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

Ancient DNA Analyses of Human Skeletal Remains fromthe Gusuku Period in the Ryukyu Islands, Japan
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, Japan2
Department of Anatomy, Faculty of Medicine, University of the Ryukyus,
Uehara 207, Nishihara, Okinawa Prefecture 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 Okinawa Island (Nakandariyama site) and Hateruma 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. Haplogroups M7a, M7b, D4, A, and B were observed in these individuals, with M7a and D4 predominant in both archaeological sites, indicating characteristics of the contemporary Okinawan population. The geographic distribution of haplogroups M7a and D4 at both sites seems to support theclaim inferred from the archaeological evidence for a relationship between the Okinawa andSakishima Islands during the Gusuku period. The fact that all these haplogroups were observedwith relatively high frequencies in the late Kaizuka, Gusuku, and contemporary Ryukyu populations 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

Introduction
Due to its geographical position and distinction as the largest of the Ryukyu Islands, Okinawa may have been an important place along
the migration route from southern Asia to theJapanese archipelago. The ﬁrst human skeletal
evidence on Okinawa dates to the Paleolithic
period (Matsuʼura, 1982). Human genetic diversity on this island has largely been shaped bymigration processes occurring after the initial
Paleolithic occupations. This was followed by
the spread of farming, which probably entailed
population movements from Kyushu Island.
Much is already known about the main demographic transitions revealed by studies of genetic
diversity in extant populations (Koganebuchi

et al., 2012; Matsukusa et al., 2010) and research
into historical, cultural, and classic anthropological sources from the Ryukyu Islands (e.g., Fukumine et al., 2006; Takamiya, 2005; Asato, 1996;
Tagaya and Ikeda, 1976). However, the consistency in the information from these myriad
sources is still debated.
Because of the Ryukyu Islandsʼ complex history and prehistory, it is not surprising that they
have often attracted the interest of population
geneticists (e.g., Jinam et al., 2012). Nevertheless, we still know very little about the genetic
structure of their human populations. To answer
questions regarding the genotypes of ancient
populations, genetic analysis of skeletons excavated from archaeological sites is the moststraightforward approach. Ancient DNA analyti-

2Ken-ichi Shinoda, Tsuneo Kakuda and Naomi Doi

Fig. 1. Geographic distribution of the Ryukyu archipelago. Map showing location of the Nakandariyama oldtomb and the Moubaru site.

cal methods developed signiﬁcantly in the lasttwo decades directly compare ancestral populations to their present-day counterparts. They permit direct reconstruction of the genetic history of
populations through time, providing a temporaldimension of the study of past human populations.
Our previous investigation of the genetic
structure of humans from the Shell Midden (Kaizuka) period on Okinawa provides proof ofgenetic continuity between the ancient and contemporary populations of this island from at least
the late Kaizuka period (Shinoda et al., 2012).
However, no genetic evidence has been reported
from the intermediate Gusuku period (thirteenth–sixteenth centuries) mainly because sufﬁcientskeletal remains for ancient DNA analysis arelacking.
During this period, agricultural production ofrice, wheat, and millet was developed and overseas trading of these goods began. All areas of
the Ryukyu Islands formed a common culture
during this time (Asato, 1996). In order to understand the history of these islands, it is important

to clarify the human genetic characteristics ofthis period. The present study aims to explain thegenetic composition of the population from the
Gusuku period, and address their genetic relationship (at a molecular level) with other Ryukyu
groups from different cultural periods.
Materials and Methods
Archaeological sites and specimens
In this analysis, we used human skeletal
remains excavated from two archaeological sites
on mainland Okinawa (Nakandariyama site) and
Hateruma Island (Moubaru site), the two southernmost and inhabited islands of Japan. The
approximate locations of these sites are shown in
Figure 1.
Nakandariyama is a cliff burial site located in
the city of Naha. It was excavated in 2003 by theCenter for Buried Cultural Properties (Okinawa
Prefecture) and comprises at least 47 individuals.
These consisted of 15 males, 10 females, 15juveniles, and 7 individuals of indeterminate
gender, ranging from infants to mature adults.

MtDNA Polymorphisms in Gusuku Period

These skeletons generally display a prolonged
upper jaw, which is characteristic of medieval
humans but different from former Kaizuka people (Doi, 2005).
Since no cemetery artifacts remain, the chronology of this site was determined based on thedesign of burial goods. All tombs except No. 7
belong to the early modern period. In contrast,
the age of tomb No. 7 is estimated to be in the
Gusuku period. In order to determine the genetic
characteristics of the Gusuku population frommainland Okinawa, eight well-preserved samples
excavated from tomb No. 7 were selected for
DNA analysis.
A sediment production project in the 1980s
identiﬁed the Moubaru site, located in the
Misyuku village in the northwest part of Hateruma Island. By counting skulls and limb bones,
the minimum number of individuals was estimated at 29 (13 adult males, 10 adult females, 6
immature individuals) (Doi, 1998). According to
14
C dating of bone samples, the remains belong
to the Gusuku period (Dr. Yoneda, personal communication). Seven well-preserved samples wereselected for DNA analysis. A list of all samples
used in this study is presented in Table 1.
Authentication methods and extraction of DNA
DNA analyses were performed at the National
Museum of Nature and Science, which is dedicated to ancient DNA analysis. In the present
study, we employed standard precautions to
avoid contamination, e.g., separation of pre- andpost-PCR experimental areas, use of disposable
laboratory wares and ﬁlter-plugged pipette tips,
treatment with DNA contamination removal
solution (DNA Away; Molecular Bio Products,
San Diego, CA, USA), UV irradiation of equipment and benches, and negative extraction and
PCR controls (Shinoda et al., 2006).
Extraction and puriﬁcation of DNA andsequence analysis were mainly performed by
T.K. and K.S. The multiplex ampliﬁed productlength polymorphism (APLP) analysis (Umetsu
et al., 2005) was conducted in different laboratories as described in the genotyping section.

3

In order to prevent contamination from postexcavation handling, the tooth samples wererinsed with DNA-decontamination agents andthen washed thoroughly with distilled water
before drying. Next, the samples were crushed
into powder using a Multi-beads Shocker (Yasui
Kikai Corporation). DNA was extracted from
0.5 g of powder from each sample using a commercial DNA extraction kit (Qiagen, DNA Investigator kit, Germany) after Protainase K digestion. In the case of the Moubaru sample, thetooth root was cut off and used for 14C dating andstable isotope analysis.
Ampliﬁcation and sequencing of HVR1
Segments of HVRs (nucleotide positions
16121-16238, HVR1-1; 16209-16291, HVR1-2;
and 160289-16366; HVR1-3) of mtDNAs, as per
the revised Cambridge reference sequence(Andrews et al., 1999), were sequenced in all
samples.
Aliquots (2 μl) of the extracts were used as
templates for PCR. Ampliﬁcations were carriedout in a reaction mixture (total volume, 15 μl)
containing 1 unit of Taq DNA polymerase (HotStarTaqTM DNA polymerase; Qiagen, Germany),
0.1 M of each primer and 100 mM of deoxyribonucloside triphosphates in 1×PCR buffer provided 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 sec; 50°C–56°C for 20 sec, and 72°C for 15
sec; and ﬁnal extension at 72°C for 1 min.
The following primers were used to amplify
HVR1-1, HVR1-2, HVR1-3:
L16120 5′-TTACTGCCAGCCACCATGAA-3′
H16239 5′-TGGCTTTGGAGTTGCAGTTG-3′
L16208 5′-CCCCATGCTTACAAGCAAG-3′
H16312 5′-ACTATGTACTGTTAAGGGTG-3′
L16269 5′-CTAGGATACCAACAAACCTA-3′
H16367 5′-ATCTGAGGGGGGTCATCCAT-3′
The PCR products were subjected to agarose
gel electrophoresis on a 1.5% gel and wererecovered by using a QIAEX II agarose gel
extraction kit (Qiagen, Germany). Aliquots of thesamples were prepared for sequencing on a Big-

Code
Name

SampleCode
NameSample

N.D.
T
T
N.D.
T
N.D.
N.D.
N.D.

N.D.
N.D.
T
N.D.
T
N.D.
N.D.

G
N.D.
N.D.
N.D.
N.D.
N.D.
G

8793
(M10)

G
G
G
N.D.
N.D.
N.D.
G
A

8701
(N,D4g)

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.
C
N.D.
N.D.
N.D.
C

C
C
C
N.D.
C
C
C
C

9293
(D4b2b)

N.D.
CRS
CRS
N.D.
N.D.
290,311,319
N.D.

N.D.
N.D.
N.D.
362
N.D.
N.D.
N.D.
N.D.

HV1-3
(1628916366)(16,000+)

T
T
T
N.D.
T
T
N.D.

T
T
T
T
T
T
T
T

9950
(B5)

N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

N.D.
N.D.
A
N.D.
N.D.
N.D.
N.D.
A

856
(D4d1b)

G
G
G
G
G
G
G

G
G
G
G
G
G
G
G

10310
(F)

G
A
A
A
G
G
G

G
G
A
A
G
G
G
G

3010
(D4)

G
N.D.
G
N.D.
N.D.
N.D.
G

N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

10397
(D5)

T
T
T
T
T
T
T

T
T
T
T
T
T
T
T

3644
(D4h)

T
N.D.
N.D.
N.D.
T
N.D.
T

N.D.
T
T
T
T
N.D.
T
C

10400
(M)

G
G
G
G
G
G
G

A
G
G
G
G
G
G
G

4048
(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.

13563
(G2)

T
T
T
N.D.
C
N.D.
C

T
C
T
T
T
T
C
T

4386
(M7a)

T
T
T
T
T
T
T

T
T
C
T
T
T
T
T

14979
(D4a)

G
N.D.
N.D.
N.D.
G
N.D.
G

G
G
G
N.D.
N.D.
N.D.
G
G

4820
(B4b)

G
G
G
N.D.
G
N.D.
G

G
G
G
G
G
G
G
G

15346
(B4c)

G
G
G
N.D.
G
G
G

G
G
G
N.D.
G
G
G
G

5147
(N9b)

All polymorphic sites are numbered according to the revised Cambridge reference sequence (Andrews et al., 1999).
CRS indicates that the sequence of the segment is identical to the revised Cambridge reference sequence.
Diagnostic polymorphisms of the PCR-Luminex analysis are emphasized by bold italic type.
N.D. denotes “not determined”.

Nakandariyama site (Okinawa Island)
1
7-6
Maxilla, Left, M3
2
7-13(2) Mandible, Right, M337-21(2) Maxilla, Right, M2
4
32
Maxilla, Left, M2
5
35
Maxilla, Right, M2
6
41
Mandible, Left, M2
756-1
Mandible, Right, M1
8
no name Maxilla, Right, M3
Moubaru site (Hateruma Island)
11
Maxilla, Left, M32
2-2
Maxilla, Right, M3
3
4
Maxilla, Right, M3
4
9-1
Mandible, Left, M3
5
10
Maxilla, Left, M1
611-1
Maxilla, Right, M3
7
11-2
Maxilla, Right, M3

No.

N.D.
N.D.
223
N.D.
N.D.
N.D.
N.D.
217,249

HV1-2(1620916291)(16,000+)

N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

HV1-1
(1612116238)(16,000+)

Sample used for DNA exreaction and the result of the analysis.

Nakandariyama site (Okinawa Island)
1
7-6
Maxilla, Left, M3
2
7-13(2) Mandible, Right, M337-21(2) Maxilla, Right, M2
4
32
Maxilla, Left, M2
5
35
Maxilla, Right, M2
6
41
Mandible, Left, M2
756-1
Mandible, Right, M1
8
no name Maxilla, Right, M3
Moubaru site (Hateruma Island)
11
Maxilla, Left, M32
2-2
Maxilla, Right, M3
3
4
Maxilla, Right, M3
4
9-1
Mandible, Left, M3
5
10
Maxilla, Left, M1
611-1
Maxilla, Right, M3
7
11-2
Maxilla, Right, M3

No.

Table 1.

N.D.
G
N.D.
N.D.
G
G
G

G
G
G
G
G
N.D.
N.D.
G

15497
(G1)

C
A
N.D.
C
C
C
C

C
C
A
A
C
C
C
C

5178
(D)

A
A
A
A
A
A
A

A
A
A
A
A
A
A
A

15524
(D4b2a)

G
N.D.
G
G
G
G
N.D.

G
G
G
N.D.
G
G
G
G

5231
(N9a)

A
A
A
N.D.
A
A
A

A
A
A
A
N.D.
A
A
A

15874
(D4e2)

T
N.D.
N.D.
N.D.
N.D.
T
T

T
T
T
T
T
T
T
T

5964
(D4e1)

not deleteN.D.
not delete
not delete
not delete
not delete
not delete

not delete
not delete
not delete
not delete
not delete
not delete
not deletedelate

8272
(B)

M
D4
D4
D4
M7a
N.D.
M7a

M7b
M7a
D4a
D4
M
N.D.
M7a
B

M
D4
D4
D4
M7a
A
M7a

M7b
M7a
D4a
D4
M
N.D.
M7a
B4

SNP
Haplogroup
PCR-Luminex

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)4
Ken-ichi Shinoda, Tsuneo Kakuda and Naomi Doi

MtDNA Polymorphisms in Gusuku Period

Dye cycle sequencing kit Ver.3.1 (Applied Biosystems, Foster City, CA, USA), which was performed using forward and reverse primers. The
primers used in the PCR ampliﬁcation were also
used in the sequencing reaction. Sequencing was
performed in both directions to enable identiﬁcation of polymorphisms or ambiguous bases byusing a single primer. The sequencing reactions
were performed on a DNA Sequencer (ABImodel 3130) equipped with SeqEd software.
Genotyping of Polymorphisms
To conﬁdently assign mtDNAs to the relevant
haplogroups, 24 haplogroup-diagnostic single
nucleotide polymorphisms (SNPs) including anine b.p. repeat variation in the non-coding cytochrome oxidase II/tRNALys intergenic region
that deﬁne major haplogroups found in Japaneseand East Asian population were analyzed bymultiplex APLP (Umetsu et al., 2005). SNPs thatdeﬁne major haplogroups were detected by useof suspension-array technology (Luminex 100;
Luminex) at the laboratory of G&G Science,
Fukushima. The methodology used for genotyping and primer sequences have been described indetail elsewhere (Itoh et al., 2005, Shinoda et al.,2012).
Results and Discussion
There are many advantages to using mtDNA.
Because it is present in high copy numbers inmammalian cells, mtDNA can most likely bedetected even in highly degraded samples, suchas the ancient ones studied here. However, hot
and humid conditions are generally unfavorable
to the preservation of DNA in human skeletal
remains, limiting the possibilities for ﬁnding
well-preserved DNA in subtropical regions suchas the Ryukyu Islands (Shinoda et al., 2012).
Due to the poor quality of mtDNA extracted
from the ancient material, it was not possible toamplify all samples.
Table 1 shows the results of PCR ampliﬁcation. Suspected false positive results stemmingfrom contamination with contemporary DNA and5other questionable data were omitted from thisstudy. In these samples, 6 out of 45 PCR ampliﬁcations were successfully analyzed. In contrast,
12 of the 15 samples were successfully typed ineach haplogroup by multiplex SNP typing assays
to simultaneously analyze important SNPs within
the mtDNA coding region. Two samples thatdeﬁned macrohaplogroup M could not be identiﬁed further to haplogroup.
In the case of multiplex analysis, to minimize
the risk of contamination and to raise the efﬁciency of PCR, very short DNA fragments (average 60–80 bp) were ampliﬁed. However, thelength of ampliﬁed HVR1 portion exceeded 100
bp in this experiment. Therefore, it is presumablethat DNA extraction products of both archaeological sites were degenerated and most of theirlengths were under 80 bp.
The haplogroup composition of the samples is
M7a (33.3%); M7b (8.3%); D4 (41.7%); A(8.3%); and B (8.3%) in total (Table 1). Owing to
the small sample size, it is difﬁcult to verify thegenetic characteristics by statistical methods,
although it is noteworthy that the M7a and D4
haplogroups from both archaeological sites were
well-represented. The distribution of mtDNA
haplogroups in this period will provide someinsight on the human population history of the
Ryukyu Islands.
Figure 2 shows the haplogroup frequencies of
the Gusuku period (present study), modern
Ryukyu, and Taiwanese aboriginal populations.
Regardless of their geographical location, theRyukyu and Taiwanese aboriginal populations
differed when compared according to their shareof haplogroups. Taiwanese aborigines do nothave haplogroup M7a and D4 that dominate theextant Ryukyu and Gusuku populations. Therefore, we assume there was no genetic connection
between the Taiwanese and Gusuku populations,who had different origins and population histories overall.
The biased distribution pattern of haplogroup
M7a in Japan may have diversiﬁed from theancestral M7 haplogroup, which was introduced
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)
Taiwan aborigines: Trejaut et al., (2005)

within the Japanese archipelago (Adachi et al.,2011). The Ryukyu Islands are candidates for theorigin of this haplogroup (Shinoda, 2007). On
the other hand, haplogroup D4 is observed innortheastern China and Korea as well as mainland Japan (Tanaka et al., 2004) and there is nobiased distribution among the mainland Japan.
Moreover, this haplogroup apparently occurred ata low frequency in the Jomon population (Shinoda and Kanai, 1999; Adachi et al., 2011). A
reasonable theory is that the Yayoi immigrants
were predominantly from haplogroup D4.
Archaeological ﬁndings indicate that travel
and trade between mainland Japan and the
Ryukyu archipelago began around the twelfth
century. There are also some indications thatwaves of immigrants moved to Okinawa frommainland Japan and reached the Sakishima area
(Asato, 1996). The geographic distribution of
haplogroups M7a and D4 in both sites seems to
support the claim inferred from the archaeological evidence that a relationship existed between
the Okinawa, Sakishima, and Hateruma Islandsin the Gusuku period.
This study identiﬁed four distinct haplogroups(M7a, M7b, D4, A, B) in the archaeological
specimens sampled from the Okinawa and Hateruma Islands, which agree well with the genetic
variation of extant Okinawan and mainland Japanese populations (Figure 2). It follows that thethese mtDNA haplogroups except M7b havebeen present in the late Kaizuka population (Shinoda et al., 2012) and that haplogroup D4 mostprobably reached the Okinawa Islands frommainland Japan at least by the end of the late
Kaizuka period.
High frequencies of haplogroups M7a and D4
are characteristic of the contemporary Okinawan
population (Tanaka et al. 2004). The distributionof these haplogroups among the late Kaizuka,
Gusuku, and modern Ryukyu populations suggests that the formation of the extinct Ryukyu
Islands population was the result of population
expansion.
The number of samples for which DNA haplogroups could be determined was small in thepresent analysis, so unfortunately, the results provided minimal insight. However, the establishment of genetic characteristics among numerous
burial sites in the Gusuku period may provide us
with extremely valuable information regarding
human migration and population dynamics in the
Ryukyu Islands. The preliminary experiment presented here proved that sufﬁcient amounts of
DNA are retained in some human skeleton samples, even though the analytical efﬁciency may
be poor. Thus, we believe it is worthwhile to continue the experiments to obtain more detailed
data on the human skeletal remains from the
Gusuku period site. Moreover, insight on the
population history of the Ryukyu Islands could
be obtained by comparing ancient mtDNA

MtDNA Polymorphisms in Gusuku Period

sequences collected from the Kaizuka sites thatare contemporaneous with the Jomon period in
mainland Japan with sequences from the lateKaizuka, Gusuku, and present-day populations.
Acknowledgments
We want to thank Mr. Katagiri of the Okinawa
Prefectural Museum and his colleagues at theCenter for Buried Cultural Properties, Okinawa
Prefecture, for permitting us to use the skeletalmaterials and for their valuable advice.
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