Hum Genet (2006) 118: 695–707
DOI 10.1007/s00439-005-0106-9

O RI GI N AL IN V ES T IG A T IO N

Shi-Lin Li Æ Toshimichi Yamamoto
Takashi Yoshimoto Æ Rieko Uchihi Æ Masaki Mizutani
Yukihide Kurimoto Æ Katsushi Tokunaga Æ Feng Jin
Yoshinao Katsumata Æ Naruya Saitou

Phylogenetic relationship of the populations within and around Japan
using 105 short tandem repeat polymorphic loci

Received: 1 July 2005 / Accepted: 1 November 2005 / Published online: 29 November 2005
 Springer-Verlag 2005

Abstract We have analyzed 105 autosomal polymorphic
short tandem repeat (STR) loci for nine East and Southeastern Asian populations (two Japanese, ﬁve Han Chinese, Thai, and Burmese populations) and a Caucasian
population using a multiplex PCR typing system. All the
STR loci are genomewide tetranucleotide repeat markersof which the total number of observed alleles and theobserved heterozygosity were 756 and 0.743, respectively,
for Japanese populations. Phylogenetic analysis for theseallele frequency data suggested that the Japanese populations are more closely related with southern Chinese
populations than central and/or northern ones.
STRUCTURE program analysis revealed the almost
clearly divided and accountable population structure at
K=2–6, that the two Japanese populations always
formed one group separated from the other populations
and never belong to diﬀerent groups at K‡3. Furthermore, our new allele frequency data for 91 loci wereanalyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian
populations with large population size (six Han Chinese,
three Japanese, two Southeast Asia) formed one distinct
S.-L. Li Æ T. Yamamoto (&) Æ T. Yoshimoto Æ R. Uchihi
M. Mizutani Æ Y. Kurimoto Æ Y. Katsumata
Department of Legal Medicine and Bioethics, Graduate Schoolof Medicine, Nagoya University, Nagoya, Japan
E-mail: yamachan@med.nagoya-u.ac.jp
Fax: +81-52-7442121
K. Tokunaga
Department of Human Genetics, Graduate School of Medicine,
The University of Tokyo, 113-0033 Tokyo, Japan
N. Saitou
Division of Population Genetics, National Institute of Genetics,
411-8540 Mishima, JapanF. JinChinese Academy of Sciences, Institute of Genetics
and Developmental Biology, 100101 Beijing, China

cluster and are closer to each other than other ethnic
minorities in east and Southeast Asia. This pattern may
be the caviar of comparing populations with greatly
diﬀering population sizes when STR loci were analyzed.
Keywords Short tandem repeat Æ Population genetics Æ
East Asian Æ Japanese Æ Phylogenetic tree Æ
Polymorphism

Introduction
The Japanese archipelago was geographically dissociated from Asian continent around 12,000 years ago after
the last glacial period (Aikens and Higuchi 1982). In theprocess of formation of the modern Japanese, there havebeen many migration events into Japan from continental
Asia. There are many studies comparing mitochondrial
DNA polymorphisms of Japanese and surrounding
populations(e.g., Horai et al. 1996; Tanaka et al. 2004;
Tajima et al. 2004). However, maternally inherited
mitochondrial DNA has diﬀerent characteristics in thecontext of its sex-speciﬁc modes of transmission compared to nuclear DNA. This restriction also applies toY-chromosomes that are transmitted paternally (e.g.,
Hammer and Horai 1995).
Genotyping technologies have remarkably improved
for many types of DNA markers recently. Especially, thenumerous short tandem repeats (STRs), also known as
microsatellites, have been used in phylogenetic analysesof extant human populations (e.g., Bowcock et al. 1994;
Brinkmann et al. 1996; Perez-Lezaun et al. 1997; Chu
et al. 1998; Rosenberg et al. 2002; Ayub et al. 2003;
Zhivotovsky et al. 2003). These STR loci are clearly
useful for studying the genetic relationships of closely
related populations. However, there has been little study
on the phylogenetic relationship around the Japanese
population using these genome-wide STR DNA markers
on autosomal chromosomes to date.

696

Takezaki and Nei (1996), by using computer simulation with microsatellite DNA loci, showed that
Cavalli-Sforza and Edwards’ (1967) chord distance DC
and Nei et al.’s (1983) DA distance generally showed thehigher correct topology (PC) values than other distancemeasures in both the inﬁnite-allele model (IAM) and the
stepwise mutation model (SMM), whether the bottleneck eﬀect exists or not. In addition, in case of SMM
such as microsatellites, they showed that high PC values(more than 80%) are obtained under the condition ofsmall branch length, small sample size (30), and a large
number of loci (100) with high heterozigosity (0.8).
Therefore, in the present study, we genotyped STR lociunder conditions as close to these as possible, andexamined the genetic relationship among human populations in east and Southeast Asia. Additionally, we
examined the genetic relationship of the worldwide
human populations by adding our data into the allele
frequency data available at a web site (http://
www.cmb.usc.edu/people/noahr//diversity.html#data).
Moreover, a novel computer program, STRUCTURE,
was recently developed for an extensive analysis of
population substructure and to identify population
outliers (Pritchard et al. 2000). We applied this program
to infer the population structure among human populations in east and Southeast Asia using the genotype
data.

Materials and methods
DNA samples
Blood samples were collected with informed consent
from Japanese living in the middle part of Honshu, theFig. 1 Geographical location of
the nine Asian populations
analyzed in the present study

main island of Japan (Nagoya), Han Chinese living in
ﬁve provinces (Shaanxi, Hunan, Guangdong, Fujian,and Beijing), Thai living in Bangkok, and Burmese living
in Yangon (see Fig. 1). The DNA was extracted fromblood samples by the usual organic extraction method orusing some commercially available kits. Japanese DNA
samples in Okinawa were previously collected forstudying HLA alleles and haplotypes (Hatta et al. 1999).
The DNA samples of Caucasian living in UK werekindly provided by Dr. Yuri E. Dubrova at the
University of Leicester. Thirty-two DNA samples ofeach population were utilized for STR genotyping. In
the DNA samples of these Han Chinese populations, apart of those of Guangdong (Huizhou region) andFujian (Putian region), and those of Shaanxi (Xi’anregion) and Hunan (Changsha region) were used for thestudies reported previously by Roubinet et al. (2004) andOota et al. (2002), respectively.
DNA ampliﬁcation and genotyping
One hundred and ﬁve tetranucleotide STR markers on
autosomal chromosome were selected from 168 STR lociin the screening set 8A (Research Genetics, Huntsville,
AL, USA) by removing tri- and dinucleotide STRs, andSTRs on X/Y-chromosome, as shown in Table 1, with
their common motifs of repeat unit. Each one of theirprimer sets is labeled with any one of three diﬀerent
ﬂuorescent-colored dyes (FAM, TET, HEX). The PCR
ampliﬁcation was performed using 48 sets of temporary
multiplex, and the PCR products were multi-loaded with
15 panels by gel electrophoresis with a ABI PRISM 377
DNA Sequencer (PE Applied Biosystems, Foster city,
CA, USA) based on a method described previously

697
Table 1 Marker designation, type of repeat unit, number of alleles, GST, and heterozygosity values (Ht) for the 105 STR markers used in
the present study
RepeatMarker
Chromosome 1
D1S1612
D1S1597
D1S552
D1S2134
D1S1665
D1S534
D1S1679
D1S518
D1S1660
D1S549Chromosome 2
D2S2976
D2S1400
D2S1394
D2S2972
D2S1328
D2S1399
D2S1391
D2S1384
Chromosome 3
D3S2387
D3S4545
D3S2432
D3S1766
D3S2460
D3S2427
Chromosome 4
D4S2366
D4S2639
D4S1627
D4S1625
D4S1652
Chromosome 5
D5S2845
D5S1470
D5S2500
D5S1505
D5S820
D5S1456
Chromosome 6
D6S1053
D6S1056
GATA184A08
D6S1277
Chromosome 7
D7S3056
D7S3051
D7S2846
D7S3046
D7S1842
D7S1823
Chromosome 8
D8S1106
D8S1477
D8S1113
D8S1132
D8S373Chromosome 9
D9S2169
D9S925
D9S1118
D9S9349 Asian populations

9 Asian + English populations

Unit

Alleles

GST

HtAlleles

GST

Ht

GGAA
GATA
GGAT
GATA
GATA
GATA
GGAA
GATA
GATA
GATA

10
8
8
13
11
15
9
9
8
10

0.030
0.019
0.025
0.028
0.019
0.022
0.013
0.015
0.019
0.018

0.798
0.706
0.685
0.765
0.722
0.791
0.839
0.780
0.802
0.752

12
8
8
13
11
15
9
9
8
10

0.040
0.020
0.027
0.050
0.020
0.028
0.013
0.025
0.023
0.018

0.812
0.710
0.692
0.785
0.713
0.804
0.839
0.794
0.805
0.755

GATA
GGAA
GATA
GATA
GATA
GGAA
GATA
GATA

15
6
8
12
8
14
8
8

0.091
0.030
0.023
0.018
0.018
0.011
0.018
0.031

0.617
0.515
0.727
0.748
0.595
0.869
0.662
0.767

16
7
8
12
9
14
8
8

*

0.101
0.037
0.021
0.019
*
0.072
0.015
0.022
0.042

0.658
0.534
0.724
0.741
0.649
0.866
0.677
0.779

GATA
GATA
GATA
GATA
GATA
GATA

19
13
10
9
9
18

0.015
0.017
0.017
0.024
0.015
0.022

0.863
0.777
0.791
0.725
0.745
0.888

20
15
12
9
9
19

0.018
0.032
0.019
0.024
0.016
0.025

0.866
0.793
0.792
0.732
0.748
0.889

GATA
GATA
GATA
GATA
GATA

8
10
8
8
7

0.019
0.013
0.021
0.020
0.013

0.769
0.814
0.781
0.721
0.586

8
10
8
8
7

0.030
0.032
0.041
0.022
0.036

0.779
0.831
0.795
0.730
0.612

GATA
GATA
GATA
GATA
GATA
GATA

9
10
11
8
8
6

0.027
0.015
0.018
0.018
0.021
0.022

0.772
0.797
0.780
0.818
0.771
0.778

9
10
11
8
8
6

0.026
0.017
0.018
0.021
0.024
0.023

0.770
0.802
0.785
0.821
0.777
0.783

GATA
GATA
GATA
GATA

8
10
11
9

0.016
0.014
0.014
0.019

0.796
0.843
0.839
0.726

8
10
12
9

0.015
0.017
0.017
0.021

0.796
0.844
0.835
0.732

GATA
GATA
GATA
GATA
GGAA
GATA

8
11
6
12
10
11

0.023
0.013
0.027
0.027
0.017
0.018

0.742
0.788
0.700
0.844
0.820
0.821

8
11
6
12
10
11

0.024
0.017
0.025
0.028
0.022
0.022

0.734
0.790
0.702
0.846
0.828
0.828

GATA
GGAA
GGAA
GATA
GATA

8
11
8
10
10

0.017
0.020
0.036
0.017
0.027

0.653
0.800
0.701
0.851
0.847

9
14
8
10
10

0.023
0.028
0.035
0.016
0.028

0.663
0.810
0.710
0.851
0.845

GATA
GATA
GATA
GATA

7
10
11
10

0.015
0.012
*
0.055
0.016

0.675
0.773
0.850
0.786

7
11
11
10

0.014
0.014
*
0.053
0.017

0.679
0.777
0.848
0.788

*

698
Table 1 (Contd.)
RepeatMarker
Chromosome 10
D10S1435
D10S1426
D10S1432
D10S677
D10S1239
D10S1213
D10S1248
Chromosome 11
D11S1984
D11S1999
D11S2000
D11S4464
Chromosome 12
D12S372
D12S391
D12S375
D12S1064
PAH
D12S395
Chromosome 13
D13S894
D13S317
D13S796
Chromosome 14
D14S1280
D14S306
D14S617
D15S822Chromosome 15
D15S643
D15S657
D15S642
Chromosome 16
D16S764
D16S753
D16S3253
D16S2624
D16S539
Chromosome 17
D17S1308
D17S1303
D17S1293
D17S1290
D17S1301
Chromosome 18
D18S877
D18S1364
Chromosome 19
D19S591
D19S586
D19S433
D19S246
Chromosome 20
D20S482
D20S470
D20S481
D20S480
Chromosome 21
D21S1432
D21S2055
Chromosome 22
D22S689
D22S683*

9 Asian populations

9 Asian + English populations

Unit

Alleles

GST

HtAlleles

GST

Ht

GATA
GATA
GATA
GGAA
GATA
GGAA
GGAA

9
7
8
9
8
12
9

0.016
0.026
0.022
0.013
0.014
0.023
0.016

0.748
0.733
0.706
0.837
0.687
0.723
0.759

9
7
8
10
8
12
9

0.015
0.028
0.021
0.016
0.023
0.024
0.018

0.746
0.738
0.709
0.840
0.700
0.734
0.764

GGAA
GATA
GATA
GATA

11
10
22
8

0.017
0.015
0.022
0.016

0.838
0.716
0.892
0.743

12
10
22
9

0.034
0.033
0.023
0.017

0.848
0.743
0.896
0.745

GATA
GATA
GATA
GATA
TCTA
GATA

7
14
7
9
9
10

0.013
0.012
0.023
0.016
0.029
0.019

0.730
0.846
0.752
0.768
0.745
0.668

7
14
7
9
9
10

0.013
0.017
0.028
0.017
0.028
0.030

0.728
0.853
0.757
0.772
0.750
0.687

GATA
GATA
GATA

7
8
11

0.021
0.017
0.012

0.623
0.801
0.808

8
8
11

0.024
0.027
0.014

0.637
0.807
0.808

GATA
GATA
GGAA
GATA

7
9
10
19

0.013
0.012
0.025
0.018

0.648
0.770
0.763
0.838

7
9
10
19

0.011
0.016
0.027
0.031

0.650
0.772
0.771
0.851

GATA
GATA
GATA

14
8
12

0.011
0.013
0.016

0.835
0.830
0.755

14
8
13

0.012
0.016
0.030

0.837
0.829
0.774

GATA
GGAA
GATA
GATA
GATA

8
10
9
8
7

*

0.111
0.015
0.016
0.023
0.038

0.704
0.762
0.741
0.706
0.789

8
10
9
8
7

0.102
0.021
0.022
0.030
0.039

0.700
0.772
0.745
0.715
0.789

GTAT
GATA
GGAA
GATA
GATA

6
9
11
16
7

0.030
0.014
0.014
0.012
0.013

0.577
0.724
0.845
0.833
0.697

7
9
11
16
7

0.037
0.020
0.016
0.016
0.013

0.596
0.723
0.848
0.835
0.698

GATA
GATA

7
10

0.037
0.019

0.697
0.843

7
10

0.035
0.022

0.696
0.840

GATA
GATA
GGAA
GATA

7
8
13
13

0.020
0.018
0.034
0.016

0.743
0.657
0.814
0.788

7
8
13
13

0.027
0.016
0.033
0.027

0.752
0.661
0.810
0.803

GATA
GGAA
GATA
GATA

10
15
9
10

0.023
0.021
0.022
0.015

0.716
0.872
0.723
0.808

11
16
9
10

0.022
0.024
0.028
0.015

0.720
0.872
0.741
0.806

GATA
GATA

9
17

0.013
0.022

0.741
0.885

9
18

0.016
0.024

0.740
0.888

GATA
GATA

9
23

0.027
0.019

0.798
0.838

9
24

0.026
0.021

0.798
0.848

*

GST>0.05
The numbers and both values were calculated separately without/with the Caucasian population. The markers are arranged according totheir location on the chromosomes

699(Mizutani et al. 2001) with minor modiﬁcation. Fragment sizes for each STR loci were determined on thebasis of known internal lane size standards using software GeneScan Analysis (Version 3.1), and their genotypes were determined by comparing the size data
analyzed by Mizutani et al. (2001). These 105 STR
markers were widely distributed in all autosomes as listed in Table 1; varying 10 loci on chr. 1–2 loci on chr. 18,
21, and 22.
Statistical analyses
Tests for Hardy–Weinberg equilibrium (HWE) were
performed using a homozygosity test (Weir 1992), a
likelihood ratio test (Chakraborty et al. 1991), and an
exact test (Guo and Thompson 1992). The observed Ht
and the unbiased estimates of expected Ht were calculated according to Edwards et al. (1992). The GST valuesand Ht were estimated using DISPAN (downloaded
from http://mep.bio.psu/downlods/dispan.zip).
The genetic distances were calculated from the allele
frequency data at all the 105 STR loci by DA (Nei et al.
1983) distance with the NJBAFD (downloaded fromhttp://iubio.bio.indiana.edu/soft/molbio/evolve/njbafd/),and DC (Cavalli-Sforza and Edwards 1967) and hW (FST)
distance (Reynold et al. 1983) with the PHYLIP 3.5c
(Felsenstein 1995), and then phylogenetic trees wereconstructed by using the Neighbor-Joining (NJ) method(Saitou and Nei 1987) using the MEGA Version 2.1(Kumar et al. 2001). Bootstrap values were obtained
based on 1,000 replications. A phylogenetic tree by using
the DA distance and NJ method with the NJBAFD was
also constructed from the allele frequency data at 91
STR loci, which are in common among 105 loci in thepresent study, in total 61 worldwide populations byadding our ten populations into 52 world populations
obtained from the literature reported previously(Rosenberg et al. 2002). However, when the allele frequency data were downloaded from http://www.cmb.
usc.edu/people/noahr//diversity.html, those for 51 populations were obtained because the data for Han
population were calculated by combining those in US
Han and northern China Han populations.
Since the data sizes were slightly diﬀerent between
both the data sets, they were matched between both
databases without contradiction by comparing the allele frequency distribution at each locus in Japaneseand Chinese in our database to those in Japanese andHan populations in the database of Rosenberg et al.
(2002).
The MDS analysis based on a 10·10 matrix of pairwise DA distance values calculated above and FST(Latter 1972) calculated with the MICROSAT Version
2.0 software at 105 STR loci in our ten populations was
performed using the SPSS 12.0 software package (SPSS
Inc., Chicago, IL, USA). Similarly, the MDS analyses
on 61·61 DA and FST distance matrix at 91 STR loci inthe 61 worldwide populations was performed.

To conduct an extensive analysis of population substructure and to identify population clusters, we also
applied the computer program STRUCTURE 2.0(Pritchard et al. 2000) which examines the populations
at the level of the individual on the basis of genotype
data at 105 loci. The program was run for 20,000 iterations after a burn in of length 20,000 with an admixture
model of correlated allele frequencies. Models in which
there are K population (where K may be the unknown
number clustering those populations) assigned from 2 to
7 in this study are assumed, which are estimated on thebasis of their individual genotypes in each population.
To more thoroughly display results produced by thegenetic clustering program STRUCTURE, we used
program DISTRUCT (downloaded from http://www-hto.
usc.edu/»noahr/distruct.html) to make detailed graphical ﬁgure, having used STRUCTURE to generate thepopulation Q-matrix, which was created by averaging
membership coeﬃcients of each cluster across individuals for each population.
Like the phylogenetic tree analysis mentioned above,
the genotype data at the 91 STR loci in our ten populations were combined with those in the 18 East Asia
populations and Uygur population out of the Central/
South Asia populations (downloaded from http://
www.cmb.usc.edu/people/noahr//diversity.html)(Rosenberg et al. 2002) by matching the size data, and
STRUCTURE and DISTRUCT analyses were also
performed in the total 29 populations.

Results
We genotyped all these 105 tetranucleotide STR loci as
described previously (Mizutani et al. 2001). A total of
320 individuals were typed from the following ten human populations: two Japanese, ﬁve Han Chinese, one
Burmese, one Thai, and one English population. The
number of alleles at each STR loci observed in these ten
populations was counted as shown in Table 1, and thetotal number of alleles and number of unique allelesobserved in each population and average heterozygosities (±SE) calculated in each population are also shownin Table 2.
Deviation from the HWE was checked using threekinds of statistical tests: homozygosity (Homo) test,
likelihood ratio (LR) test, and exact (Ex) test. The
numbers of loci at which the allele frequency distributions were signiﬁcantly deviated from HWE (P<0.05)
with the three kinds of tests are summarized in Table 3.
Only two loci at which the distributions were signiﬁcantly deviated from HWE (P<0.05) with all the threetests were observed in more than one population:
D2S1400 in the Okinawa and Bangkok populations, andD14S617 in the Shaanxi and Hunan populations. The
mean of the total number of loci signiﬁcantly deviatedfrom HWE was 25.5 loci (8.1%).
These values were comparatively low even though
the number (64) of chromosomes analyzed in each

700
Table 2 Total number ofalleles, number of uniquealleles, and average
heterozygosities (±SE) for theten human populations

Values in parentheses indicate
percentage of unique alleles ineach population

Population
Japan
NagoyaOkinawa
China
Beijing
Shaanxi
Hunan
FujianGuangdongSoutheast Asia
Bangkok
YangonEuropeEngland
10 populations

Total alleles

Unique alleles

Average heterozygosity (±SE)

750
763

12 (1.60%)
17 (2.23%)

0.7698±0.0082
0.7627±0.0082

765
760
747
751
766

16 (2.09%)
6 (0.79%)
8 (1.07%)16 (2.13%)
6 (0.78%)0.7800±0.0071
0.7667±0.0078
0.7706±0.0078
0.7648±0.0093
0.7637±0.0098

761
787

18 (2.37%)
20 (2.54%)

0.7774±0.0081
0.7774±0.0081

795
1084

27 (3.40%)

0.7988±0.0059
0.7732

Table 3 The number of loci observed as signiﬁcant deviations from Hardy-Weinberg equilibrium (HWE) (P<0.05) with three tests at the
105 STR loci in the ten populations in the present study
Test

Japanese
NagoyaChineseOkinawa

Beijing

Southeast Asian
Shaanxi

Hunan

FujianGuangdong Bangkok

H only
1
5
5
2
3
6
4
L only
1
4
1
1
1
5
0
E only
2
0
0
2
1
0
0
H and L
0
0
0
0
0
0
2
H and E
0
1
0
1
2
2
1
L and E
4
1
1
8
4
1
5
H and L and E 2
3
2
4
7
1
3
H
3
9
7
7
12
9
10
L
7
8
4
13
12
7
10
E
8
5
3
15
14
4
9
H or L or E
18 (5.7%) 22 (7.0%) 14 (4.4%) 35 (11.1%) 38 (12.1%) 20 (6.3%) 29 (9.2%)

YangonCaucasianEngland

3
3
6
2
2
0
3
0
0
1
0
0
1
1
2
5
3
2
4
51
9
9
9
12
10
3
13
9
5
34 (10.8%) 28 (8.9%) 17 (5.4%)

H Homozigosity test, L Likelihood ratio test, E Exact testValues in parentheses indicate percentage of total loci signiﬁcantly deviated from
HWE in each population

population was relatively small compared with thenumber of alleles (locus mean=10.3). These ﬁndings
indicate that there was no contingent event, sampling
error, or population substructure. Accordingly, it wasconsidered that these allele frequency data would be
reliable and the whole data were used for the following
analyses.
For statistical properties for the 105 STR loci analyzed in the present study, their GST as a measure ofallelic diversity and Ht for each locus were calculated asshown in Table 1. The GST value averaged over all lociwas 0.0254 in the ten populations, and 0.0209 in the nine
Asian populations, close to the values reported previously in a study on northern Pakistan populations (GST
values: 0.03; Mansoor et al. 2004), but about six times
less than the values on worldwide populations (0.15;
Ayub et al. 2003) using microsatellite markers. In comparison between the nine and the ten populations, GST
values of the latter were slightly higher than those of theformer at almost all the loci (0.004 on the average), but
extremely high at only one locus (0.054 higher;
D2S1328), which indicates the extremely diﬀerent
allele frequency distribution between East Asians andCaucasians at the locus. The 105 STR loci showed signiﬁcantly higher heterozygosities in the English population than those in all the East Asian populations. The
Southeast Asian populations had a higher level of variation than the Japanese and Chinese populations.
A phylogenetic tree, based on DA distance using the
NJ method, provides strong evidence for the closest
relationship among the two regional Japanese populations (bootstrap: 100%), and also for closer relationshipbetween the Japanese and Southern Han Chinese populations (Fujian and Guangdong) (bootstrap: 84%) asshown in Fig. 2. The Beijing population located in
Northern China is clustered with the two central Han
Chinese including Hunan and Shaanxi populations. The
branching pattern in which the Southern Chinese andJapanese populations were in diﬀerent clusters from theNorthern and Central Chinese populations was supported by the somewhat high bootstrap values of 67%,
suggesting that authenticity of this pattern might beconsidered. The Southeast Asian populations were notclustered with each other, while the Thai population wasclose to the Chinese and Japanese populations with highbootstrap values of 98%.

701

Fujian93

Guangdong84

Okinawa
67

100

NagoyaShaanxi

98

Hunan

36

Beijing40

Bangkok
YangonCaucasian
0.02

Fig. 2 A Neighbor-joining (NJ) tree showing the relationships often human populations examined in the present study on the basis
of DA distances calculated from the allele frequencies at 105
tetranucleotide STR loci. The scale for the distance is shown
bottom left. Bootstrap values are provided at each fork of branches
as italic numbers

We also constructed two other NJ trees using diﬀerent genetic distances, DC and FST. These phylogenetic
trees showed the same topology as that on the basis of
DA distance at all with slightly diﬀerent branch lengths
between each population (trees not shown), which suggested that the phylogenetic relationship among these
ethnic and/or regional populations is authentic.
Multidimensional scaling analysis (MDS) of pairwise
DA distance values revealed groups of genetically related
populations (Fig. 3), same as the phylogenetic tree asmention above. One of the most characteristic ﬁndingsof the present analysis was that the Japanese groups had
Fig. 3 Multidimensional
scaling (MDS) plot of ten
human populations analyzed in
the present study, based on DA
genetic distances calculated
from 105 autosomal
tetranucleotide STR locisomewhat closer genetic aﬃnities with the southernChinese populations than northern Chinese populations,and another was that the distribution was slightly different from their geographical relationship. The low‘‘stress’’ value (0.23) of the MDS plot indicated a goodﬁt between the two-dimensional graph and the original
distance matrix. The MDS of pairwise FST distanceshowed two groups, the Japanese populations and theChinese and Southeast Asian populations. The distance
between the former group and the latter group was almost equal to that between the former group and theEnglish, and that between the latter and the English, andthe shape linking the two groups and English was analmost regular triangle despite the lower stress value(0.16) (ﬁgure not shown).
K values (number of ‘‘populations’’) were assigned
from 2 to 6 for STRUCTURE and DISTRUCT programs in Fig. 4a. At K=2, the two clusters, namely
English and Asians, can be clearly seen. At K=3,
however, the two Japanese populations were primarily
separated from the other Asian populations. At K=4,
the Beijing population is separated from other nonJapanese Asian populations (yellow color), but this‘‘population’’ component also slightly appeared in other
Asian populations.
The new ‘‘population’’ (light blue color) coming intoview at K=5 occupied the major portion in Central
Chinese populations (Hunan and Shaanxi), and distributed at some degrees in Southern Chinese andSoutheast Asian populations. At K=6, Southern Chinese(Guangdong and Fujian) was newly occupied by new‘‘population’’ (purple color), and it also constituted alittle part of the other Asian populations. Interestingly,
the ‘‘population’’ (green color) dominated in English

702

Fig. 4 Population sub-structures of (a) the ten populations in thisstudy and of (b) the combined 29 populations with Rosenberg
et al.’s 19 populations are estimated by genotype data of 105 and 91
tetranucleotide STR markers, respectively, using the DISTRUCT
program with the assistance of the STRUCTURE program. Each

population is separated by a vertical line, which is sectioned into K
colored segments that represent the proportion of membership ofeach pre-deﬁned population in K clusters. The populations arelabeled below the ﬁgure, with their regional aﬃliations above the
ﬁgure

703
10
18

63

Daur(As5)
Yakut(As15)

Oroqen(As4)N
Xibo(As8)
Henzhe(As7)She(As12)
Dai(As10)
29
Cambodians(As16)5
Lahu(As11)
28
Tujia(As1)S
Mongola(As6)17
Miaozu(As3)22
Yizu(As2)44
5 29
Naxi(As13)
Tu(As14)
72
Yangon(SEA2)
49
Bangkok(SEA1)
45 3
22
Beijing(HC3)
Shaanxi(HC1)
Hunan(HC2)94
89Guangdong(HC5)35
Fujian(HC4)
Han(As17)
60
76
Japanese(As18)
73
Nagoya(J2)98
Okwaina(J1)74
NewGuinea(Oc1)
Melanesian(Oc2)100
85
Pima(Am1)
Maya(Am2)
100
Colombia(Am3)82
40
Karitiana(Am4)96
48
Uygur(As9)100
Hazara(P3)Enndgla(EU1)
Burusho(P8)Pathan(P6)63
54
Balochi(P2)43
72Briahu(P1)40
Makrani(P4)37Sindhi(P5)81Palestinian(ME3)62
Bedouin(ME1)
37 50
Druze(ME2)
Mozabite(Af7)
Adygei(EU8)
43
Russians(EU9)21
78
Tuscan(EU6)
Kalash(P7)3459Sardinian(EU4)22
Bergamo(EU5)
French(EU2)26
Orcadians(EU7)23Basques(EU3)62
58
Sa n (Af5)75
Mb uti-Pymies (Af2)Biaka Pymies(Af1)71
Yoruba(Af4)100
Bantu(Af6)95
Mandenka(Af3)
31
35
8

I

II

III

IV
Surui(Am5)

V

VI

0.05

Fig. 5 Neighbor-joining (NJ) tree for 61 worldwide humanpopulations including the present study data and the datapreviously reported (Rosenberg et al. 2002) on the basis of DA
distances calculated from the allele frequencies at 91 tetranucleotide STR loci. The scale for the distance is shown bottom left.
Bootstrap values are provided at each fork of branches as italic
numbers. Our own data are the following ten populations: two
Japanese populations in pink-colored circles (J1: Okinawa, J2:
Nagoya), ﬁve Han Chinese populations in red-colored ellipses(HC1: Shaanxi, HC2: Hunan, HC3: Beijing, HC4: Fujian, HC5:
Guangdong), two Southeast Asian populations in orange-coloredellipses (SEA1: Bangkok, SEA2: Yangon), and one European
population in white-colored pentagon (EU1: England). The
remaining 51 population data were from Rosenberg et al. (2002):
18 Asian populations in yellow colored circles (As1: Tujia, As2:
Yizu, As3: Miaozu, As4: Oroqen, As5: Daur, As6: Mongolia, As7:
Hezhen, As8: Xibo, As9: Uygur, As10: Dai, As11: Lahu, As12:

She, As13: Naxi, As14: Tu, As15: Yakut, As16: Cambodian, As17:Han, As18: Japanese), two Oceania populations in light bluecolored squares (Oc1: New Guinea, Oc2: Melanesian), ﬁve native
American populations in orange-colored triangles (Am1: Pima,
Am2: Maya, Am3: Colombia, Am4: Karitiana, Am5: Surui), eight
Pakistani populations in light green-colored squres (P1: Brahui, P2:
Balochi, P3: Hazara, P4: Makrani, P5: Sindhi, P6: Pathan, P7:
Kalash, P8: Burusho), eight European populations in white-coloredpentagons (EU2: French, EU3: Basques, EU4: Sardinian, EU5:
Bergamo, EU6: Tuscan, EU7: Orcadians, EU8: Adygei, EU9:
Russians), three Middle-East populations in purple-colored starlike shapes (ME1: Bedouin, ME2: Druze, ME3: Palestinian), andseven African populations in gray colored two small triangles (Af1:
BiakaÆPygmies, Af2: MbutiÆPygmies, Af3: Mandeka, Af4: Yoruba,
Af5: San, Af 6: Bantu, Af7: Mozabite). Arrows in various colors
pointed some populations to help the explanations in Discussion

slightly existed only in the Burmese population at all theK values. Furthermore, at K=6, the Japanese populations slightly included ‘‘population’’ (purple color) thatis dominant in the southern Chinese populations.

Japanese and the present southern Han Chinese sharesome common features with each other. Therefore, there
is a possibility that the southern Chinese contributed
more to the present day Japanese population than thenorthern Chinese.
This pattern is somewhat diﬀerent from some previous results using classic markers (Saitou et al. 1994; Nei
1995; Omoto and Saitou 1997) and HLA markers(Saitou et al. 1992; Hatta et al. 1999; Bannai et al. 2000),
where Japanese were more closely related to North East
Asian populations. The reason for this discrepancy is
not clear.
Hanihara (1991) proposed the dual structure modelon peopling of Japanese. According to this model,
Ainu and Okinawa Japanese (Ryukyuan) originatedfrom Jomonese, of which origins have been argued to

Discussion
The phylogenetic relationship of Japanesewith other Asian populations
In the present study, the phylogenetic tree (Fig. 2) and
MDS scattergram (Fig. 3) showed that the Japanese
populations were somewhat closer to the southernChinese populations than the northern and central
Chinese populations. This suggests that both the present

704

Fig. 6 Multidimensional scaling (MDS) plot of 61 worldwide
human populations, based on DA genetic distances calculated
from 91 autosomal tetranucleotide STR loci. Population ID’s were

the same as in Fig. 5. Arrows in various colors pointed some
populations to help the explanations in Discussion

give rise to southern part of East Asia (Hanihara 1991).
Contemporary Okinawa Japanese, however, are genetically much closer to Mainland Japanese than Ainu
(Omoto and Saitou 1997; Tajima et al. 2002). In theanalysis using six radiation groups from mtDNA phylogenetic network, the frequency patterns were similar
between Cantonese and Ryukyu Japanese, and between
Korean and mainland Japanese (Oota et al. 1999).
Furthermore, the analyses using HLA genes andhaplotypes suggested a recent gene ﬂow to Okinawa
from south China (Hatta et al. 1999; Bannai et al.
2000; Tokunaga et al. 2001).
STRUCTURE analysis for 18 east Asian populations reported previously (Rosenberg et al. 2002)
showed that Japanese shared a greater degree of similarity with small ethnic minority populations ofNorthern China (Daur, Hezhen, and Oroqen) thanwith southern Chinese populations, and that Japanese
were closer to Han of northern China than Han people
migrated to the USA. Accordingly, if membershipshared with small ethnic groups from northern China
and Japanese in Rosenberg et al. (2002) was the sameas membership in blue in the present study, the modern

Japanese may consist of people who originated from
northern China and blended with people aﬀected bythose from southern part of China while migrating
through the Korean peninsula, and not aﬀected bythose from around Beijing. To obtain more information, STRUCTURE and DISTRUCT analyses werealso performed using the genotype data at the 91 STRloci in the total 29 populations by combining the datain our ten populations with those in the 18 East Asia
populations and Uygur population out of the Central/
South Asia populations (Rosenberg et al. 2002). As
shown in Fig. 4b, the characteristic memberships of thethree populations in Japan (in yellow) were also observed in the small ethnic minority populations ofNorthern China (Daur, Hezhen, Oroqen, and Mongolia),
same as in the southern Han Chinese population(Guangdong and Fujian) at somewhat higher ratio
than the other populations (K=4–7). However, since
Ainu Japanese and Korean were not analyzed in thisstudy, this may be only a speculation. Further study isneeded to clear up the relationship between Okinawa
Japanese and the other Japanese populations including
Ainu.

705

Combined phylogenetic analysis with published data
We also examined the phylogenetic relationship of thesenine East and Southeast Asian populations newly
examined in this study and the already publishedworldwide population data (Rosenberg et al. 2002). The
NJ tree based on DA genetic distance and the MDS
scattergram were constructed from the allele frequency
data at 91 STR loci for the total of 61 worldwide populations, as shown in Figs. 5 and 6, respectively. The
reasons why we reanalyzed a part of their data byadding our data are: (1) Rosenberg et al. (2002) did notpresent any phylogenic tree, (2) Rosenberg et al. (2002)
analyzed their data by mixing di-, tri-, and tetranucleotide repeat STR markers with extremely diﬀerent
mutation rates, and (3) The genetic distances DA and DC
show more correct topology than other distance measures (Rogers’ DR: 1972, Nei’s DS: 1972, Latter’s FST:1972 and so on) in SMM under such conditions where
the number of samples are about 10–30 using about 100
STR markers with about 0.80 of the heterozygosities,
whether the bottleneck eﬀect exists or not, according toa simulation study (Takezaki and Nei 1996).
Although a NJ tree construction and MDS analysis
based on FST were performed, in fact, however, since the
NJ tree showed very similar topology only with diﬀerent
blanching lengths, and the MDS plots were also very
similar with those from 377 loci reported previously(Zhivotovsky et al. 2003) but with lower 0.16 in stressvalue, only the NJ tree and MDS plots based on DA
distance are shown here.
Six major clusters (I–VI) can be recognized both in
the NJ tree (Fig. 5) and in the MDS scattergram(Fig. 6). The nine East and Southeast Asian populations
examined in the present study all belong to cluster II.
Japanese (As18) and Han (As17) populations studied by
Rosenberg et al. (2002) are also included in cluster II(pointed by two blue arrows in Figs. 5 and 6), and theJapanese (As18) located closer to Nagoya (J2) andOkinawa (J1), both in the NJ tree and in the MDS
scattergram.
The 15 East Asian small ethnic populations formedcluster I, and divided into two sub-clusters (N and S) in
which the former included some small ethnic groups ofnorthern China (Daur, Oroqen, Xibo, and Hezhen) andYakut in Siberia, while the latter included those ofsouthern China such as She, Dai, Tujia, and Lahu. It is,however, ambiguous whether the cluster of a small
ethnic group including Yizu, Naxi, and Tu distributed in
the cluster I or II because of its low bootstrap values andlong branch length. The lengths of branches in the
populations of cluster I were more than twice longer
than those of cluster II. This observation suggested thatthe cluster II populations are very close with each other,and that the cluster I populations are aﬀected by bottleneck eﬀect or depend on the small number of sampling.
Interestingly, even though the Yakut population(As15 marked by a black arrow in Figs. 5 and 6)

inhabits near the Lake Baikal and in the basin of the
Middle Lena River in Siberia, it belonged to cluster I-N
in the NJ tree, but was very close to cluster II in the
MDS scattergram. The pattern observed in the MDS
scattergram is consistent with Matsumoto (1988) who
concluded that the origin of Japanese is near the Lake
Baikal because of the very similar allele distribution
estimated from immunogloblin phenotypes (Km andGm) after examining many East Eurasian populations.
However, since the Yakut population belonged to cluster I-N in the NJ tree, its origin remains ambiguous.
The other clusters consisted of Oceanian (cluster
III), Native American (cluster IV), non-Asian Eurasian(cluster V), and African (cluster VI), respectively.
Interestingly, however, the NJ tree shows that Uygur(northwestern China) and Hazara (Pakistan) populations did not belong to the six major clusters, instead
located between clusters IV and V. In the MDS scattergram, the Uygur population (As9) located in theborder of clusters V and IV, while the Hazara population (P3) located in the Eurasia cluster (V) at theclosest position to the Asian cluster (II). These two
populations are marked with two red arrows both in
Figs. 5 and 6. Positions of the Uygur population inthese ﬁgures suggests that its ancestral population wasshared with that of native American and admixed withEurasian populations later. Hazara population can be
explained as the descendants of Middle-East Asian whoslightly admixed with some East Asian populations.
The latter possibility was supported by a study on Ychromosomal DNA variation in Pakistan (Qamar et al.
2002).
More interestingly, Kalash (P7) and Tuscan (Eu6)ethnic groups made a sub-cluster with Italian andFrench ethnic groups (Bergamo, Sardinian, French,
Basques, and Orcadians) in the Eurasian cluster V in the
NJ tree, and they are located at the closest position witheach other in the MDS plot (pointed by two orange
arrows in Figs. 5 and 6). These results may be consistent
with Kalash people’s oral tradition that they are thedescendants of the Alexander the Great’s army (Lines
1999).
Since Y-STR haplotypes and mtDNA were transmitted along only male and female lineages, respectively,
no test can be performed for deviation in samplings,
especially for small number of samples in small population size. However, in the case of autosomal markers,
at least tests for HWE exist to conﬁrm the deviation
because of their biparental inheritance. In the present
study, after the samples collected from each Asian
population were conﬁrmed as no deviation from HWE
at almost all autosomal STR loci, those reliable andreasonable allele frequency data could be provided
for not only the phylogenetic tree analysis, but also
the structure analysis as those populations by the
STRUCTURE–DISTRUCT program, but not that asindividuals by only Structure program (Rosenberg et al.
2002). Accordingly, the results of the distance based
methods (NJ tree and MDS plots based on DA distance706

as a SMM model) as one of bottom-up procedures could
be very similar to one of the model based methods(conﬁguration of the memberships constructed by
STRUCTURE–DISTRUCT program) as one of topdown procedures. In short, Japanese (both Hondo- andOkinawa Japanese) are isolated and distinguishable
from other east and Southeast Asian population, andslightly more aﬀected by southern part of Chinese thanby northern or middle part of Chinese. However, withregard to studies on the peopling of Japanese or theorigin of Japanese, further studies are necessary withthese methods in the present study using more detailed
samples from other ethnic, regional, or national populations within and around Japan such as Ainu, Koreans,
Mongolia, Eskimo, and so on.
Acknowledgements We are grateful to Prof. Sirirurg Songsivilai,
Dr. Hla Hla Htay, and Dr. Yuri E. Dubrova for providing Thais,
Burmese, and Caucasian DNA samples, respectively. We also
thank Dr. Naoko Takezaki for helpful technical assistance andcomments. This work was in part supported by Grants-in-Aid for
Scientiﬁc Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (12012204 to TY and
11307008 to YK).

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