Hum Genet (2003) 113 : 24–33
DOI 10.1007/s00439-003-0945-1

O R I G I N A L I N V E S T I G AT I O NAtsushi Tajima · Cheih-Shan Sun · I-Hung Pan ·Takafumi Ishida · Naruya Saitou · Satoshi Horai

Mitochondrial DNA polymorphisms in nine aboriginal groups of Taiwan:
implications for the population history of aboriginal Taiwanese

Received: 22 November 2002 / Accepted: 28 February 2003 / Published online: 10 April 2003
© Springer-Verlag 2003

Abstract Mitochondrial DNA (mtDNA) polymorphisms
in the D-loop region and the intergenic COII/tRNALys 9-bp
deletion were examined in 180 individuals from all nineaboriginal Taiwanese groups: Atayal, Saisiat, Bunun, Tsou,
Rukai, Paiwan, Ami, Puyuma, and Yami. A comparisonof 563-bp sequences showed that there were 61 different
sequence types, of which 42 types were specific to respective aboriginal groups. D-loop sequence variation andphylogenetic analysis enabled the 180 aboriginal lineages
to be classified into eight monophyletic clusters (designated C1–C8). Phylogeographic analysis revealed thattwo (C2 and C4) of the eight clusters were new characteristic clusters of aboriginal Taiwanese and accounted for
8.3% and 13.9% of the aboriginal lineages, respectively.
From the estimated coalescent times for the two uniqueclusters, the mtDNA lineages leading to such clusters wereinferred to have been introduced into Taiwan approximately
11,000–26,000 years ago, suggesting ancient immigrationsof the two mtDNA lineages. Genetic distances, based on

net nucleotide diversities between populations, revealed
three distinct clusters that were comprised of northern
mountain (Atayal and Saisiat), southern mountain (Rukai
and Paiwan), and middle mountain/east coast (Bunun,
Tsou, Ami, Puyuma, and Yami) groups, respectively. Furthermore, phylogenetic analysis of 16 human populations(including six other Asian populations and one African
population) confirmed that the three clusters for aboriginal Taiwanese had remained largely intact. Each of theclusters (north, south, and middle-east coast) was characterized by a high frequency of a particular lineage (C4,C2, and 9-bp deletion, respectively). This may result fromrandom genetic drift among the aboriginal groups after asingle introduction of all the mtDNA lineages into Taiwan, but another plausible explanation is that at least
three genetically distinct ancestral populations have contributed to the maternal gene pool of aboriginal Taiwanese.

Introduction
Electronic database information: the accession numbers for thedata in this article are as follows:
DDBJ/EMBL/GenBank databases,
accession nos. AB093682–AB093861
A. Tajima · S. Horai (✉)
Department of Biosystems Science,
Graduate University for Advanced Studies (Sokendai),
240-0193 Hayama, Kanagawa, Japan
Tel.: +81-46-8581575, Fax: +81-46-8581544,
e-mail: horai@soken.ac.jp
C.-S. Sun
Taidon Hospital, Taidon, Taiwan
I-H. Pan
National Taiwan University, Taipei, Taiwan
T. Ishida
Department of Biological Sciences, Graduate School of Science,
University of Tokyo, Tokyo, Japan
N. Saitou
Division of Population Genetics, National Institute of Genetics,
Mishima, Japan

Taiwan is a group of islands situated in the South China
Sea and has a population of more than 22 million according to the census data in 2000. The population of Taiwan
is largely comprised of early immigrants from mainland
China from the 17th century and recent immigrants afterWorld War II, whereas the aboriginal population accounts
for around 1.5% of the total population (Jin et al. 1999;
Lin et al. 2000). On the basis of linguistic, cultural, andgeographical features of such aboriginal people, they canbe divided into nine ethnic groups: the Atayal, Saisiat,
Bunun, Tsou, Rukai, Paiwan, Ami, Puyuma, and Yami.
As shown in Fig. 1, six groups inhabit the central mountain area of the main island of Taiwan: the Atayal andSaisiat in the north, the Bunun and Tsou in the middle,
and the Rukai and Paiwan in the south. The Ami and
Puyuma inhabit the plains of the eastern coast surroundedby high mountains and the Pacific Ocean, whereas the
Yami live on Lanyu Island, located off the eastern coast of
the main island. Although there are several hypotheses regarding the origin of these aboriginal people, none has

25Fig. 1 Geographic distribution of the nine aboriginal groups in
Taiwan. The approximate boundaries for these groups are indicated by dotted lines. Insert Map showing an expanded view of thesurrounding regions of Taiwan in east and southeast Asia

gained general acceptance (Melton et al. 1998; Jin et al.
1999).
One of the common characteristics of nine aboriginal
Taiwanese groups is that they are included in the Austronesian language group, although their languages can befurther classified into four subgroups: Atayalic, Paiwanic,
Tsouic, and Malayo-Polynesian (Table 1; Ruhlen 1987).
From a linguistic viewpoint regarding the worldwide diversification of the Austronesian language group, Taiwan
can be treated as the homeland of the Austronesian languages (Blust 1984–1985; Diamond 1988; Bellwood 1991;
Gray and Jordan 2000). In other words, the Austronesiancolonization in the Pacific and other regions including
Madagascar is thought to have originated in Taiwan (orsouthern mainland China) approximately 6,000 years ago,although opposition to this has been expressed in recent
studies (Oppenheimer 1998; Richards et al. 1998; Su et al.
2000; Oppenheimer and Richards 2001; Lum 2001). Thus,
an understanding of the temporal and geographic origins
of aboriginal Taiwanese may shed light on questions involved in the Neolithic expansion of Austronesian-speaking populations.
Studies of the genetic variation in human populations
allow us to reconstruct their evolutionary history. In particular, mitochondrial DNA (mtDNA) has been widely used
to investigate the genetic relationships among closely related populations because of its unique genetic properties,
including its high mutation rate and maternal inheritance.
Previous studies of mtDNA polymorphisms in aboriginal
Taiwanese population have contributed, to some degree,
to clarifying their genetic background (Melton et al. 1995,
1998; Sykes et al. 1995). However, such genetic studies
have sampled only four (the Atayal, Bunun, Paiwan, andAmi) of the nine aboriginal Taiwanese groups, and therefore comprehensive analyses of all the nine aboriginal
groups are necessary to depict the entire scenario regarding the formation of the aboriginal Taiwanese population.
In the present study, we have examined mtDNA polymorphisms in the major noncoding (D-loop) region andintergenic COII/tRNALys 9-bp deletion (Cann and Wilson

Table 1 Summary statistics for mtDNA polymorphisms within aboriginal Taiwanese groups
Population

Linguistic affiliationa

No. ofindividualskb

hc (mean±SE)

Sd

π (%)e(mean±SE)

AtayalSaisiat
Bunun
Tsou
Rukai
PaiwanAmi
PuyumaYami
total

Atayalic
Paiwanic
Paiwanic
TsouicTsouicPaiwanic
Paiwanic
Paiwanic
Malayo-Polynesian

20
20
20
20
20
20
20
20
20
180

9
9
11
10
14
8
13
11
10
61

0.816±0.073
0.884±0.049
0.921±0.035
0.905±0.037
0.916±0.055
0.842±0.060
0.937±0.035
0.916±0.041
0.895±0.043
0.973±0.004

23
26
25
26
29
20
27
30
22
67

1.16±0.27
1.35±0.28
1.15±0.25
1.21±0.26
1.20±0.25
1.17±0.29
1.10±0.25
1.51±0.30
1.36±0.29
1.36±0.25

aAccording to Ruhlen (1987), in which all are members of Austronesian groupbNumber of mtDNA sequence types

cHaplotype diversity
dNumber of polymorphic sites
eNucleotide diversity (%)

261983) in order to investigate the genetic background andrelationships among all nine aboriginal Taiwanese groups.
These analyses have revealed that the maternal gene poolof the aboriginal Taiwanese consisted of eight mtDNA
lineage clusters, two of which are rare in other Asian populations. Phylogeographic analysis of such two uniquelineages has provided new insights into the population
history of the aboriginal Taiwanese.

Nucleotide diversity within and between populations (dX anddXY, respectively) and net nucleotide diversity between populations(dA; Nei and Li 1979) were computed with the SENDBS program(provided by N. Takezaki), by using Kimura’s two-parameter correction. A phylogenetic tree for human populations was constructedfrom the matrix of pairwise dA distances by the NJ method. At
the intrapopulational level, haplotype diversity (h: equivalent toheterozygosity for diploid genes; Nei 1987) was calculated with
ARLEQUIN software ver 2.001 (Schneider et al. 2001). Statisticalcomparison was performed by using the χ2-test; P<0.05 was considered significant.

Materials and methods
Subjects and DNA samples
We examined a total of 180 unrelated individuals from nine aboriginal Taiwanese groups (20 each from the respective groups).
Genetic studies related to this research have previously establishedthe presence of serum protein polymorphisms (Umetsu et al. 1994,
1995; Yuasa et al. 2001), red cell enzyme polymorphisms (Jin et al.
1995, 1999), and the prevalence of human T-lymphotropic retrovirus (Ishida et al. 1993) in all of the nine aboriginal groups. All
the individuals gave their informed consent prior to their inclusionin this study. Total DNA was isolated from the buffy coat in bloodby treatment with sodium dodecyl sulfate and proteinase K andsubsequent phenol/chloroform extraction.
Amplification and direct sequencingof the D-loop region of mtDNA
A fragment of the D-loop region of mtDNA was amplified withprimer pair A and E (see below) by the polymerase chain reaction(PCR), as described elsewhere (Fucharoen et al. 2001; Qian et al.
2001). The amplified PCR products were purified on MicroSpin
S-400 HR columns (Amersham Biosciences, Piscataway, N.J., USA)and were directly sequenced with primers B–E. Nucleotide sequences of the primers were as follows: A, 15897-5′-GTATAAACTAATACACCAGTCTTGT-3′-15921; B, 15985-5′-AGCACCCAAAGCTAAGATTC-3′-16004; C, 16204-5′-AGCAAGTACAGCAATCAACC-3′-16223; D, 16451-5′-GCGAGGAGAGTAGCACTCTT-3′-16432; E, 100-5′-CAGCGTCTCGCAATGCTATCGCGTG-3′-76. The notation of Anderson et al. (1981) was used
for numbering of bases. Sequencing reactions were performed with
the Dye Terminator Cycle Sequencing FS Kit (Applied Biosystems, Foster City, Calif., USA) according to the manufacturer’s instructions. Nucleotide sequences of both strands were determinedwith an automated DNA sequencer (ABI model 377).
Typing of the COII/tRNALys intergenic deletion
The mtDNA 9-bp deletion in the COII/tRNALys intergenic region
was examined by the PCR method as described by Horai et al.
(1996). The amplified fragments (100 bp or 91 bp in length) encompassing the intergenic region were separated by electrophoresis on 4% agarose gels, followed by staining with ethidium bromide for typing.
Data analyses
A total of 563 nucleotide sites (positions 16048–16569 followedby positions 1–41 (numbering according to Anderson et al. 1981)
were compared to estimate the number of nucleotide substitutions
per site between individual sequences under the two-parametermodel of nucleotide substitutions (Kimura 1980). On the basis of
the estimated number of nucleotide substitutions, a phylogenetic
tree for the mtDNA sequences was constructed by using the neighbor-joining (NJ) method (Saitou and Nei 1987). The reliability of
the NJ tree was analyzed by the bootstrap method (Felsenstein1985) with 1,000 replications.

Results and discussion
Sequence diversities in the D-loop region of mtDNA
Nucleotide sequences of 563-bp fragments of the D-loop
region were determined for 180 individuals from nine aboriginal Taiwanese groups. There were 61 distinct types ofsequence defined by 67 polymorphic sites, as summarized
in Fig. 2. A total of 42 types was unique to their respective
groups, whereas 19 were shared by 105 individuals (58.3%
of the aboriginal population) among two or more groups.
Fourteen of the 42 unique types were shared by two or
more individuals within each group (a total of 47 individuals; 26.1% of the aboriginal population), whereas each of
the remaining 28 types was found in only one individual.
As shown in Table 1, haplotype diversities (h) at the sequence type level varied in the various aboriginal Taiwanese groups, ranging from 0.816 for the Atayal to0.937 for the Ami. The total diversity for aboriginal Taiwanese was 0.973, which was lower than corresponding
estimates for most of the other east and southeast Asian
populations (e.g., Yao et al. 2002b). The relatively low
haplotype diversities both for each aboriginal group andfor the entire population are consistent with recent findings regarding polymorphisms of HLA genes (Lin et al.
2000; Chu et al. 2001) and several serum protein loci
(Yuasa et al. 2001). At the nucleotide level, however, nucleotide diversities (π) for the nine aboriginal groups werein the range of 1.10% (for the Ami) to 1.51% (for the
Puyuma; Table 1). The overall diversity for the entire aboriginal population (1.36%) was the same magnitude asthat for east Asian populations (1.34%; Horai et al. 1996),
indicating a high level of DNA variation in the contemporary aboriginal Taiwanese population.

Fig. 2 Nucleotide sequence differences in the D-loop region of 
mtDNA for 180 aboriginal Taiwanese. The positions of 67 polymorphic sites observed are numbered according to the publishedreference sequence for human mtDNA (Anderson et al. 1981), as
indicated by an uppercase letter (A, T, G, or C) below the verticalfive-digit numerals. For other sequences (types 1–61), only the differences from the reference sequence are indicated, whereas dotsshow identity with the reference. The number of individuals foreach sequence type from nine aboriginal Taiwanese groups is represented right, where abbreviated population names are as follows:
ATA Atayal, SAI Saisiat, BUN Bunun, TSO Tsou, RUK Rukai, PAI
Paiwan, AMI Ami, PUY Puyuma, YAM Yami. The total number(N) for each type is shown in the column of the extreme right.
Boxed letters right Phylogenetic clusters characterized in Fig. 3

27

28

Two unique lineages of mtDNA in aboriginal Taiwanese
Figure 3 shows a phylogenetic tree for the 180 sequences of
the D-loop region from nine aboriginal Taiwanese groups.
This unrooted NJ tree revealed that the 180 mtDNA lineages could be classified into eight monophyletic clusters(named C1–C8), although the lineages from different aboriginal groups were intermingled in each cluster. All of theeight clusters except C1 possessed either specific combinations of three or four shared polymorphic sites (C2, C3,
C6, C7, and C8) or single unique polymorphisms (C4 andC5; Fig. 2). In particular, an A-to-C transversion at 16220
and a T-to-C transition at 16298 were unique polymorphisms among 15 individuals in C2, whereas a T-to-Ctransition at 16297 was specific to C4. Thus, most of these
phylogenetic clusters seem to retain the ancestral polymorphic state before genetic diversification within and

Fig. 3 Phylogenetic tree for
180 mtDNA sequences fromnine aboriginal Taiwanesegroups. Bootstrap values (morethan 50%) are attached to theinternal branches. The scale
for the distance is shown bottom left. The eight monophyletic clusters in the tree areindicated by brackets withcluster numbers (C1–C8). The
individual sequences at the tipsof branches are represented by
the abbreviations of population
names (as in Fig. 2) followedby the three-digit numerals forthe sample numbers

among clusters, thereby suggesting several distinct maternal lineages in the aboriginal Taiwanese population.
To ensure the robustness of the phylogenetic clusters
further, we inferred the mtDNA haplogroup status of theclusters from their respective D-loop sequence motifs.
According to mtDNA haplogroup nomenclature based oncomplete mtDNA sequences from Asian populations (Kivisild et al. 2002), clusters C1, C3, C6, C7, and C8 would
correspond to haplogroups F*, F1a, B5a, B4*(+B4b), andB4a, respectively, whereas C5 contained several other haplogroups such as haplogroup D belonging to Eurasian superhaplogroup M. Thus, these haplogroup assignments revealthat most of the clusters appear to be monophyletic at the
haplogroup level, although the present cluster identification in the NJ tree is somewhat arbitrary. Moreover, because two haplogroups (B and F) belong to Eurasien super-haplogroup N (Kivisild et al. 2002; Yao et al. 2002a),29
Table 2 Frequency distribution of mtDNA phylogenetic clusters in nine aboriginal Taiwanese groups
Population

Frequency (%)
C1

AtayalSaisiat
Bunun
Tsou
Rukai
PaiwanAmi
PuyumaYami
averageπ (%)a±SE

C2

C3

C4

C5

C6

C7

C8

9-bp deletion(C6+C7+C8)

40
–
–
30
20
–
5
5
10
15
–
–
40
25
10
–
10
20
15
–
–
5
30
20
30
–
50
25
5
15
–
10
20
10
15
45
–
35
10
10
35
–
5
5
10
–
20
–
10
30
–
–
40
40
15
–
–
5
50
–
–
30
30
5
15
25
10
15
–
20
10
30
–
–
25
15
20
5
15
20
40
12.8
8.3
8.3
13.9
26.1
6.1
9.5
15.0
30.6
0.393±0.136 0.177±0.123 0.610±0.230 0.404±0.126 0.791±0.126 0.182±0.087 0.603±0.211 0.329±0.136

aNucleotide diversity (%) within cluster. Standard error (SE) of each diversity is calculated by the bootstrap method (500 replications)the two super-haplogroups contribute to the maternal genepool of modern aboriginal Taiwanese. In general, haplogroups B, F, and M constitute the majority of mtDNAs
in southeast Asian populations (Schurr and Wallace 2002),
and hence the present findings indicate that aboriginal
Taiwanese have some degree of genetic affinity with southeast Asian populations in terms of mtDNA haplogroup
distribution.
On the other hand, we were not able to determine mtDNA
haplogroups for clusters C2 and C4, which accounted for
8.3% and 13.9% of the aboriginal mtDNA lineages, respectively (Table 2), because they had no characteristic
sets of D-loop mutations required for assignments. On thebasis of the previous findings of mtDNA polymorphisms
in aboriginal Taiwanese (Melton et al. 1998), two of 28
aboriginal sequences apparently belong to clusters C2(sample PAI16: A16220C-T16298C-T16311C-T16362C)
and C4 (ATA13: T16086C-G16129A-T16297C-T16324C).
These two clusters remain monophyletic in an NJ tree
rooted with two African orthologus sequences (accession
nos. D38112 and AF381988) from mtDNA haplogroup
L1a (Maca-Meyer et al. 2001; data not shown). Thus, theinability to assign mtDNA haplogroups for the two clusters implies the presence of unique haplogroups or new
branches of previously defined haplogroups in aboriginal
Taiwanese groups. The currently available sequence datain the DDBJ/EMBL/GenBank databases reveal no identical sequence types belonging to C2 (types 5–7) and C4(types 12–20) in other human populations, whereas the related lineages to each cluster have been found at very low
frequencies. For example, each of two haplotypes, viz.,
haplotype 119 (C16168T-A16220C-A16265G-T16298CT16362C; C2) and 81 (T16086C-T16297C-T16324C; C4)
in Sykes et al. (1995) has been found in one Philippine.
Thus, the two phylogenetic clusters are not specific to aboriginal Taiwanese but are rare in other Asian populations.
Cluster C2 was observed in four (the Tsou, Rukai, Paiwan, and Puyuma) of the nine aboriginal Taiwanese groups,
whereas all of the nine groups except Tsou exhibited C4 atvarious frequencies (Table 2). Nucleotide diversities forthe coalescence of the two clusters (C2 and C4) were estimated as 0.177±0.123% and 0.404±0.126%, respectively(Table 2). Based on complete mtDNA sequences fromthree human individuals (Horai et al. 1995), we calculated
an average substitution rate for the 563-bp mtDNA segment examined in this study as 7.8×10–8/site per year. Assuming this rate under a panmictic population model withconstant effective population size, we estimated coalescent times for the two clusters as 11,000±7,900 years forC2 and 26,000±8,100 years for C4. These estimates forthe clusters would be linked to their arrival times in Taiwan if there were little or no diversity within the respective founding lineages. As described above, the mtDNA
sequences related to the two clusters have been infrequently observed in neighboring Asian populations. Therefore, it is plausible that the genetic diversification withinclusters C2 and C4 occurred mainly in Taiwan, but notprior to settlement of individuals carrying the clusters in
Taiwan. This interpretation suggests an ancient genetic
isolation of the mtDNA lineages belonging to the twoclusters in the aboriginal Taiwanese. The implications forthis genetic isolation will be discussed later in Genetic relationships among aboriginal Taiwanese groups.
Distribution of COII/tRNALys intergenic 9-bp deletion
A deletion polymorphism in a 9-bp tandem repeat of the
mtDNA COII/tRNALys intergenic region is one of thecharacteristics not only of Asians (Horai and Matsunaga
1986; Stoneking and Willson 1989; Ballinger at al. 1992;
Harihara et al. 1992), but also of the Asian descendents,
such as Pacific populations (Hertzberg et al. 1989; Hagelberg and Clegg 1993; Lum et al. 1994) and Native Americans (Schurr et al. 1990; Ward et al. 1991; Torroni et al.
1992; Horai et al. 1993). In particular, the geographic distribution of this deletion in the Pacific region has been associated with past range expansions of Austronesian-language speakers (Melton et al. 1995, 1998; Redd et al. 1995;
Sykes et al. 1995).

30Consistent with the preliminary findings in six aboriginal Taiwanese groups (Horai 1991), the aboriginal groups
exhibited the mtDNA 9-bp deletion at various frequencies, ranging from 10% for the Atayal and Rukai to 50%
for the Bunun (Table 2). These differences among the aboriginal groups were statistically significant at the 5% level(P=0.039; χ2-test), indicating an uneven distribution ofthis deletion among aboriginal Taiwanese. The averagefrequency in aboriginal Taiwanese was 30.6%, which wasslightly lower than those from previous studies in four(the Atayal, Bunun, Paiwan, and Ami) of the entire aboriginal group (41.5%, Melton et al. 1995; 36%, Sykes etal. 1995). A total of 55 aboriginal Taiwanese with the 9-bp
deletion appeared in the three clusters C6, C7, and C8 on
the basis of D-loop sequences (Fig. 3). The nucleotide sequences (Fig. 2) revealed that all the individuals with thedeletion showed a specific combination of two polymorphisms (T16189C and T16519C). Thus, this deletion event
appears to have occurred once on the background of mtDNA
carrying such a characteristic set of mutations.
A unique combination of three transitions (T16217C,
A16247G, and C16261T) with the 9-bp deletion has beenreferred to as the “Polynesian motif” because of its highfrequencies in Polynesian populations (Melton et al. 1995;
Redd et al. 1995). With reference to the evolutionary history of Polynesian populations and/or Austronesian-language speakers, much attention has been turned to where
and when this motif emerged in Asia-Pacific region (e.g.,
Richards et al. 1998). In this survey, we have found mtDNA
sequence types carrying only one (T16217C in cluster C7)
or two (T16217C and C16261T in C8) of the three mutations in aboriginal Taiwanese population with the 9-bp
deletion. However, there is no A16247G mutation among
the aboriginal Taiwanese (Fig. 2). The lack of the Polynesian motif in the aboriginal Taiwanese groups agrees withprevious findings (Melton et al. 1995, 1998; Sykes et al.
1995), indicating that the motif itself is unlikely to havearisen in Taiwan.

Genetic relationships among aboriginal
Taiwanese groups
To investigate genetic relationships among the nine aboriginal Taiwanese groups, we estimated net (dA) nucleotide diversities between each pair of the aboriginal groups.
Table 3 shows the dA distances and interpopulational (dXY)
and intrapopulational (dX or dY) diversities among the nineaboriginal groups. The dA distances ranged from 0.038%
to 0.226% (between the Atayal and Paiwan). On the basisof the geographic distribution of the aboriginal groups in
Taiwan (Table 3), the smallest values (0.038%) were foundin two combinations between the middle mountain groups
and east coast groups (the Bunun-Ami and the TsouPuyuma), suggesting close genetic relationships between
these aboriginal groups.
Based on the dA distances, we constructed a phylogenetic tree for nine aboriginal Taiwanese groups by the NJ
method (Fig. 4). The tree showed that there were four
clades of two aboriginal groups with bootstrap probabilities over 50%: the Atayal-Saisiat (92%), the Tsou-Yami(69%), the Bunun-Ami (55%), and the Rukai-Paiwan(54%). Furthermore, the two clades of the Bunun-Ami andTsou-Yami formed a larger cluster, together with the
Puyuma. The remaining two clades (the Atayal-Saisiat
and the Rukai-Paiwan) correspond to northern and southern mountain groups, respectively. It is worth noting thathigh frequencies of the unique lineage cluster C2 arefound in the southern (Rukai and Paiwan) groups, andthose of another cluster (C4) in the northern (Atayal andSaisiat) groups (Table 2). On the other hand, the large
cluster of five aboriginal groups suggests close genetic
affinities between middle mountain groups and east coastgroups including the Yami. In spite of the heterogeneous
distribution of the mtDNA 9-bp deletion among nine aboriginal Taiwanese groups, higher frequencies of the deletion (over 30%) are common to all of the five aboriginal
groups (Bunun, Tsou, Ami, Puyuma, and Yami) in the

Table 3 Estimates of interpopulational (dXY), intrapopulational (dX or dY), and net (dA) nucleotide diversities among nine aboriginal Taiwanese groups
Population

Central mountain groupsa
North

AtayalSaisiat
Bunun
Tsou
Rukai
PaiwanAmi
PuyumaYami

East coast groups
MiddleSouth

On coast

Off coast

AtayalSaisiat

Bunun

Tsou

Rukai

PaiwanAmi

PuyumaYami

1.161b
0.042
0.170
0.190
0.152
0.226
0.150
0.110
0.203

1.297
1.348
0.145
0.192
0.129
0.163
0.139
0.116
0.124

1.325
1.394
1.149
0.070
0.144
0.160
0.038
0.060
0.125

1.377
1.473
1.252
1.214
0.162
0.187
0.112
0.038
0.076

1.331
1.401
1.318
1.368
1.197
0.107
0.098
0.053
0.213

1.391
1.421
1.319
1.378
1.290
1.169
0.130
0.113
0.197

1.282
1.365
1.165
1.271
1.249
1.266
1.104
0.066
0.151

1.445
1.544
1.388
1.399
1.406
1.451
1.372
1.507
0.107

1.464
1.479
1.381
1.365
1.493
1.462
1.384
1.542
1.362

aThe classification is based on the places inhabited by the aboriginal Taiwanese groups(Chu et al. 2001)

bAll values are multiplied by 100. The figures on the diagonal (bold)refer to dX (or dY), those above the diagonal give dXY, and thosebelow the diagonal represent the values of dA=dXY–(dX+dY)/2

31

Fig. 4 Neighbor-joining (NJ) tree, showing the relationships ofthe nine aboriginal Taiwanese groups on the basis of dA distanceswith Kimura’s two-parameter correction. The numbers shown for
the internal branches are the bootstrap probabilities. The scale forthe genetic distance is shown below

Fig. 5 Neighbor-joining (NJ) tree for the nine aboriginal Taiwanese and other Asian and African populations, on the basis of dA
distances estimated from the nucleotide sequences of 482-bp fragments of the D-loop region (positions 16129–16569 followed bypositions 1–41 in the reference sequence of Anderson et al. 1981).
The data sources for the seven other human populations are as follows: five east Asians (Horai et al. 1996), Thais Fucharoen et al.
2001), and Africans (Horai and Hayasaka 1990; Vigilant et al.
1991). Bootstrap probabilities (over 50%) are attached to the internal branches. The scale for the genetic distance is shown below

middle mountain and east coast regions including the
Lanyu Island (Table 2). Thus, the present observations imply that the aboriginal groups inhabiting the middle
mountain-east coast regions including the Lanyu Island
share some extent of a common maternal ancestry. Thus,
the phylogenetic relationships among the nine aboriginal
groups are likely to fit with their geographic distribution(north, south, and middle-east coat), although the bootstrap support is not so strong.
To infer phylogenetic relationships among the nineaboriginal Taiwanese groups and other human populations,
we constructed an NJ tree for a total of 16 populations(Fig. 5), combined with the published data for five east
Asian (Horai et al. 1996), Thai (Fucharoen et al. 2001) and
African (Horai and Hayasaka 1990; Vigilant et al. 1991)
populations. The tree indicated that the three population
clusters (north, south, and middle-east coat) for aboriginal
Taiwanese remained largely intact, except that the middleeast coat cluster lacked the Ami. The nine aboriginal
groups clustered further with Taiwan Han and Thais (bootstrap value 63%), but there appeared to be two separate
sub-clusters, which consisted of the aboriginal Taiwaneseand Taiwan Han-Thai, respectively. The genetic difference between aboriginal Taiwanese and Taiwan Han at the
population level is consistent with those in the phylogenetic analyses undertaken with HLA loci (Lin et al. 2000;
Chu et al. 2001), although they have been neighbors on
Taiwan for the past four centuries.
The three population clusters (north, south, and middle-east coat) in aboriginal Taiwanese also appear to correspond to the geographic distribution of three mtDNA
lineages: C4, C2, and the 9-bp deletion (C6–C8), respectively. This implies that at least three genetically distinct
human populations were involved in the prehistoric settlement of Taiwan, although the three lineages (C2, C4, and9-bp deletion) account only for 52.7% of the aboriginal
gene pool studied. Another explanation for the population
relationships is that a single introduction of all three mtDNA
lineages into Taiwan was coupled with subsequent isolation and random genetic drift among the aboriginal groups.
One way of evaluating these scenarios is to determine ancestral source populations of these three mtDNA lineages,
especially the geographic origins of the unique lineage
clusters C2 and C4. However, the infrequent distributionof the two unique lineages outside Taiwan prevents us
from verifying these in such a way. The divergence times
for the clusters C2 (11,000±7,900 years) and C4 (26,000±
8,100 years) suggest that mtDNA lineages leading to thetwo clusters were separately brought into Taiwan. Thus,
these postulated human migrations may favor, even if
only partially, the former interpretation. There is no discrepancy in dating between this suggestion and the earliest human remains in Taiwan (Lien 1981); modern humans probably settled here 20,000–30,000 years before
present (BP). Paleogeographic data (Voris 2000) havesuggested that Taiwan was connected to the Asian continent by the exposed continental shelf when sea levels wereat 120–75 m below present-day levels, during the last glacial
period. Subsequently, at approximately 11,000 years BP

32(50 m below the present levels), there were no land connections between Taiwan and the continent, and evidently,
Taiwan has been isolated since then. The deglaciation accompanied by sea-level rise at the end of the Pleistocene
would result in the characteristic geographic distributionof the two mtDNA lineages (C2 and C4) to Taiwan. At
any rate, we should be able to characterize the unique
mtDNA lineages more minutely when obtaining complete
mtDNA sequences from such lineages and extensive geographic sampling from neighboring human populations.
Linguistic and cultural evidence for Austronesian-language speakers (Blust 1984–1985; Diamond 1988; Bellwood 1991) has been mostly used to interpret the maternal genetic background of aboriginal Taiwanese to date
(Melton et al. 1995, 1998; Sykes et al. 1995). In otherwords, the population history of aboriginal Taiwanese issupposed to date back to 6,000–4,000 BC (Melton et al.
1998), although there is no genetic evidence that excludes
the possibility of the earlier arrival of modern humans in
Taiwan. The present phylogeographic analysis of the two
unique lineage clusters allows us to infer that modern humans carrying the lineages migrated into Taiwan around
11,000–26,000 years ago, suggesting a longer history of
the aboriginal people in Taiwan. It should be noted thatcomparative analyses of the geographic distribution of otherdistinct mtDNA lineages (clusters C1, C3, C5, C6, C7,
and C8: haplogroups B, F, and M) in east and southeast
Asia are necessary to address the questions as to when andhow the respective lineages were brought into Taiwan.
These analyses should also be useful for clarifying theprehistoric dispersal of the Austronesian-speaking populations. This is because the analyses would help us to test
two opposing views of aboriginal Taiwanese as a sourceof the Austronesian expansion (Melton et al. 1995, 1998;
Sykes et al. 1995), and an isolated population of Austronesian-language speakers/southeast Asians (Oppenheimer
1998; Su et al. 2000). To attain a deeper understanding of
the peopling of Taiwan, the maternal genetic view has
also to be integrated with wider knowledge from other genetic studies, such as Y-chromosome haplotype analysis,
and many different disciplines.
Acknowledgements We thank Ms. Naoko Anaguchi for technical assistance. This study was supported in part by Grants-in-Aid
from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan to S. Horai.

References
Anderson S, Bankier AT, Barrell BG, Bruijn MH de, Coulson AR,
Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier
PH, Smith AJ, Staden R, Young IG (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–
465
Ballinger SW, Schurr TG, Torroni A, Gan YY, Hodge JA, Hassan
K, Chen KH, Wallace DC (1992) Southeast Asian mitochondrial
DNA analysis reveals genetic continuity of ancient Mongoloid
migrations. Genetics 130:139–152
Bellwood P (1991) The Austronesian dispersal and the origin of
languages. Sci Am 265:70–75

Blust R (1984–1985) The Austronesian homeland: a linguistic perspective. Asian Perspect 26:45–67
Cann RL, Wilson AC (1983) Length mutations in human mitochondrial DNA. Genetics 104:699–711
Chu CC, Lin M, Nakajima F, Lee HL, Chang SL, Juji T, Tokunaga
K (2001) Diversity of HLA among Taiwan’s indigenous tribes
and the Ivatans in the Philippines. Tissue Antigens 58:9–18
Diamond JM (1988) Express train to Polynesia. Nature 336:307–
308
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Fucharoen G, Fucharoen S, Horai S (2001) Mitochondrial DNA
polymorphisms in Thailand. J Hum Genet 46:115–125
Gray RD, Jordan FM (2000) Language trees support the expresstrain sequence of Austronesian expansion. Nature 405:1052–
1055
Hagelberg E, Clegg JB (1993) Genetic polymorphisms in prehistoric Pacific islanders determined by analysis of ancient bone
DNA. Proc R Soc Lond [Biol] 252:163–170
Harihara S, Hirai M, Suutou Y, Shimizu K, Omoto K (1992) Frequency of a 9-bp deletion in the mitochondrial DNA among
Asian populations. Hum Biol 64:161–166
Hertzberg M, Mickleson KN, Serjeantson SW, Prior JF, Trent RJ(1989) An Asian-specific 9-bp deletion of mitochondrial DNA
is frequently found in Polynesians. Am J Hum Genet 44:504–
510
Horai S (1991) A genetic trail of human mitochondrial DNA. In:
Mukohata Y (ed) New era of bioenergetics. Academic Press,
Tokyo, pp 273–299
Horai S, Hayasaka K (1990) Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrialDNA. Am J Hum Genet 46:828–842
Horai S, Matsunaga E (1986) Mitochondrial DNA polymorphismin Japanese. II. Analysis with restriction enzymes of four orfive base pair recognition. Hum Genet 72:105–117
Horai S, Kondo R, Nakagawa-Hattori Y, Hayashi S, Sonoda S,
Tajima K (1993) Peopling of the Americas, founded by four
major lineages of mitochondrial DNA. Mol Biol Evol 10:23–
47
Horai S, Hayasaka K, Kondo R, Tsugane K, Takahata N (1995)
Recent African origin of modern humans revealed by completesequences of hominoid mitochondrial DNAs. Proc Natl Acad
Sci USA 92:532–536
Horai S, Murayama K, Hayasaka K, Matsubayashi S, Hattori Y,
Fucharoen G, Harihara S, Park KS, Omoto K, Pan IH (1996)
mtDNA polymorphism in east Asian Populations, with specialreference to the peopling of Japan. Am J Hum Genet 59:579–
590
Ishida T, Pan IH, Horai S, Saitou N, Sun CS (1993) Seroepidemiological survey of human T-lymphotropic retrovirus among indigenous populations in Taiwan. Int J Epidemiol 22:927–930
Jin F, Saitou N, Ishida T, Sun CS, Pan IH, Omoto K, Horai S(1995) Genetic polymorphism of ADA in Taiwan aboriginal
populations: new ADA variants detected by isoelectric focusing method. Anthropol Sci 103:227–234
Jin F, Saitou N, Ishida T, Sun CS, Pan IH, Omoto K, Horai S(1999) Population genetic studies on nine aboriginal ethnic
groups of Taiwan. I. Red cell enzyme systems. Anthropol Sci
107:229–246
Kimura M (1980) A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide
sequences. J Mol Evol 16:111–120
Kivisild T, Tolk HV, Parik J, Wang Y, Papiha SS, Bandelt HJ,
Villems R (2002) The emerging limbs and twigs of the east
Asian mtDNA tree. Mol Biol Evol 19:1737–1751
Lien CM (1981) On the occurrence of fossil Homo sapiens in Taiwan. Bull Dept Archaeol Anthropol 42:53–74
Lin M, Chu CC, Lee HL, Chang SL, Ohashi J, Tokunaga K, Akaza
T, Juji T (2000) Heterogeneity of Taiwan’s indigenous population: possible relation to prehistoric Mongoloid dispersals. Tissue Antigens 55:1–9

33
Lum JK (2001) The colonization of Remote Oceania and thedrowning of Sundaland. In: Jin L, Seielstad M, Xiao C (eds)
Genetic, linguistic and archaeological perspectives on humandiversity in southeast Asia. World Scientific, Singapore, pp 147–
170
Lum JK, Rickards O, Ching C, Cann RL (1994) Polynesian mitochondrial DNAs reveal three deep maternal lineage clusters.
Hum Biol 66:567–590
Maca-Meyer N, González AM, Larruga JM, Flores C, Cabrera VM(2001) Major genomic mitochondrial lineages delineate early
human expansions. BMC Genet 2:13
Melton T, Peterson R, Redd AJ, Saha N, Sofro AS, Martinson J,
Stoneking M (1995) Polynesian genetic affinities with Southeast Asian populations as identified by mtDNA analysis. Am J
Hum Genet 57:403–414
Melton T, Clifford S, Martinson J, Batzer M, Stoneking M (1998)
Genetic evidence for the proto-Austronesian homeland in Asia:
mtDNA and nuclear DNA variation in Taiwanese aboriginal
tribes. Am J Hum Genet 63:1807–1823
Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New YorkNei M, Li WH (1979) Mathematical model for studying genetic
variation in terms of restriction endonucleases. Proc Natl Acad
Sci USA 76:5269–5273
Oppenheimer S (1998) Eden in the East: the drowned continent ofsoutheast Asia. Phoenix, London
Oppenheimer S, Richards M (2001) Fast trains, slow boats, and theancestry of the Polynesian islanders. Sci Prog 84:157–181
Qian YP, Chu ZT, Dai Q, Wei CD, Chu JY, Tajima A, Horai S(2001) Mitochondrial DNA polymorphisms in Yunnan nationalities in China. J Hum Genet 46:211–220
Redd AJ, Takezaki N, Sherry ST, McGarvey ST, Sofro AS, Stoneking M (1995) Evolutionary history of the COII/tRNALys intergenic 9 base pair deletion in human mitochondrial DNAs fromthe Pacific. Mol Biol Evol 12:604–615
Richards M, Oppenheimer S, Sykes B (1998) mtDNA suggests
Polynesian origins in Eastern Indonesia. Am J Hum Genet 63:
1234–1236
Ruhlen M (1987) A guide to the world’s languages, vol 1. Classification. Stanford University Press, Stanford
Saitou N, Nei M (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4:
406–425
Schneider S, Roessli D, Excoffier L (2001) Arlequin ver. 2.001: asoftware for population genetic analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland
Schurr TG, Wallace DC (2002) Mitochondrial DNA diversity insoutheast Asian populations. Hum Biol 74:431–452

Schurr TG, Ballinger SW, Gan YY, Hodge JA, Merriwether DA,
Lawrence DN, Knowler WC, Weiss KM, Wallace DC (1990)
Amerindian mitochondrial DNAs have rare Asian mutations athigh frequencies, suggesting they derived from four primarymaternal lineages. Am J Hum Genet 46:613–623
Stoneking M, Wilson AC (1989) Mitochondrial DNA. In: Hill A,
Serjeantson S (eds) The colonization of the Pacific: a genetic
trail. Clarendon, Oxford, pp 215–245
Su B, Jin L, Underhill P, Martinson J, Saha N, McGarvey ST,
Shriver MD, Chu J, Oefner P, Chakraborty R, Deka R (2000)
Polynesian origins: insights from the Y chromosome. Proc Natl
Acad Sci USA 97:8225–8228
Sykes B, Leiboff A, Low-Beer J, Tetzner S, Richards M (1995)
The origins of the Polynesians: an interpretation from mitochondrial lineage analysis. Am J Hum Genet 57:1463–1475
Torroni A, Schurr TG, Yang CC, Szathmary EJ, Williams RC,
Schanfield MS, Troup GA, Knowler WC, Lawrence DN,
Weiss KM, Wallace DC (1992) Native American mitochondrial
DNA analysis indicates that the Amerind and the Nadene populations were founded by two independent migrations. Genetics
130:153–162
Umetsu K, Yuasa I, Suzuki T, Sun CS, Pan IH, Ishida T, Saitou N,
Horai S (1994) Polymorphisms of complement component I
and C1R subcomponent of C1 in nine aboriginal Taiwanese
populations. Hum Biol 66:339–348
Umetsu K, Yuasa I, Harada A, Suzuki T, Pan IH, Ishida T, Saitou
N, Horai S (1995) Orosomucoid phenotyping with monoclonal
antibodies: polymorphic occurrence of ORM1*Q0 in aboriginal
Taiwanese populations. Hum Hered 45:181–185
Vigilant L, Stoneking M, Harpending H, Hawkes K, Wilson AC(1991) African populations and the evolution of human mitochondrial DNA. Science 253:1503–1507
Voris HK (2000) Maps of Pleistocene sea levels in southeast Asia:
shorelines, river systems and time durations. J Biogeography
27:1153–1167
Ward RH, Frazier BL, Dew-Jager K, Pääbo S (1991) Extensive
mitochondrial diversity within a single Amerindian tribe. Proc
Natl Acad Sci USA 88:8720–8724
Yao YG, Kong QP, Bandelt HJ, Kivisild T, Zhang YP (2002a)
Phylogeographic differentiation of mitochondrial DNA in Han
Chinese. Am J Hum Genet 70:635–651
Yao YG, Nie L, Harpending H, Fu YX, Yuan ZG, Zhang YP(2002b) Genetic relationship of Chinese ethnic populations revealed by mtDNA sequence diversity. Am J Phys Anthropol
118:63–76
Yuasa I, Umetsu K, Ago K, Sun CS, Pan IH, Ishida T, Saitou N,
Horai S (2001) Population genetic studies on nine aboriginalethnic groups of Taiwan. II. Serum protein systems. Anthropol
Sci 109:257–273