Journal of Human Genetics (2012) 57, 787–795& 2012 The Japan Society of Human Genetics All rights reserved 1434-5161/12
www.nature.com/jhg

ORIGINAL ARTICLE

The history of human populations in the JapaneseArchipelago inferred from genome-wide SNP datawith a special reference to the Ainu and the
Ryukyuan populations
Japanese Archipelago Human Population Genetics Consortium (Consortium members: Timothy Jinam1,18,
Nao Nishida2,19, Momoki Hirai3,19, Shoji Kawamura3,19, Hiroki Oota4,19, Kazuo Umetsu5,19, Ryosuke Kimura6,19,
Jun Ohashi7,19, Atsushi Tajima8,19, Toshimichi Yamamoto9,19, Hideyuki Tanabe10,19, Shuhei Mano11,19,
Yumiko Suto12,19, Tadashi Kaname13, Kenji Naritomi13, Kumiko Yanagi13, Norio Niikawa14, Keiichi Omoto15,19,
Katsushi Tokunaga2,19 and Naruya Saitou1,16,17,19)
The Japanese Archipelago stretches over 4000 km from north to south, and is the homeland of the three human populations;
the Ainu, the Mainland Japanese and the Ryukyuan. The archeological evidence of human residence on this Archipelago goesback to 430 000 years, and various migration routes and root populations have been proposed. Here, we determined close to
one million single-nucleotide polymorphisms (SNPs) for the Ainu and the Ryukyuan, and compared these with existing data
sets. This is the first report of these genome-wide SNP data. Major findings are: (1) Recent admixture with the Mainland
Japanese was observed for more than one third of the Ainu individuals from principal component analysis and frappe analyses;(2) The Ainu population seems to have experienced admixture with another population, and a combination of two types ofadmixtures is the unique characteristics of this population; (3) The Ainu and the Ryukyuan are tightly clustered with 100%bootstrap probability followed by the Mainland Japanese in the phylogenetic trees of East Eurasian populations. These results
clearly support the dual structure model on the Japanese Archipelago populations, though the origins of the Jomon and the
Yayoi people still remain to be solved.
Journal of Human Genetics (2012) 57, 787–795; doi:10.1038/jhg.2012.114; published online 8 November 2012
Keywords: admixture; Ainu; Japanese Archipelago; population; Ryukyuan; SNPINTRODUCTION
The origins of the people living in the Japanese Archipelago havebeen studied for a long time (for review, see Suzuki1 and Saitou2).
The standard theory based on craniofacial data is the dual structure

model proposed by Hanihara.3 According to this model, the firstmigrants to the Japanese Archipelago came from somewhere in
Southeast Asia in the Upper Paleolithic age, who were ancestors of
the Jomon people. The second wave of migration took place later in

1Department of Genetics, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan; 2Department of Human Genetics, Graduate School of
Medicine, The University of Tokyo, Tokyo, Japan; 3Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan;
4Laboratory of Anatomy and Physical Anthropology, Kitasato University School of Medicine, Sagamihara, Japan; 5Department of Forensic Medicine, Yamagata University Schoolof Medicine, Yamagata, Japan; 6Transdisciplinary Research Organization for Subtropical and Island Studies (TRO-SIS), University of the Ryukyus, Nishihara, Japan; 7Graduate
School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan; 8Department of Human Genetics, Institute of Health Biosciences, The University ofTokushima Graduate School, Tokushima, Japan; 9Department of Legal Medicine and Bioethics, Nagoya University School of Medicine, Nagoya, Japan; 10Department of
Evolutionary Studies of Biosystems, School of Advanced Sciences, Graduate University for Advanced Studies (SOKENDAI), Hayama, Japan; 11Department of Mathematical
Analysis and Statistical Inference, The Institute of Statistical Mathematics, Tachikawa, Tokyo, Japan; 12Research Center for Radiation Emergency Medicine, National Institute ofRadiological Sciences, Chiba, Japan; 13Department of Medical Genetics, Graduate School of Medicine, University of the Ryukyus, Nishihara, Japan; 14Health Sciences Universityof Hokkaido, Tobetsu, Japan; 15Department of Anthropology, Faculty of Science, The University of Tokyo, Tokyo, Japan; 16Department of Biological Sciences, Graduate School of
Science, The University of Tokyo, Tokyo, Japan and 17Division of Population Genetics, National Institute of Genetics, Mishima, Japan
18Current address: Division of Human Genetics, National Institute of Genetics, Mishima, Japan.
19Members of the ‘Asian Archival DNA Repository Consortium’.
Correspondence: Dr N Saitou, Division of Population Genetics, National Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan.
E-mail: saitounr@lab.nig.ac.jp
or Dr K Tokunaga, Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
E-mail: tokunaga@m.u-tokyo.ac.jp
or Dr K Omoto, Faculty of Science, Department of Anthropology, The University of Tokyo, Tokyo 113-0033, Japan.
E-mail: kocolias@msg.biglobe.ne.jp
Received 17 March 2012; revised 27 August 2012; accepted 30 August 2012; published online 8 November 2012

History of Japanese Archipelago populations
T Jinam et al788

the Yayoi period, and the people came in this time from Northeast
Asia. The indigenous Jomon people and the new migrants in and after
the Yayoi period gradually mixed with each other. This model
provides a reasonable explanation for the morphological similarity
between the Ainu people of Hokkaido, the northernmost main islandof the Japanese Archipelago, and the Ryukyuan (or Okinawan) people
in the Southwest Archipelago, despite a large geographical distance.
The similarity of these peoples was already noticed 101 years ago by
von Baelz4 as the Ainu–Ryukyuan common origin theory.
A series of studies on genetic polymorphisms of classic markers,
such as blood groups, serum proteins and red cell enzymes, werecarried out for human populations in the Japanese Archipelago fromthe 1960s to the 1970s, and the Ainu and the Ryukyuan populations
were also studied (for example, Misawa and Hayashida;5,6 Omoto andHarada;7 Nakajima et al.;8 Omoto et al.9). Omoto10,11 computed
genetic distances among various populations of the world, and byconstructing a phylogenetic tree he concluded that the Ainu
population may have originated in East Asia, in spite of theirunique morphological characters somewhat resembling West
Eurasians. Omoto et al.9 estimated genetic distances among the
Ainu, the Ryukyuan, the Mainland Japanese and the Chinese
populations. Although they did not show a phylogenetic tree,
distance relationships indicated a clustering of the Ainu and the
Ryukyuan if we apply the neighbor-joining method.12 Omoto13
constructed a phylogenetic tree for various populations of theMainland Japan, the Ainu and the Ryukyuan, and showed that the
Ainu and the Ryukyuan were clustered together. Seven serum protein
polymorphism data were used for this tree construction, and this tree
was the first one to suggest the genetic similarity between the Ainu
and the Ryukyuan. Nei14 constructed a neighbor-joining12 tree of
15 Eurasian populations based on DA distances15 computed fromallele frequency data of 18 polymorphic classic markers. The Ainu andthe Ryukyuan clustered with 62% bootstrap probability, followed by
the Mainland Japanese population.
Omoto and Saitou16 constructed an unrooted tree of three
populations in the Japanese Archipelago (Ainu, Ryukyuan and
Mainland Japanese) and Korean from allele frequency data of 25
classic polymorphic marker loci, and showed that Ainu and
Ryukyuan clustered with 85% and 74% bootstrap probabilities
when DA distances15 and Dst distances17 were used, respectively.
These probabilities were considerably higher than the random
expectation (33% ¼ 1/3), and they considered these results aspartially supporting the dual structure hypothesis of Hanihara.3
This is because the Ainu–Ryukyuan clustering was expected fromthe contrasting genetic backgrounds of Jomon vs Yayoi, irrespective of
their origins. Saitou18 later constructed a distance-based phylogenetic
network19 of these four populations using DA distance values
obtained by Omoto and Saitou,16 and found that the length of split
separating Ainu and Ryukyuan populations from Mainland Japaneseand Korean was much longer than that separating Mainland Japaneseand Ryukyuan from the rest of two populations. It confirmed a treelike structure of the four populations shown by Omoto and Saitou.16
Mitochondrial DNA and Y chromosomal DNA examinations
became popular for human population studies from the 1980s, anda series of papers were published for human populations of theJapanese Archipelago.20–28 All of these studies showed some genetic
similarity between the Ainu and the Ryukyuan populations. However,mitochondrial DNA and Y chromosomes are both non-recombining,
and the genetic information that can be extracted is limited.
Meanwhile, Tokunaga and his colleagues29–32 conducted DNA
typings of highly polymorphic human leukocyte antigen loci for
Journal of Human Genetics

human populations of the Japanese Archipelago, and found aclustering of the Ainu and the Ryukyuan populations.
The situation surrounding DNA polymorphism study of humanpopulation drastically changed with the sequencing of the humangenome.33 A genome-wide cataloging of single-nucleotide polymorphisms (SNPs) were carried out for four human populations
including the Japanese living in Tokyo.34 Nishida et al.35 also
conducted an SNP typing of 400 Mainland Japanese individuals,
and Yamaguchi-Kabata et al.36 produced and studied SNP data for7000 individuals living in the various locations of the JapaneseArchipelago. They clearly demonstrated the genetic differences
between the Mainland Japanese and the Ryukyuan populations.
Worldwide surveys of SNP typing were carried out under the
Human Genome Diversity Project (HGDP-CEPH)37 including theMainland Japanese, and the human genetic diversity was analyzed formany human populations in Asia under the HUGO Pan-Asian SNP
Consortium,38 including the Mainland and Ryukyu Japanese.
However, the Ainu individuals were not included in any of thesestudies.
One of us (Keiichi Omoto) carried out a series of studies on thegenetic polymorphism of many human populations in Asia, andDNA samples have been preserved under his and Momoki Hirai’s
leadership at the University of Tokyo Kashiwa Campus. We recently
formed ‘Asian Archival DNA Repository Consortium’ to keep andutilize these precious DNA samples. This paper is an outcome ofactivities of this Consortium. The availability of archival DNA in the
Ainu population as well as the advancement of high throughput SNP
genotyping for this study allowed us to examine in closer detail thegenetic substructure and the evolutionary history of the humanpopulations in the Japanese Archipelago. We conducted bothindividual-based analyses and population-based analyses, andwill suggest that the two major waves of migrations during the
paleolithic–Jomon period and the Yayoi and the later period produced
the unique features of human populations in the Archipelago.

MATERIALS AND METHODS
Sample data, ethical approval and SNP genotyping
Blood samples of the Ainu people were collected in Biratori Town, Hidaka
District of Hokkaido in the early 1980s for analysis of DNA by a group fromThe University of Tokyo, and have since been archived there. A study to use
these samples was approved by the Research Ethics Committee of The
University of Tokyo. These Ainu DNA samples were the same used in previous
studies on mitochondrial DNAs,20,21,23,24 on Y chromosome,24,26,28 and on
human leukocyte antigen types.29–32 Two of us (Naruya Saitou and Timothy
Jinam) recently visited Biratori Town, and explained these molecularanthropological studies in the past as well as the current study to therepresentatives of the Ainu people living in that area. DNA samples of the
Ryukyuan from the Okinawa main island were collected from 2004 to 2008 by
a group from University of the Ryukyus. A study to use these samples wasapproved by the Ethics Committee of University of the Ryukyus.
A total of 36 Ainu and 38 Ryukyuan samples were genotyped using theAffymetrix genome-wide SNP 6.0 microarray platform (Affymetrix, Santa
Clara, CA, USA). All genotyping experiments and their computational analyses
were conducted at the Department of Human Genetics, The University ofTokyo. In addition to the Ainu and the Ryukyuan populations, SNP genotype
data, generated using the same method, from 200 Mainland Japanese (first set)
mostly from the Kanto area35 were used. These three groups (Ainu, Ryukyuan
and Mainland Japanese) form the Japanese Archipelago population data set,
which was further augmented with HapMap data34 from four populations,
namely Yorubans from Africa, Americans of European origins, Han Chinese
from Beijing (CHB) and Japanese from Tokyo (JPT). The list of these sevenpopulations is shown in Table 1.

History of Japanese Archipelago populations
T Jinam et al789

Data filtering and quality checks
SNPs of the mitochondrial DNA, X chromosome and Y chromosome were
excluded from the initial SNPs numbering 906 600. Duplicate SNPs and those
without a dbSNP ID were also filtered out, resulting in a total of 868 257
remaining SNPs. Individual samples with poor genotyping performances were
further filtered out based on the Affymetrix contrast quality control (cQC)
threshold of 0.04, as recommended by the manufacturer. Three Ryukyuan and
two Mainland Japanese samples were omitted based on this criterion. However,in the Ainu population, only 13 out of 36 individuals passed the cQC
threshold. This was probably due to the degradation of DNA quality of thearchival samples. To maximize the number of Ainu individuals to be used fordownstream analysis, further SNP filtering was done based on confidence
scores for each SNP generated during genotype calling using the Affymetrix
Birdseed Ver2 algorithm.
In general, SNPs with a confidence score 40.1 are more likely to have failed
genotype calling (that is ‘no calls’). By visually inspecting genotype cluster
graphs of random SNPs with confidence scores ranging from 0.1 to 0.004, a
more stringent cutoff of 0.008 was used to exclude under-performing SNPs
while retaining the maximum number of individuals. Thus, based on thiscriterion, 212 448 SNPs were omitted from the set of 36 Ainu individuals,
resulting in 656 237 remaining SNPs. The SNP data in the Ainu and all otherpopulations that were generated using the Affymetrix Genome-Wide 6.0 Assay
were further filtered to remove SNPs with call rate o95% and that deviate
from Hardy–Weinberg equilibrium (Po0.001). For example, 449 SNP lociwere further excluded from the Ainu data, resulting in 655 788 SNP loci (seeSupplementary Table S1). The filtering steps were done on each population
separately and the number of SNPs filtered out is shown in Supplementary
Table S1. After merging SNP data from all populations, the final number of
SNPs in the seven population data sets was 641 314.

Merging with other population data
We also included SNP data from 30 other East Asian populations available
from public databases in addition to the Japanese Archipelago and HapMap
population data sets. These included the HGDP-CEPH data set, which consistsof 650 000 SNPs from 51 worldwide populations37 and the Pan-Asian SNP(PASNP) data set, which consists of 54 794 SNPs from 73 populations in
Asia.38 The number of SNPs that overlap between the Japanese ArchipelagoHapMap 7-population data sets with the HGDP-CEPH panel was 114 001.
After applying filters (excluding SNPs with o95% genotype call rate andminor allele frequency o1%) in the merged data set, there were 101 562 SNPs
remaining. For merging the Japanese Archipelago-HapMap population data
sets with the PASNP data, 15 526 overlapping SNPs from both data sets wereextracted and merged. After applying the same filtering criteria as above, thenumber of remaining SNPs was 14 997. The combination of the JapaneseArchipelago, HapMap, HGDP-CEPH and PASNP data sets yielded only 4237
overlapping SNPs. All filtering and merging steps were carried out using
PLINK software.39 Supplementary Table S2 shows the list of 16 and 14
populations used from HGDP-CEPH and PASNP data sets, respectively.

Data analysis
Subsequent analysis was carried out using different combinations of the above
data sets. For the merged data from all data sets, only populations in East Asia
(Supplementary Table S2) were used for analysis. We first conductedindividual-based analyses. Principal component analysis (PCA), using thesmartpca program in the EIGENSOFT software package,40 was our main
strategy. The program frappe41 was used to represent alternative views of
population structure and admixture patterns. A maximum-likelihood method
is used in this program, and it is computationally more efficient than
STRUCTURE.42 Population-based phylogenetic trees were constructed byusing CONTML (a maximum-likelihood method for allele frequency data43
was used) for SNP allele frequency data and NEIGHBOR (the neighbor-joining
method12 was used) for Dst distance17 matrices computed from SNP allele
frequency data by using GENDIST programs, all from the PHYLIP package44
with 5000 bootstrap replicates. Neighbor-net networks45 were also constructedfrom Dst distance matrices using the software SplitsTree 4.46

RESULTS
Individual-based analyses based on over 640 000 SNP data
The PCA result for the individual SNP data of the seven populations
listed in Table 1 is shown in Supplementary Figure S1. The African,
the West Eurasian and the East Eurasian (five East Asian populations)were located at the apexes of the triangle in this figure. Because
African populations are known to be more distantly related from thetwo Eurasian populations,47–49 the second PCA was conducted
after excluding the African population (Supplementary Figure S2).
Now the first principal component (PC1) separates the West Eurasianand the five East Asian populations, whereas the PC2 separates the
Ainu population and the remaining four East Asian populations.
Interestingly, the Ainu individuals are linearly aligned, suggestingvarying degrees of recent admixture with the mainland Japanese
population. It also appears that the population genetically closest to
the Ainu is the Ryukyuan, despite the fact that these two populations
are geographically located on the northernmost and southernmost
poles of the Japanese archipelago, respectively.
When the West Eurasian individuals were further eliminated in thethird PCA (Figure 1a), the unique feature of the Ainu individuals andthe relationship of the other three populations became prominent.
The PC1 and PC2 of this figure explained 1.8% and 0.6%,
respectively, of the total variances among the SNP data of 356
individuals. The Mainland Japanese individuals and the HapMap
Japanese (JPT) individuals clustered together, as expected. Many Ainu
individuals were located at the left side and the Han Chinese in
Beijing (HapMap CHB) were distributed at the rightmost side,
whereas the Ryukyuan and the Mainland Japanese were sandwiched
between them. The PC1 and PC2 coordinates were well correlatedwith Ryukyuan, Mainland Japanese and Han Chinese individuals.
This pattern suggests the existence of the south (Ryukyuan) to thenorth (Han Chinese in Beijing) geographical cline. However,
Ainu individuals, distributed in the northernmost Japanese Archipelago, were found to be closer to the Ryukyuan individuals than to
the Mainland Japanese, consistent with the result shown in
Supplementary Figure S2.
This finding clearly supports the genetic similarity between the
Ainu and the Ryukyuan, in spite of their large geographical distancewith each other within the Japanese Archipelago.
Another interesting pattern was the substantial interindividual
variation among the Ainu individuals compared with those of theother three populations. Three Ainu individuals were within thecluster of the Mainland Japanese population, whereas the other five
Ainu individuals in a red circle constituted a distinct cluster.

Table 1 Basic information of three populations in the JapaneseArchipelago and the four populations of the HapMap data set
No. ofSampledAverage

Populationindividuals

locationheterozygosity

Reference

Ainu
Ryukyuan

36
35

Hokkaido, Japan
Okinawa, Japan

0.220
0.238

This study
This study

Mainland JapaneseJapanese (JPT)198
45

Tokyo, JapanTokyo, Japan

0.242
0.240

Ref. 35

Han Chinese (CHB)
European (CEU)42
89

Beijing, China
Utah, USA

0.242
0.265

Ref. 34

Yoruban (YRI)

89

Ibadan, Nigeria

0.284

Ref. 34

Ref. 34

Ref. 34

Abbreviations: CEU, Americans of European origins; CHB, Han Chinese from Beijing;
JPT, Japanese from Tokyo; YRI, Yorubans from Nigeria.

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History of Japanese Archipelago populations
T Jinam et al790

Figure 2 The result of frappe analysis from k ¼ 2 to k ¼ 5 for the same
individuals of five populations used for the PCA analysis shown in
Figure 1a.

Figure 1 (a) A PCA plot of individuals for the three populations in theJapanese Archipelago (Ainu, Ryukyuans and the Mainland Japanese),
HAPMAP Japanese (JPT) and HAPMAP Chinese (CHB). (b) APCA plot for
36 Ainu individuals only.

To further examine this pattern, PCA was performed only for the 36
Ainu individuals (Figure 1b). Now, the PC1 and PC2 explained 6.1%
and 5.3% of the total variances, respectively. Interestingly, the highheterogeneity similar to that of Figure 1a was reproduced. Thisfinding indicates that the wide variation observed for the Ainu
individuals in Figure 1a was not because of the coexistence of otherthree populations, but because of the interindividual relationshipinherent to the Ainu population. This result may be caused by recent
admixtures involving two different source populations.
To see whether this was the case, we calculated the allele-sharing
distances between the Ainu and the Mainland Japanese individuals,
and compared these with the PC1 coordinates of the Ainu individuals. There was a clear positive correlation (r2 ¼ 0.542) between theallele-sharing distances and the PC1 coordinates (Supplementary
Figure S3). The three Ainu individuals within the Mainland Japanesecluster had the smallest allele-sharing distances, and conversely, theAinu individuals located farthest from the Mainland Japanese on
the PC1 axis tend to have greater allele-sharing distances with theMainland Japanese population. These observations suggest that theallele sharing between the Ainu and the Mainland Japanese was theresult of relatively recent and continuing episodes of gene flow
between the two populations. If this is the case, another high degree
Journal of Human Genetics

of variation within the Ainu individuals regarding PC2 may also be
explained by the gene flow from a human population geneticallydistinct from the Mainland Japanese. In particular, the five Ainu
individuals in the red circle having the highest PC2 coordinates may
be affected by recent admixture events.
The result of the frappe analysis is shown in Figure 2. When k ¼ 2,
the two ancestry components corresponds to one (dark blue), which
is 100% in 16 Ainu individuals and the other (orange), which is thehighest (B90%) in all the CHB individuals. Interestingly, 20 Ainu
individuals showed varied proportions of the orange-colored ancestry
component, again suggesting the recent admixture. Most of the
Ryukyuan and the Mainland Japanese individuals showed about 30%
and 20% blue-colored component, respectively. This difference wasconsistent with the PCA results in Figure 1a, in that the Ainu wascloser to the Ryukyuan than to the Mainland Japanese. The three
Ainu individuals who were within the Mainland Japanese cluster inFigure 1a are located at the rightmost columns of the Ainu
population, and they also showed B20% blue-colored component,
similar to the Mainland Japanese.
As k was increased to 3, the orange-colored component at k ¼ 2
divided into two (orange and magenta). Now, all the CHB individuals
are almost full of magenta ancestry component, and the Mainland
Japanese and JPT individuals also consist of B30% magenta-coloredancestry component. There are two Japanese individuals, one in the
Mainland Japanese and the other in HapMap JPT, who have 470%
magenta component. These two individuals are also outliers in the
PCA analysis shown in Figure 1a. If we consider blue- and magentacolored ancestry components as the Jomon and the Yayoi factors, theintermediate orange-colored component is not easy to comprehend.
It is possible that this does not correspond to a real ancestral
population, but an artificially inferred component corresponding to
the long-term admixture between the Jomon and the Yayoi genetic
components. At k ¼ 4, the five outlier Ainu individuals observed in
the PCA plot (those in red circles in Figures 1a and b) weredifferentiated from the rest of the Ainu, as indicated in the purple
color. The Ryukyuan-specific ancestral component appeared at k ¼ 5.
This is again not easy to interpret, and could be an artificially inferredcomponent.
Generally speaking, the frappe results appear to be consistent with
the PCA analysis in terms of the two patterns: (1) varying amountsof admixture in the Ainu with the Mainland Japanese and (2) the
possible presence of another source population, which contributed to
the genetic structure of the Ainu. A high correlation between PC1
coordinates of Figure 1a and proportions of the blue-colored ancestry
from the frappe analysis (k ¼ 2) shown in Supplementary Figure S4
confirms the pattern (1). The pattern (2) can be supported by a highHistory of Japanese Archipelago populations
T Jinam et al791

correlation between the purple component frequencies for k ¼ 4 andPC2 coordinates in Figure 1a for the Ainu individuals, as shown in
Supplementary Figure S5.
Individual-based analyses merged with HGDP-CEPH and PASNP
data sets
We now move to analyses combined with individuals belonging to the
16 HGDP-CEPH populations. Figure 3a shows the PCA result. The
overall distribution of individuals in this figure indicates an L shape.
Ainu and Yakut individuals are at the two extremes. The Ryukyuan
and the Mainland Japanese populations were located as if they
were pulled by the Ainu population, whereas the remaining East
Asian populations were closer to northern East Asian populations(Yakut, Mongolian and Oroqen). This clear dichotomy with theL-shaped constellation remains when we further added individuals of
the 14-population PASNP data sets (Figure 3b). The Ainu population
is located at the one extreme followed by the Ryukyuan, whereasanother extreme is now the Uyghur population. The Uyghurs are
known to be an admixed population with West Eurasians, so as theYakuts to a lesser extent.38 This fact indicates that the Ainu, and thetwo other populations in the Japanese Archipelago (Ryukyuan and
Mainland Japanese) to a lesser degree, contain genetic components
neither found in the other East Eurasians nor West Eurasians.
Another notable addition in Figure 3b is the Korean, who are locatedFigure 3 PCA plots of the individuals for the three populations in theJapanese Archipelago (Ainu, Ryukyuans and the Mainland Japanese) andother Asian populations. (a) Result with the HGDP-CEPH data set(see Supplementary Table S2 for the list of populations). (b) Result with the
HGDP-CEPH data set and the PASNP data set (see Supplementary Table S2for the list of populations).

between the Mainland Japanese and the Han Chinese in the northernpart of China. This result is consistent with that of Tian et al.50
The result of the frappe analysis for populations corresponding to
Figure 3a is shown in Supplementary Figure S6. When k ¼ 2, thedichotomy pattern is quite similar with that of Figure 2, andadditional East Eurasian populations showed similar blue/orange
frequencies as in the Han Chinese. As k increased to 3, the light-blue
ancestry component, which was almost exclusively found in the Yakutindividuals, diverged from the orange component under k ¼ 2. The
green ancestry component further divided from the orange one at
k ¼ 4, and this was dominant in the Ryukyuan and the Mainland
Japanese. The five ‘outlier’ Ainu individuals in Figure 1 were fully
covered by the violet ancestry component at k ¼ 5. The fraction of thiscomponent was high only in the Ainu. The same situation continues
from k ¼ 2 to k ¼ 6 for the blue ancestry component. This resultclearly indicates a unique position of the Ainu population in East
Asia. Supplementary Figure S7 shows the result of the frappe analysis
for populations corresponding to Figure 3b. The overall pattern issimilar to that of Supplementary Figure S6.
Population-based analysis
Individual-based analyses presented in the previous section deciphered the complex structure of each population, especially for the
Ainu. Populations are still realistic units of modern human evolution,
and we thus constructed phylogenetic trees and networks of
populations.
We first used SNP data of the Ainu, the Ryukyuan, the Mainland
Japanese and the Han Chinese in Beijing (see Table 1), andconstructed an unrooted maximum-likelihood tree (Supplementary
Figure S8). The Ainu and the Ryukyuan populations clustered with
100% bootstrap probability, and this is consistent with the PCA result(Figure 1a). The overall pattern is similar to the tree shown by Omoto
and Saitou16 based on only 25 polymorphic loci of classic genetic
markers, if we equate the Han Chinese in Beijing in the tree ofSupplementary Figure S8 with the Korean in their tree; the branchgoing to the Ainu population is quite long compared with that to the
Ryukyuan, whereas the branch going to the Mainland Japanese had
zero length.
A neighbor-joining tree was then constructed for the 29 humanpopulations in East Asia, using the merged SNP data with the HGDPCEPH and PASNP data sets to see the relationships between the three
populations in the Japanese Archipelago with other worldwide
populations (Figure 4a). Three populations (Japanese, Mainland
Japanese and Ryukyu) listed in Supplementary Table S2 were excluded
to avoid redundancy of populations. The Korean population is now
phylogenetically closest to the human populations in the JapaneseArchipelago, though the bootstrap probability to support the clustering of these four populations was 94% (bootstrap value not shown).
Four populations (Hezhen, Daur, Oroqen, and Mongolians) that
distribute in the northeast Asia as well as the Yakut, the Xibo and the
Uyghur formed one cluster in this tree, whereas three populations(Tu, Naxi and Yi) in the southern China formed one cluster, and thesetwo clusters are phylogenetically closer to the Korean and the JapaneseArchipelago cluster. In terms of genetic distances, however, some Han
Chinese populations (CHB in Beijing and Han-Tw) were closer topopulations in the Japanese Archipelago. These smaller genetic
distances may be contributed to a smaller random genetic driftcaused by large population sizes of these Han populations.
We then selected 14 populations in Figure 4a and constructed aneighbor-joining tree (Figure 4b). Because sample sizes of many
ethnic minorities in China were small, we merged the data for
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History of Japanese Archipelago populations
T Jinam et al792

Figure 4 Phylogenetic trees for the three Japanese populations and other
Asian populations. (a) A neighbor-joining tree for the three Japanese
populations and other Asian populations listed in Supplementary Table S2.
(b) A maximum-likelihood tree for the three Japanese populations with
Northeast Asians (Hezhen, Daur, Oroqen, Mongolian), Koreans, Han Chineseand populations from central China (Tu, Naxi, Yi). All bootstrap values
shown with arrows were obtained from 5000 replications.

Hezhen, Daur, Oroqen and Mongolians as the northeast Asian
population, and three populations in southern China (Tu, Naxi
and Yi) were also merged. The Ainu and the Ryukyuan were clusteredwith 100% bootstrap probability, followed by the Mainland Japanese.
The three populations in the Japanese Archipelago clustered with the
Korean with 100% bootstrap probability. The Ainu population has along branch and is clearly different from the other populations in thisfigure, confirming the unique phylogenetic position of this population in East Asia. The very short, almost nonexistent branch leading
to the Mainland Japanese, as well as its intermediate position between
the Ainu-Ryukyu and the cluster for the remaining populations
suggest that the Mainland Japanese was formed by the result of
admixture between these two ancestral population sources, symbolized as the Jomon and the Yayoi. The northeast Asian population was
phylogenetically closest to the Korean–Japanese Archipelago population cluster, followed by the populations in southern China. The three
Han Chinese populations in Beijing, Taiwan and Shanghai clustered
together with 92% bootstrap probability.
Phylogenetic networks were also constructed for the same genetic
distance matrices used for constructing trees in Figures 4a and b, asshown in Supplementary Figures S9 and S10, respectively. The overall
pattern of Supplementary Figure S9 was similar to that of Figure 4a,
except for the intermediate position of the Yakut, which was not only
close to the Uyghur but also close to the four northeast Asian
populations (Oroqen, Daur, Hezhen and Mongolian). There are twointeresting reticulations in Supplementary Figure S10. Although thesouthern Chinese populations are close to the three Han Chinese
population cluster, they are also close to the northeast Asian
population. The Korean is also located in an intermediate position,
close to the Japanese Archipelago populations, but also phylogenetically close to the Han Chinese.
Journal of Human Genetics

DISCUSSION
Genetic heterogeneity of the Ainu population
The s.d. (8.3 " 10 #3) of allele-sharing distances for the Ainu population is B10 times higher than those (0.75 " 10 #3 and 0.85 " 10 #3)
for the Ryukyuan and the Mainland Japanese, respectively. One
possible factor for this variation may be degradation of DNA qualityof the Ainu individual samples after preservation of almost 30 years.
We thus classified 36 Ainu individuals into two categories(high or low) in terms of Affymetrix Contrast QC (CQC) values.
Supplementary Figure S11 shows the distribution of these twocategories of individuals on the PCA plot shown in Figure 1a. Thereis no clear correlation between these two categories and the locationsof the Ainu individuals. Furthermore, we performed PCA separately
using 13 Ainu individuals who passed the Affymetrix CQC threshold,and the remaining 23 who did not pass the threshold. Supplementary
Figure S13 shows that in both cases, the same scattered pattern wasobserved. We also examined effects of SNP genotyping call heterogeneity among SNP loci. Because the genotyping call rate threshold of
95% was used to produce the PCA plots shown in Figure 1a, weused 90 and 100% call rates and conducted PCA, as shown in
Supplementary Figures S12A and S12B. These three PCA results are
essentially the same. We further retrieved the top 100 SNPs
contributing to PC1 and PC2 in Figure 1a, and a total of 200 SNPs
were obtained. After pruning for linkage disequilbrium, 109 SNP lociwere left. We used the Digi Tag2 method51 for 96 SNP loci randomly
selected from the 109 SNP loci for technical simplicity. The DNA
samples used were 36 Ainu, 33 Ryukyuan and 22 Mainland Japanese.
SNP call rates for Ainu, Ryukyuan and Mainland Japanese were 0.950,
0.985 and 0.999, respectively. Although the call rate for Ainu wassomewhat lower than those for other two populations, theproportions of identical SNP typing with those obtained by using
Affymetrix ver. 6.0 were 99.4%, 99.8% and 99.9% for Ainu, Ryukyuan
and Mainland Japanese, respectively. We thus conclude that SNP
genotypes estimated by using Affymetrix ver. 6.0 were almost identicalwith those estimated by using the Digi Tag2 method for all the three
populations. These results indicate that the DNA degradation was not
the reason for the variation among the Ainu individuals.
Interestingly, the average heterozygosity (0.220) of the Ainu
population was the least among the seven populations listed inTable 1, whereas those of the other four East Asian populations weremore or less the same (B0.24). This feature is not consistent with ahigh degree of variation on allele-sharing distances for the Ainu
population, if it is a panmictic and isolated population with no geneflow with the surrounding populations. As we showed in this study,
however, the Ainu population seems to have experienced gene flow
with two different populations, the Mainland Japanese and the yet
unknown population in the north. If gene flow happens to occur, theheterogyzosity of admixed individuals should be in the middle ofthose of the two original populations. To see whether this is the case,the 28 Ainu individuals (three individuals within the Mainland
Japanese and five outliers in the red circle in Figure 1a were notincluded) were divided into two; more admixed and less admixed.
When the Ainu individuals were separated according to the normalized PC1 coordinate 0.75 of Supplementary Figure S4, the average
heterozygosity of the more admixed 13 Ainu individuals was 0.223,
whereas that for the less admixed 15 Ainu individuals was 0.201.
When Ainu individuals were separated according to the normalized
PC2 coordinate 0.3 of Supplementary Figure S5, average heterozygosities of the less admixed (18), more admixed (10) and outlier (5)Ainu individuals became 0.213, 0.201 and 0.163, respectively. In bothcases, the putative admixed Ainu individuals showed the intermediate

History of Japanese Archipelago populations
T Jinam et al793

heterozygosities, and this further confirms that the Ainu population
has experienced admixture with the two surrounding populations. In
fact, we came to know through discussion with the Ainu people that
there were some Mainlaind Japanese individuals who married Ainu
people in Biratori Town when blood collection was conducted. These
genetically non-Ainu people might have been included in the ‘Ainu’samples we used. Another information from the Ainu representativesof the Biratori Town was that some Sakhalin Ainu people migrated tothat town after the World War II. There is a possibility that the five
outliers in the red circle in Figure 1a are Sakhalin Ainu people.
Another possible reason for the low average heterozygosity in the
Ainu compared with other populations is ascertainment bias in which
SNPs included in the Affymetrix 6.0 genechip were chosen based on
their polymorphism in only a few ascertained populations.
Possible mother population for the alternative admixture eventswith the Ainu population
Unlike admixture with the Mainland Japanese, it is difficult toascertain the other potential source of admixture in the Ainu without
a proper source population. Previous studies did support the ideaof contact with Northern populations, which may have contributed
to the genetic diversity in the Ainu. Archeological data point to an
introduction of a distinct culture, which was quite different from theSatsumon culture, by the Okhotsk people into Hokkaido during the7th–10th centuries.52 The cultural contact with these northernpeoples seemed to continue until recently. Genetic studies using
mitochondrial DNA24 and human leukocyte antigen loci32 support
this idea by showing close affinities between the Ainu and the Nivkhi
who live in the Sakhalin Island and the Amur River region. It would
therefore be interesting to collect samples from populations from thatarea in future studies to have a clearer view of the relationships
between the Ainu and Northeast Asian populations.
Perspectives
We still have no clear clues on homelands of the Jomon and the Yayoi
people, who constituted the two major genetic components ofthe modern human populations in the Japanese Archipelago.
It should be noted that Omoto53 conducted a pioneering study on
the phylogenetic relationship of the Ainu population consideringvarious degrees of admixture. When a 60% admixture with theMainland Japanese was assumed for the modern Ainu population, theancestral Ainu population was clustered with Sahulian (Papuan andAustralian).53 This sort of simulations based on the real data isneeded.
We should also integrate evidences from various research fields
such as archeology and morphology. There is a long history of
craniofacial studies on Jomon, Yayoi and historical populations in theJapanese Archipelago.1,3,54 Although metric characters were mostly
used, studies on nonmetric characters are promising,55–58 for they are
expected to be more under genetic controls than metric ones.
Hanihara’s analysis58 suggested the existence of gene flows between
the Hokkaido Jomon people and the Okhotsk people. An applicationof the computed tomography images for measuring nonmetric cranial
variations in living humans was already started,59 and genome-wide
association studies of morphological characters are waiting to be
initiated. In fact, the genetic background of shovel-shaped incisors
found frequently in East Asian individuals was recently deciphered,60
and the nonsynonymous polymorphism on the EDAR gene showed
the greatest difference in SNP genotype frequencies between the
Mainland Japanese and Ryukyuan clusters,36 followed by anothernonsynonymous polymorphism on the ABCC11 gene that is

responsible for the earwax phenotypic differences.61 Similar
statistical analyses will be conducted in the near future using
the genome-wide SNP data produced by this study. Genomic
DNA polymorphisms are not restricted to SNPs, but also include
insertion–deletion type polymorphisms such as microsatellite
polymorphisms (for example, Li et al.62).
Ancient DNA data are very important for understanding theevolutionary history of the present-day organisms. Because oftechnical difficulties, most of the ancient DNA data available
regarding the origin of peoples in the Japanese Archipelago aremitochondrial DNAs,63–71 with exceptions of the ABCC11 gene72 andthe ABO blood group gene.73 If the genome-wide nuclear DNA
polymorphism data can be obtained for ancient DNA samples foundfrom the Japanese Archipelago, we will be able to have a much wider
scope on the history of peoples in this Archipelago.
Conclusion
We demonstrated that the Ainu are genetically closer to the Ryukyuan
than they are to the Mainland Japanese in this study. The close
association between the Ainu and the Ryukyuan, despite their current
geographical locations, which is at the two opposing ends of theJapanese Archipelago, may be interpreted as having a shared common
ancestry probably dating back to the Jomon period. The population
tree in Figure 4b also places the Mainland Japanese in an intermediate
position between the Ainu/Ryukyuan and the Continental population
clusters. This observation, coupled with the very short external branchin the Mainland Japanese, strongly suggests that they are the result of
admixture between the two genetically distinct ancestors, namely the
Jomon people and the Yayoi ancestors. Our analysis revealed a great
genetic variation within the individuals of the Ainu group, brought
about by admixture with the mainland Japanese and possibly anotherpopulation from Northeast Asia. Figure 5 depicts a plausible timecourse of the human populations in the three regions of the JapaneseArchipelago based on the findings of this study, though many featuresare still speculative. In conclusion, our results support the more

Figure 5 A scenario of the evolutionary history of the human populations in
the three regions of the Japanese Archipelago based on the results of thisstudy and archeological evidences.52 The Northern, the Central and theSouthern populations, corresponding to the Ainu, the Mainland Japaneseand the Ryukyuan in the present day, were assumed to diverge
simultaneously, sometime during the Jomon period, although we do nothave a precise time estimate. The admixture of the indigenous Jomon
people and Yayoi migrants was assumed to occur sometime after the Yayoi
period started 3000 years ago.76 Vertical arrows designate gene flows inhistorical times, but their timings and frequencies are rather speculative.
Journal of Human Genetics

History of Japanese Archipelago populations
T Jinam et al794

than 100-year-old hypothesis of von Baelz4 that the Ainu and the
Ryukyuan have shared genetic ancestry, and the admixture hypothesis(for example, Torii74 and Kanaseki75) that the mainland Japanese are
the result of admixture between the ancestral Yayoi people and theindigenous Jomon people.
The SNP genotype data determined in this study are available uponrequests to corresponding authors, under the conditions of collaboration with us and with an appropriate approval of human genomic
DNA research ethics committee of institutions to which researchers
involved in the data analyses belong.
ACKNOWLEDGEMENTS
We thank all people who donated their DNAs to this study, especially for the
Ainu and the Ryukyuan people. We appreciate the effort of Department of
Promotion of Measures for Ainu, Biratori Town, for arrangement of themeeting with Ainu representatives. We also thank Ms. Yuko Ogasawara,
Natsumi Baba, Rieko Hayashi and Minae Kawashima for technical assistance.
We would also like to acknowledge the HUGO Pan-Asian SNP Consortium forproviding the SNP genotype data. This work was supported in part by aStrategic Research Project to Naruya Saitou from SOKENDAI, and Grant-inAids for Scientific Research (A) (23247040), (A) (22247036) and (B) (21370108)
to Hirofumi Matsumura (Katsushi Tokunaga and Naruya Saitou are coinvestigators), Shoji Kawamura and Hiroki Oota, respectively, from Japan
Society for the Promotion of Science.

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