecycle sequencing kit (Applied Biosystems, Foster
City, CA, USA), which was performed using forward and reverse primers. The primers that wereused in the PCR amplification were also used in
the sequencing reaction. Sequencing was performed in both directions so as to enable identification of polymorphisms or ambiguous bases
using a single primer. The sequencing reactions
were performed on a DNA Sequencer (ABImodel no., 3130) equipped with SeqEd software.
Data analysis
The nucleotide diversity and the mean numberof pairwise differences between the mitochondrial D-loop sequences were computed using theArlequin software package version 3.0 (Excoffieret al., 2005), considering Tamura and Nei distances and a gamma parameter value of 0.26
(Mayer et al., 1999). The differences (Raymondand Rousset, 1995) between the population of theold tomb of Suubaru and the other populations
were also computed using the Arlequin software.
Neighbor-joining (NJ) trees were constructed on
the basis of the pairwise Fst values by using theMega 3.0 program (Kumar et al., 2004) in orderto study the relationships between the populations. The haplogroup status was tentatively assigned on the basis of a search for an HVR1

motif specific to a haplogroup and by matchingor almost matching the values with the mitochondrial mtDNA haplotypes in the global database.
The haplogroup status was further characterizedon the basis of other specific mutations in the
HVR2 motif.
Results and discussion
In this study, we assessed the mtDNA variations in samples obtained from the old tomb of
Suubaru. MtDNA was extracted from the teeth ofeach skeleton, and sequencing was successfully
performed for 15 of the 16 samples. DNA wasindependently extracted from 3 teeth of same individual (tomb 15) in order to assess the reproducibility and authenticity of the results. As
shown in table 1, the HVR1 sequences determined for these 3 samples completely coincided.
Thus, the authenticity of the results was confirmed.
In general, hot and humid conditions are unfavorable to the preservation of DNA in humanskeletal remains; hence, the possibility of finding
well-preserved DNA in a tropical region such as
Okinawa is low. However, our success rate (94%)
in this regard was reasonably higher than theusual success rate associated with obtaining otherancient materials, i.e., 50–70%. This can be attributed to the fact that the bones excavated in
the present study were well preserved and thatthese samples were relatively recent when compared with ancient DNA.
The fact that we identified 7 different haplotypes among the skeletal remains of 13 individuals (corresponding to a haplotype diversity of
53.8%) indicates that the genetic characteristicsof the Suubaru population were relatively diverse(Table 1). Identical haplotypes were observed for
6 individuals, and these haplotypes were classified into 2 groups; the remaining 7 individuals
had unique haplotypes. Since mtDNA is maternally inherited, the finding that some of the studied individuals shared the same haplotype suggests the possibility that they were maternally related. This would not be very surprising, sinceDNA Analysis of the Old Tomb of Suubaru
Table 1.
Tomb No.

15

Changes in the nucleotides of mtDNA and the haplogroups observed in the old tomb of Suubaru.
Sample No.

HVR1 Sequence (+16000)

HVR2 sequence

Haplogroup

1
2
14
692
151
128
164
172
856
857
858
261
803
579
611
649

209, 223, 324
209, 223, 324, 344
223, 298, 327
209, 223, 324
223, 290, 319
223, 278, 362, 384
232A, 304, 311, 344
209, 223, 324
223, 269, 278, 362
223, 269, 278, 362
223, 269, 278, 362
N.D.
209, 223, 324, 344
209, 223, 324, 344
209, 223, 324
216, 223, 362

CRS
CRS
193, 195, 249d
CRS
N.D.
CRS
N.D.
150
200, 260
N.D.
N.D.
N.D.
N.D.
N.D.
CRS
N.D.

M7a
M7a
C
M7a
A4
G2
F1b1a
M7a

1
2
6
9
11
14
15
23
25
33
36
38

G2a2b1
N.D.
M7a
M7a
M7a
D/G

Table 2. Diversity indices of the mtDNA HVR1 haplogroup for the skeletal remains obtained from the old tombof Suubaru and those of other populations.
Site

n

Gene
diversity

Nucleotide
diversity

Mean number of
pairwise differences

Suubaru
Jomon (Kanto)
Yayoi (Kuma-Nishioda)
Aboriginal Taiwanese

13
67
31
28

1.00/0.030
0.91/0.024
0.85/0.051
1.00/0.010

0.020/0.012
0.018/0.010
0.012/0.007
0.022/0.012

3.897/2.089
3.429/1.775
2.241/1.268
4.161/2.133

The references for the population are as follows:
Kanto Jomon: Shinoda and Kanai (1999), Shinoda (2003)
Yayoi: Shinoda (2004)
Aboriginal Taiwanese: Tajima et al. (2003)

the burial ground was used by a rather small
group of individuals. The above results suggestthat these cemeteries were used over a long period and that the studied individuals were related
across generations.
Genetic variation within the populations wasdetermined by calculating the sequence diversity,
nucleotide diversity, and the mean number of
pairwise differences (Table 2). These factors
were also calculated for the ancient Japanese andcontemporary aboriginal Taiwanese populations.
The Suubaru population exhibited values of 0.02
for nucleotide diversity and 3.89 for the mean
number of pairwise differences, which werehigher than the values obtained in the case of theother Japanese ancient populations (Table 2) andlower than those obtained in the case of the aboriginal Taiwanese population. This result indi-cates the existence of a relatively high degree ofgenetic variation among the Suubaru people.
The haplogroups of the Suubaru population
were classified on the basis of variations in the
HVR1 and HVR2 sequences of mtDNA. It wasdifficult to define the haplogroups determined forthe skeletal remains obtained from tomb 38 because they contained unusual motifs. Nevertheless, these samples were classified under haplogroup D or G because of the presence of mutations at sites 16223 and 16362. Finally, 6 haplogroups were detected in the ancient samples,
haplogroup M7a being the most prevalent. Thechanges observed in the nucleotides in the Dloop regions of the mtDNA and the haplogroupsof each individual are shown in Table 1.
The mtDNA haplogroup distribution often
exhibits spatially-varying frequency patterns

16

Ken-ichi Shinoda and Naomi Doi
Table 3.

Estimated frequencies of the mtDNA haplogroups among regional populations.

Haplogroup

NorthernChinese(n125)Korea(n694)MainlandJapanese(n1312)

Ryukyuans(n326)

D4
D5
G
M7a
M7b
M7c
M8
M9E
M10
CZ
A
B4
B5
F
N9a
N9b
Y
R
Other

35.2
6.4
5.6
0
2.4
2.4
6.4
3.2
3.2
1.6
4.0
9.6
1.6
7.2
3.2
0
1.6
1.6
4.8

28.8
3.5
5.8
3.4
3.4
3.7
2.4
3.2
4.7
3
7.8
4.8
4.7
4.9
3.9
0.2
2.2
0
9.8

32.6
4.8
6.9
7.5
4.8
0.8
1.4
0
1.3
1.8
6.9
7.7
4.3
5.3
4.6
2.1
0.4
0.1
6.7

34.0
2.7
1.6
24.2
4.7
0.5
0
2.1
0
0.3
8
11.7
2.4
2.1
0.3
4.3
0.5
0
0.5

TaiwanTaiwan Han
Aborigine
Chinese(n640)(n221)
1.5
4.8
0
0
9
9
0
11.4
0.4
0
0
17.1
5.9
26.7
1.2
0
1.4
2.9
8.7

14.0
6.3
0
0.5
12.0
4.3
0
0
0
0
0.5
24.5
6.7
19.2
0
0
1
7.7
3.3

SouthernChinese(n78)
14.1
5.1
1.3
0
7.7
2.6
2.6
0
2.6
0
0
25.6
1.3
23.1
1.3
0
0
2.6
10.1

Suubaru
old tomb(n13)
19
(D/G)
45
0
0
0
0
0
9
8
0
0
0
0
0
0
0
0

The references for the population are as follows:
Northern Chinese: Yao et al. (2002)
Korean: Lee et al. (2006)
Mainland Japanese: Tanaka et al. (2004)
Ryukyu: Umetsu et al. (2005)
Taiwan aborigines: Trejaut et al. (2005)
Taiwan Han Chinese: Horai et al. (1996), Tsai et al. (2001)
Southern Chinese: Yao et al. (2002)(Forster 2004). To determine the genetic characteristics of the Suubaru population, we compared
their mtDNA data with that of populations in geographically related areas. The data are listed inTable 3. Haplogroups A, C, D, G, Y, and Z occur
frequently in the Northeast Asian populations,whereas haplogroups B and F are predominant inSoutheast Asians; haplogroups C, Y, or Z haverarely been found in Southeast Asian populations(Kivisild et al. 2002).
All the haplogroups known to occur in theSoutheast and Northeast Asian populations, i.e.,
C, D, G, A, and F were detected in the Suubaru
population. However, most of the haplogroups
that were found at the Suubaru site are dominant
in Northeast Asian populations. Therefore, it canbe assumed that the buried people studied here
did not mainly originate from Southeast Asia andTaiwan but rather from Northeast Asia, and they
may have migrated into the study area via Japan-

ese mainland and Okinawa Island.
The results of our phylogenetic analysis basedon the Fst values show that the Suubaru population shares a relationship with mainland Okinawa(Figure 3). Archaeological findings indicate thattravel and exchange of goods between the Japanese mainland and Ryukyu Archipelago began in
about the 12th century (Asato, 1996). Further,some findings have suggested that a large numberof immigrants migrated to Okinawa from mainland Japan and further to the Sakishima area.
These immigrants are assumed to be the direct
descendants of the Okinawa population that immigrated after the Gusuku period. Moreover, thefact that haplogroup D4, which is dominant in
mainland Japan, was absent among the haplotypes determined in the present study indicatesthat people who emigrated from Okinawa Island
after the Gusuku period were probably native tothis island. Archaeological evidence suggests that

DNA Analysis of the Old Tomb of Suubaru

Fig. 3.

17

Neighbor-joining tree based on the Fst values determined for 8 populations.

the people who first settled in Yonaguni-jimamight have emigrated from Taiwan and thePhilippines. However, in this study, we could notestablish a genetic relationship between the aboriginal Taiwanese and the Suubaru people. Thus,
it appears that the influx of emigrants from the
Okinawa mainland to Sakishima area had large
influence at the Gusuku period.
It is therefore possible that the similarities in
the haplogroup composition between the Suubaru
population and the contemporary population of
the main island of Okinawa is the result of the arrival of immigrants, who almost replaced the indigenous population and brought along withthem a new haplogroup distribution.
Determination of the kinship among the numerous individuals buried at a single burial site
may provide extremely valuable insights into thesocial structure during ancient times. Furthermore, obtaining DNA data from people inhabiting a single region over a prolonged time period
may enable estimation of the movement ofgroups and the population dynamics in the region.
Our study of the old tomb of Suubaru unraveled the genetic characteristics of the westernmost island of Japan after the Gusuku period.
However these conclusions must be regarded as
tentative since they are based on small sample
sizes, even though the analysis have been highly
efficient. Thus, we believe that further experiments to obtain more detailed data on the humanskeletal remains from the Ryukyu Archipelago

are important. We hope to pursue this matter infuture studies.
Acknowledgments
We thank Mr. Katagiri of the Center for Buried
Cultural Properties, Okinawa Prefecture, and hiscolleagues for their valuable advice and for permitting us to use the skeletal materials. This research was supported in part by a Grant-in-Aid
for Scientific Research (No. 17107006 and17570195) from the Ministry of Education, Culture, Science and Technology of Japan.
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