Bull. Natl. Mus. Nat. Sci., Ser. D, 35, pp. 25–34, December 22, 2009

Mitochondrial DNA diversity and population differentiation in
ethnic groups in Laos
Ken-ichi Shinoda1, Kazuo Umetsu2 and Yoshiharu Sekino3
1

Department of Anthropology, National Museum of Nature and Science,
3–23–1 Hyakunincho, Shinjuku-ku, Tokyo 169–0073, Japan2
Department of Forensic Medicine, Faculty of Medicine, Yamagata University,
2–2–2 Iida-Nishi, Yamagata-shi, Yamagata 990–9585, Japan3
Department of Liberal Arts, Musashino Art University, 1–736 Kodaira-shi, Tokyo 187–8505, Japan

Abstract Laos is a country inhabited by more than 100 indigenous ethnic groups and subgroups, many of which permeate neighboring countries. Hence, studying the genetic history of theethnic populations in Laos can provide crucial clues to the peopling of Southeast Asia. Among theethnic groups, the Lao Lum (lowland Lao) is the largest and most widespread. The Kham are believed to be the earliest inhabitants of Laos. The Lao Sun live at high altitudes in northern Laos.
Ethnohistorical data suggest that the Lao Lum migrated to Southeast Asia from the north and arrived in present-day Laos by the 10th century. The Kham migrated from the north, probably fromthe Yangtze River basin, in prehistoric times. The Mon, individuals of one of the Lao Sun subgroups, migrated to Laos from southern China and share their ethnicity with the Muong of Vietnam.
To address this hypothesis, we obtained sequences of hypervariable segment (HVS) 1 and HVS2
from a total of 70 samples of individuals belonging 3 ethnic groups. Along with these, we used 16
diagnostic variants found in the coding regions and compared these with sequences obtained fromother Southeast Asian populations. Among the individuals analyzed by HVS1 and HVS2 sequencing, we observed 37 different haplotypes defined by 82 polymorphic sites. The mitochondrial DNA(mtDNA) data show that the Lao Sum display a close affinity for both the North Vietnamese andthe Thai, whereas the Lao Lum display a close affinity with the original indigenous groups ofSoutheast Asia. The Kham are unique because they exclusively share a large number of haplogroups with original inhabitants of the south of Southeast Asia and the north of Southeast Asia.
This is the first systematic study on the genetic structure of Laos’s populations. The study aims at
studying the matrilineal ancestry of the people of Laos and providing more data for evaluating thegenetic structure of East Asians.
Key words : Mitochondrial DNA, Haplogroup, Laos, Ethnic group, Population genetics

Introduction
Largely on the basis of geography, Southeast
Asia is commonly divided into 2 regions: the islands and the mainland. The islands comprise Indonesia, east Malaysia, and the Philippines,
whereas the mainland comprises Burma (Myanmar), Cambodia, Laos, Thailand, Vietnam, andwest Malaysia. Because of its geographical position, Southeast Asia is considered to have been
an important route used by the first modern hu-

mans who migrated from the southern part ofAsia to the northeast of Asia. Individuals belonging to the so-called “Australo-Melanesian” race
arrived in the southeast of Asia about 50,000
years ago (Barker et al., 2007). They were thefirst inhabitants of both Sahul and the ancient
continent of Sundaland, which formed most of
the islands and the mainland of Southeast Asia.
Moreover, recent migrations have been by individuals of the Austronesian populations, who are
believed to have left Taiwan or formerly the coast

26

K. Shinoda, K. Umetsu and Y. Sekino

of South China about 6,000 years ago; these populations have spread throughout the Pacific via
the coastal region of Southeast Asia (Bellwood,1997).
Hence, the population history of the indigenous people of Southeast Asia may provide crucial insights into human migration in East Asiaand the Pacific region. The origin and genetic
affinities of the Southeast Asian population havebeen examined from many perspectives in anthropological studies (Barker et al., 2007; Matsumura et al., 2008).
Recently, because of molecular anthropological studies, there has been significant progress inunderstanding the peopling of the Southeast
Asian islands (Hill et al., 2006, 2007; Li et al.,2007; Soares et al., 2008; Moodley et al., 2009).
However, little is known regarding the peoplingof the mainland because of the lack of fundamental DNA data. There have been reports on mitochondrial DNA (mtDNA) data from Thailand
and Vietnam (Fucharoen et al., 2001; Irwin et al.,2008; Lertrit et al., 2008; Patcharee et al., 2008);
however, increasingly volatile political situations
in some countries, including Laos and Myanmar,
pose difficulties in obtaining DNA data.
Although there are 49 officially recognizedethnicities in Laos, more than a hundred indigenous ethnic groups and subgroups inhabit its
landscape, many of which permeate neighboring
countries but retain their own culture. In thisstudy, we investigated genetic relationships
among 3 ethnic groups whose samples we wereable to obtain. In 2007, one of the authors (Y.S.)
conducted an ethnological survey among individuals inhabiting the region along the MekongRiver in Laos, and obtained DNA samples from 3
ethnic groups.
The first group is Lao Lum (lowland Lao), a
diverse group that accounts for 66% of the population and comprises groups that live at lower altitudes, profess Buddhism, and practice wet-rice
cultivation. Among the 3 groups, the Lao Lum isthe largest in number and the most widespread. It
is believed that they migrated to Southeast Asia
from the north and arrived in present-day Laosby the 10th century. The second group comprises
the Kham. Individuals in this group live at medium altitudes, practice slash-and-burn agriculture,and profess animism. It is believed that they are
the earliest inhabitants of Laos who migrated
from the north (probably from the Yangtze Riverbasin) during prehistoric times. The third group
Lao Sun includes the Punoi, Pusan, and Mon
people. It is believed that the Mon people migrated to Laos from southern China and share theirethnicity with the Muong of Vietnam. They liveat high altitudes in northern Laos and also use
slash-and-burn methods of farming.
The present-day natives of mainland Southeast
Asia can be classified according to their linguistic affiliations. The indigenous languages of Laoscan be categorized into 4 major groups: the Daicor Tai-Kadai language, Mon-Khmer (a subgroupof the Austro-Asiatic language family), TibetoBurman (a subgroup of the Sino-Tibetan language family), and Hmong-Mien. The Lao Lum
and Kham speak Tai-Kadai, whereas the Mon
speak Mon-Khmer (Lewis, 2009).
mtDNA has been extensively studied in recent
yeas as part of population genetics studies. However, no genetic investigations have been carriedout for the population of Laos. The present study
is the first to provide mtDNA sequencing dataand haplogroup frequencies in 3 Lao ethnic
groups. Their genetic affinities have been studied
and compared with other Southeast Asian populations. This study will be useful not only for understanding the complex history of the peoplingof the mainland of Southeast Asia but also for
improving our knowledge on the distribution of
mtDNA variation in the mainland Southeast
Asian population.
Materials and Methods
Cheek swabs of 70 anonymous, maternally unrelated individuals were collected. These samples
included those of individuals belonging to the 3
ethnic groups (locations shown in Figure 1): 12
Lao Lum, 27 Kham, and 31 Lao Sun (Mon, 23
samples; Punoi, 2 samples; Pusan, 6 samples).

Genetic structure of ethnic groups in Laos

27

Fig. 1. The map of East and Southeast Asia. The circles indicate the sampling points of the present study andadditional published data points used in the analyses.

Appropriate informed consent was obtained fromall participants. This study was also formally approved by the relevant national level administrative bodies in Laos. These samples were exported
to Japan, and DNA was extracted using the Puregene buccal cell DNA isolation kit (Gentra Systems, Minneapolis, Minnesota). This study was
performed in keeping with the ethic guidelines of
the Japan Anthropological Society.
Direct sequencing of PCR products from theD-loop region of mtDNA
A segment of hypervariable region (HVR) 1(nucleotide positions 16121 to 16402, according
to the revised Cambridge reference sequence(rCRS); Andrews et al., 1999) and HVR2 (128 to408) was sequenced for all samples. Aliquots (2m l) of the extracts were used as templates forpolymerase chain reaction (PCR). Amplifications
were carried out in a reaction mixture (total volume, 25 m l), containing 1 unit of Taq DNA polymerase (HotStarTaq™ DNA polymerase; Qiagen), 0.1 m M of each primer, and 100 m M deoxyribonucleoside triphosphates (dNTPs) in 1
PCR buffer (supplied by the manufacturer). The

PCR conditions were as follows: incubation at
95°C for 15 min; 30 cycles of heat treatment at
94°C for 20 s, 50–56°C for 20 s, and 72°C for 15s; and a final extension step at 72°C for 1 min.
The following primers were used to amplify the
HVR1 and HVR2 region: (L16120) 5-TTACTGCCAGCCACCATGAA-3, (H16403) 5-TTGATTTCACGGAGGATGGTG-3, (L127) 5-AGCACCCTATGTCGCAGTAT-3, and (H409) 5TGTTAAAAGTGCATACCGCC-3. The PCR
products were subjected to agarose gel electrophoresis on a 1.5% gel and were recovered byusing a QIAEX II agarose gel extraction kit (Qiagen, Germany). Aliquots of the samples wereprepared for sequencing using a BigDye cycle
sequencing kit (Applied Biosystems, Foster City,
CA, USA), which was performed using forward
and reverse primers. The primers that were used
for PCR amplification were also used in the sequencing reaction. Sequencing was performed
using a single primer in both directions so as toenable identification of polymorphisms or ambiguous bases. The sequencing reactions were
performed on a DNA sequencer (ABI model no.
3130) equipped with the SeqEd software.

28

K. Shinoda, K. Umetsu and Y. Sekino

PCR-amplified product length polymorphism
analysis
In this study, a simple and rapid amplified
product length polymorphism (APLP) method
has been employed for the detection of mtDNA
haplogroups (Umetsu et al., 2001, 2005). Using
APLP, diagnostic polymorphic sites can be examined directly. Thus, with this method, it is possible to avoid ambiguity (caused by insufficient
enzymatic digestion) with respect to the actual
mutation site, which is one of the disadvantagesof the RFLP method.
The amplification reaction was conducted in a
DNA thermal cycler 9700 (Applied Biosystems,
Foster City, CA, USA) by using 4 sets of 9-plex
PCR assays. Multiplex A was designed to analyze the target sites 10398A, intergenic COII/tRNALys 9-bp deletion, 5178A, 3010A, 14979C,
8020A, 13104G, 11215T, and 11959G; Multiplex
B, to analyze target sites 10400T, 8793C, 4833G,
8200C, 3394C, 14178C, 3970T, 5417A, and
13183G; Multiplex C, to analyze target sites
3594T, 11969A, 11696A, 6455T, 4386C,
12811C, 15487T, 8684T, and 1736G; Multiplex
D, to analyze target sites 10873T, 8994A, 4580A,
10550G, 12308G, 1719A, 15607G, 7028C, and12612G; and Multiplex E, to analyze target sites
12705T, 8563G, 15535T, 12882T, 11084G,
14318C, 752T, 2766T, and 9296T.
Each reaction was performed in a total volumeof 20 m L containing 5–15 ng genomic DNA, optimum concentrations of each primer (Umetsu etal. 2005), and reagents of the multiplex PCR kit(Qiagen, Hilden, Germany). The concentration of
the primers was adjusted to obtain balanced
amounts of PCR products. After an initial denaturation step of 15 min at 95°C, 32 amplification
cycles were carried out, each consisting of 10 s at
94°C, 10 s at 52°C, and 5 s at 72°C. This was followed by a final extension step of 3 min at 72°C.
If the analysis of mitochondrial single nucleotide
polymorphisms (mtSNPs) failed because of abase substitution in a primer or a problem with
the DNA quality, the mtSNP would be analyzed
by a single PCR with the corresponding primer
set.

The sizes of the PCR products ranged from 50
to 151 bp. A 2 mL aliquot of the PCR productwas separated by electrophoresis in a 6.5 cm native polyacrylamide gel (10%T, 5%C), containing 375 mM Tris-HCl buffer (pH 8.9), with running buffer (12.5 mM Tris and 96 mM glycine;pH 8.3). DNA bands were detected fluorographically after staining with SYBR Gold (Molecular
Probes, Eugene, OR, USA).
Data analysis
The 278-bp HVS1 sequence and the 283-bp
HVS2 sequence were edited and aligned against
the rCRSs using the Genetyx-Mac software (version 15). The nucleotide diversity and the mean
number of pairwise differences between the mitochondrial D-loop sequences were computed
using the Arlequin software package (version
3.0; Excoffier et al., 2005) based on Tamura-Nei
distances and a gamma parameter value of 0.26
(Mayer et al., 1999). The length polymorphismsof the A and C stretches in region from 16180 to
16188 (triggered by the substitution of T with C
at position 16189) were ignored in this analysis.
The differences between the population were also
computed using the Arlequin software (Raymondand Rousset, 1995). Neighbor-joining (NJ) trees
were constructed on the basis of the pairwise Fst
values by using the Mega 3.0 program (Kumar etal., 2004) in order to study the relationships between the populations.
For haplotypes defined in the region from16121 to 16402, the haplotype diversity withingroups was estimated by 1iÍx2i, where xi is therelative frequency in the sample of the ith haplotype (Torroni et al., 2001). Comparative data,
often comprising HVS1 sequence data (Sykes etal. 1995; Betty et al. 1996; Fucharoen et al.,
2001; Oota et al. 2002; Yao et al., 2002a,b,c;Tajima et al. 2004; Trejaut et al. 2005; Irwin etal., 2008), were obtained from the literature. The
data used included samples from Thailand, Taiwan (aboriginal population), the Philippines, Han
Chinese, and China and Vietnam (aboriginal
populations). The geographical locations of these
populations are shown in Figure 1.

Genetic structure of ethnic groups in Laos
Table 1.

29

Sequence variation in the 70 Laos individuals analyzed in the present study
Mutations in the segmentsa

Haplo- Haplotype groupEthnic Groupc

HVS 1
16121–16402 (16000)

HVS 2
128-408b

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

A*
A1
B4*
B4*
B5
B5
B5
C
C
D4b2b
D4e
D4j
D5a
D5a
F*
F*
F*
F1

223, 235, 290, 311, 319, 362
126, 223, 234, 235, 290, 319
182C, 183C, 189, 217, 261, 357
179, 182C, 183C, 189
183C, 189, 262, 266A
182C, 183C, 189, 266A
182C, 183C, 189,197G,
189, 223, 298, 327, 389
189, 223, 298, 327
145, 223
223, 362
223, 362
182C, 183C, 189, 223, 362
182C, 183C, 172, 189, 223, 362
192, 288, 304, 309, 389
260, 298, 355, 362
260, 298, 355, 362
129, 162, 172, 304

152, 183, 198, 233, 234, 309C, 315C
153, 234, 309C
309C, 315C
150, 310, 315C
209, 315C
209 ,252, 315C
210, 310, 315C
207 ,249d, 309C
N.D.
195, 309C
146, 227, 269, 309C, 315C
227, 309C
205, 309C
150, 309C, 315C
N.D.
204, 207, 249d, 279, 309C, 315C
204 ,207, 249d, 309C, 315C
249d, 309C

19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37

F1
F1
F1
F1
F1
F1
M*
M*
M*
M*
M*
M*
M*
M*
M7b
M9
N*
N*
N9a

129, 162, 172, 304
129, 172, 304
183C, 189
129, 162, 172, 304, 311
129, 304
189, 304
129, 223, 272
271
129, 166, 217, 223, 298A ,319, 381
183C, 189, 223, 311
129, 203, 223, 271
223, 311, 362, 381
214A, 223, 256, 298
215, 223, 274
129, 192, 223, 297
158, 223, 234, 311, 362
167 ,223, 249(R) 182C, 183C, 189, 311, 389, 398
223, 257A, 261, 311

249d, 309C, 315C
152, 249d, 309C
206, 249d, 309C
152, 249d, 309C
N.D.
146, 249d, 309C, 315CC, 326
152, 249d, 309C, 316
151, 309C
152, 309C, 315C
146, 150, 152, 310, 315C
143, 146, 151,309C
215, 309C
152, 309C
130, 143, 150, 309C, 315C
150, 179, 198, 309C
150, 152, 153, 309C
152, 227, 309C, 315C
184, 189, 309C, 315CC, 366
150, 227, 309C, 315C

Lao
Kham
Lum

Lao
Sun
2 (Pusan)1 (Punoi)
2 (Mon)1
4 (Mon)5 (Mon)1
1
1 (Mon)1
1 (Mon)1
1
1 (Mon)3
1 (Mon)1
2 (Puni,
Pusan)
6
1

4
1 (Mon)1 (Mon)2

3
1 (Pusan)
1
1
2
3
1
4

1
1 (Pusan)
11112 (Mon)a
All polymorphic sites are numbered according to the revised Cambridge Reference Sequence (Andrews et al.
1999). CRS denotes that the sequence of the segment is identical to the revised Cambridge reference sequence, and N.D.
indicates ‘Not Determined’. The suffix A indicates a transversion, and d indicates a deletion. Deletions are recorded at
the last possible site.
b
Nucleotide change at position 263 in the segment 128–408 was observed in all specimens, therefore, it is omitted.

Results and Discussion
The sequence variation in HVS1 and HVS2
along with haplogroup classifications by PCRAPLP methods is shown in Table 1. HVS1 andHVS2 sequences were used to gain informationon the internal population diversity. There were

37 distinct types of sequences defined by 82
polymorphic sites. Of these, 35 were unique totheir respective populations, whereas only 2(types 20 and 23) were shared by 3 Lao Lum individuals and 7 Kham individuals. Among the 35
unique types, 11 (31.4%) were shared by morethan 2 individuals within each population, where-

30

K. Shinoda, K. Umetsu and Y. Sekino

as the remaining 24 types (68.6%) were observedin a single individual. Sequencing analysis and
APLP testing indicated that mtDNA haplotypes
in the Laos samples fell into the 2 major nonAfrican haplogroups, M and N. However, 8 haplotypes could not be further classified into specific haplogroups of M that have been identified as
M* in Table 1.
The 278-bp sequences of HVS1 from the 70
individuals were aligned and compared, and thenumber of nucleotide substitutions between eachpair of sequences was estimated. Nucleotide diversity among the 70 individuals was estimatedto be 0.02, which is slightly higher than the corresponding value estimated for the Taiwanese,
East Asian, and Oceanian populations (0.014 to
0.019), and lower than that estimated for thesouth Vietnamese population (0.022) (Trejaut etal., 2005).
The haplotype diversity (H) and nucleotide diversity (p) estimated for each group are presented in Table 2. In the ethnic populations of Laos,
H ranges from 0.878 to 0.91, and p from 0.018 to
0.02. These diversity ranges are similar to thoseobserved in other ethnic groups in East Asia (Yaoet al., 2002a; Wen et al., 2005). As shown in
Table 2, the proportion of population-specifichaplotypes ranges from 63% to 75%. The proportion of population-specific haplotypes is generally high in the Laos populations; this suggeststhat these ethnic groups continue to maintaintheir own ethnicity with a lesser degree of genetic influence from other ethnic groups. Significant
differences based on haplotype frequencies wereobserved among all population pairs at a confidence level of 0.05. Tajima’s D values of these
populations were not significantly different from0 at a confidence level of 0.05, indicating no sigTable 2.
Population Size
Lao Lum
Kham
Lao Sun1227
31

Divesity Indices of Lao populations calculated from HVS1 region

Number Number of
Haplotypeofuniquediversity
haplotype haplotype
11
12
16

nal for demographic expansions. Because thesample sizes in the data of Lao Lum were relatively small, the differences observed here may
be because of insufficient sampling or potential
regional differences, and hence should be considered with caution. However, the mismatch in distribution was unequivocally multimodal (data notshown), thus contradicting the hypothesis of population growth and pointing toward the strong effects of drift and/or small sample sizes.
Phylogenetic analysis conducted using coding
region information clustered the observed haplotypes into 16 distinct haplogroups and subhaplogroups (Table 1). The 2 familiar and widespread Southeast Asian mtDNA haplogroups are
haplogroups B and R9, the latter encompassing
haplogroup F. Haplogroup F1a, which is common
and widespread in Southeast Asia, is found largely in all 3 groups, especially the Kham. Although
haplogroup B is only present at low frequencies
in the Lao Lum groups and absent in the Kham
population that we sampled, it has a high frequency in the Lao Sun group. This haplogroup,
especially haplogroup B4, has been observed athigh frequencies in the islands in Southeast Asia.
Thus, the frequency of this haplogroup reflects
the genetic affinity of this haplogroup to the islands in Southeast Asia.
Although, 3 basic haplogroups (B, D, and F)
accounted for more than 75% of the variation observed in the 3 ethnic groups, 20% of the individuals were classified into haplogroups M* and N*.
Because the diagnostic variants in the coding regions are mainly used for identifying the East
Asian population, it is difficult to classify theSoutheast Asian population further. Judging fromthe HVS1 and HVS2 sequences, the unclassified
haplogroups M and N, which are found in Laos,

7 (63.6%)
9 (75.0%)
12 (75.0%)

0.903
0.878
0.91

Nucleotide
diversotyGenediversity

Mean numberof pairwise
differences

Tajima’sD

0.0190.011
0.0180.010
0.0200.010

0.9850.040
0.9120.030
0.9400.022

5.3942.796
5.0432.527
5.5572.744

1.294
0.559
0.519

Genetic structure of ethnic groups in Laos

appear to be only very distantly related to eachother. If the remaining unclassified M* and N*
haplogroups do represent indigenous haplogroups, then it would imply that about onefourth of the modern inhabitants of Laos cantrace their maternal ancestry back to the first
anatomically modern inhabitants of Southeast
Asia.
Although haplogroup N9a is widespread in
mainland Southeast Asia and may have a southern origin, it is absent in the Lao Lum and Kham.
Haplogroup M7b, which also is widespread inSoutheast Asia, is only found in Lao Lum. The
frequencies of haplogroup F in mainland and island Southeast Asia are the highest in Kham.
Haplogroup E is the most common indigenous
haplogroup in island Southeast Asia. It is also
common in Taiwanese aboriginals but is almost
absent in China (Hill et al., 2007). Moreover, it isalso absent in the samples examined in the present study. This means that there is only some genetic connection between the island areas and inTable 3.

31

land regions like Laos. In the present study, theresults of the mtDNA analyses demonstrate thatthe populations of the islands in Southeast Asia
have a different ancestry from most of the Laos
population. The high frequencies of haplogroups
in the north, such as A, C, and D (Yao et al.
2002a; Kong et al. 2003), found in the Lao Sun
samples were consistent with their Chinese orVietnamese origins.
To clarify the genetic characteristics of the regional population, the distribution of haplogroupfrequencies among the sampled groups was compared to information available from other Asian
populations in nearby regions (Figure 1). The
populations in the nearby regions show great genetic and linguistic heterogeneity; further, thehaplogroup frequencies varied among these populations (Table 3). Genetic differences (Fst values) among these 12 populations are shown in
Table 4. With regard to the population of Laos,
Fst values are highest between Kham and allother populations; this indicates that the Kham is

The Haplogroup Distribution Frequencies (%) in the 12 Populations Considered in the Present Study

Haplo Lao-group Lum

Kham

Lao
Sun

Thaia

Vietnam Vietnam Aboriginal(north)b (south)c Taiwanesed

Philip-pineeSouth
China(Han)fKunming(Han)g

Daih

Vah

A
B4
B5
C
D4
D5
E
F
M*
M7b
M7c
M9
N*
N9a
Y
n

0
0
0
0
3.7
3.7
0
59.3
29.6
0
0
0
3.7
0
0
27

9.7
6.5
29
3.2
3.2
3.2
0
22.6
16.1
0
0
0
0
6.5
0
31

2.8
14.1
15.3
4
2.8
1.1
0
28.7
14.2
4.5
2.8
6.8
0.6
2.3
0
177

1.7
10.7
12.4
4
2.8
1.1
0
22
15.8
14.1
3.4
0.6
6.3
5.1
0
187

0
25.4
8.5
0
0
0
16
10.6
12.8
6.4
12.8
0
4.3
0
3.2
94

0.8
22.7
2.5
1.7
15.1
5
0
23.6
8.4
6.7
2.5
0.8
7.6
2.5
0
119

4.7
11.7
7
4.7
18.6
4.6
0
23.3
2.1
16.3
0
0
0
7
0
43

3.2
11.3
8.1
9.7
8.1
4.8
0
24.1
4.9
11.3
1.6
0
9.7
3.2
0
60

1.7
1.7
1.7
0
20.7
1.7
0
39.6
26
0
0
5.2
1.7
0
0
70

0
8.3
8.3
8.3
8.3
0
0
33.3
8.3
8.3
0
8.3
8.3
0
0
12

0
20
5.7
2.9
0
0
0
11.4
20
11.4
0
0
0
5.7
22.9
35

0
25.9
5.9
0
0
4.8
11.4
26.6
1.9
9
9
0
2.9
1.2
1.4
640

a: Data from Fucharoen et al. (2001)
b: Irwin et al. (2008)
c: Oota et al. (2002)
d: Trejaut et al. (2005)
e: Phillippine include Luzon (Sykes et al. 1995) and Philippine (Tajima et al. 2004)
f: South China (Han) included Guangdong and Hong Kong Han Chinese. (Betty et al. 1996, Yao et al. 2002c)
g: Yao et al. (2002c)
h: Yao et al. (2002b)

32

K. Shinoda, K. Umetsu and Y. Sekino
Table 4.
Lao
Lum

Lao Lum
Kham
Lao SunThai
N. Vietnam
S. Vietnam
TaiwanPhillippin
South China
KunmingDai
Va

00.050180.00103
0.02856
0.021830.01449
0.01035
0.03746
0.01561
0.02182
0.03659
0.00407Kham

00.12560.077890.09458
0.150810.134610.179210.114940.134170.11176
0.02345

Fig. 2.

LaoSun

00.00673
0.01633
0.042810.07258
0.06671
0.05695
0.042020.02825
0.07815

Pairwise Fst values between each pair of population
Thai

00.00859
0.021890.03529
0.04665
0.02530.027290.01418
0.04824

N.
S.
Vietnam Vietnam

00.00587
0.039640.039160.02367
0.01733
0.00356
0.06147

00.042070.021120.02530.03935
0.028450.09742

Taiwan Phillippin

00.020420.024910.04069
0.029240.1101700.03983
0.06357
0.045310.13081

South
China

KunmingDai00.0068 00.00602 0.00396 00.05991 0.06513 0.06705

Va0

Neighbor-joining tree based on the Fst values determined for 12 populations.

genetically distinct from the other populations
living in Southeast Asia.
These genetic affinities are reflected in the NJ
trees based on both the pairwise Fst values (Figure 2). This analysis of the haplogroup profiles
presents a general view of the genetic differences. As demonstrated in Figure 2, the aboriginal Taiwanese and Philippine samples showed aclose genetic relationship, and it stands to reason
that the South Han Chinese and Kunming Han
Chinese populations are closely related to eachother. Further, populations in the northern part ofSoutheast Asia and the southern part of South
China displayed a relatively short genetic distances. As expected, the Lao Sun was also in-cluded in this cluster. This is in agreement withthe hypothesis that these populations are descendants of the immigrants from southern China or
Vietnam.
On the other hand, Kham and Va are separated
from all other populations by a large genetic distance. Further, it is evident that the Lao Lum occupies an intermediate position. It is interesting
to note that the Va are regarded as the earliest inhabitants of southwest Yunnan, and they speak
the Mon-Khmer language that belongs to thefamily of south Asian languages (Du and Yip,
1993). Yao et al. (2002b) pointed out that the Va
may receive genetic contributions from north or
northwest China, possibly during the southwest

Genetic structure of ethnic groups in Laos

migration of the ancient Di-Qiang tribe. The maternal genetic structure of the Kham and Va population bears the signatures of both southern andnorthern populations. Both ethnic groups haverelatively high frequencies of F1 lineages (south)and D lineages (north). In addition, the Kham
contains relatively high frequencies of indigenous Southeast Asian M* lineages. According to
ethnological data, individuals of the Kham population are believed to have migrated from the
Yangtze River basin in prehistoric times. It is also
likely that they acquired genetic contributions
during the southwest migration from their ancestral area.
The aim of this study was to gain insights into
variations in mtDNA and determine the originsof the 3 ethnic groups in Laos. In particular, wewanted to test the hypothesis based on ethnological data that states that the 3 ethnic groups stemfrom a different ancestral population. In somecases, the mtDNA analyses revealed clear resultson this issue. However, the origin and development of populations of ethnic groups are very
complex processes. Therefore, hypotheses thatare based on different data sets or different methods of analysis should contradict each other forthe reconstruction of the population history. In
this study, we focused on the 3 ethnic populations of Laos to infer genetic relationships
among the individuals. It is necessary to conduct
further studies for many of the remaining ethnic
groups in order to reconstruct a more realistic depiction of the peopling of Laos as well as that ofSoutheast Asia as a whole.
Acknowledgments
We are grateful to all the volunteers who donated DNA samples for this study. This research
was supported in part by a Grant-in-Aid for Scientific Research (No. 20370096) from the Ministry of Education, Culture, Science and Technology of Japan.

33

References
Andrews R.M., Kubacka I., Chinnery P.F., Lightowlers
R.N., Turnbull D.M., and Howell N. (1999) Reanalysis
and revision of the Cambridge reference sequence forhuman mitochondrial DNA. Nature Genetics, 23: 147.
Barker G., Barton H., Bird M., Daly P., Datan I., Dykes
A., Farr L., Gilbertson D., Harrison B., Hunt C., Higham T., Kealhofer L., Krigbaum J., Lewis H., McLaren
S., Paz V., Pike A., Piper P., Pyatt B., Rabett R.,
Reynolds T., Rose J., Rushworth G., Stephens M.,
Stringer C., Thompson J., and Turney C. (2007) The“human revolution” in lowland tropical Southeast Asia:
the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo). Journal of
Human Evolution, 52: 243–261.
Bellwood P. (1997) Prehistory of the Indo-Malaysianarchipelago. University of Hawaii Press, Hawaii.
Betty D.J., Chin-Atkins A.N., Croft L., Sraml M., andEasteal S. (1996) Multiple independent origins of the
COII/tRNALys intergenic 9-bp mtDNA deletion in aboriginal Australians. American Journal of Human Genetics, 58: 428–433.
Du R. and Yip V.F. (1993) Ethnic groups in China. Science Press, Beijing.
Excoffier L., Laval G., and Schneider S., 2005. Arlequin(version 3.0): An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1: 47–50.
Fucharoen G., Fucharoen S., and Horai S. (2001) Mitochondrial DNA polymorphisms in Thailand. Journal of
Human Genetics. 46: 115–125.
Hill C., Soares P., Mormina M., Macaulay V., Meehan W.,
Blackburn J., Clarke D., Raja J.M., Ismail P., Bulbeck
D., Oppenheimer S., and Richards M. (2006) Phylogeography and ethnogenesis of aboriginal Southeast
Asians. Molecular Biology and Evolution. 23:
2480–2491.
Hill C., Soares P., Mormina M., Macaulay V., Clarke D.,
Blumbach P.B., Vizuete-Forster M., Forster P., Bulbeck
D., Oppenheimer S., and Richards M. (2007) A Mitochondrial stratigraphy for island Southeast Asia. American Journal of Human Genetics, 80: 29–43.
Irwin J., Saunier, J.L., Strouss K.M., Diegoli T.M., Strurk
K.A., O’Callaghan J.E., et al. (2008) Mitochondrial
control region sequences from a Vietnamese population
sample. International Journal of Legal Medicine, 122:
257–259.
Kong Q. P., Yao Y. G., Liu M., Shen S. P., Chen C., Zhu
C. L., Palanichamy M. G., and Zhang Y. P. (2003) Mitochondrial DNA sequence polymorphisms of five ethnic populations from northern China. Human Genetics,
113: 391–405.
Kumar S., Tamura K., and Nei M. (2004) MEGA3: Inte-34

K. Shinoda, K. Umetsu and Y. Sekino

grated software for molecular evolutionary genetics
analysis and sequence alignment. Brief Bioinformatics,
5: 150–163.
Lertrit P., Poolsuwan S., Thosarat R., Sanpachudayan T.,
Boonyarit H., Chinpaisal C., and Suktitipat B. (2008)
Genetic history of Southeast Asian populations as revealed by ancient and modern human mitochondrial
DNA analysis. American Journal of Physical Anthropology, 137: 425–440.
Lewis M.P. (2009) Ethnologue: Languages of the world,
Sixteenth edition. Dallas, Texas.
Li H., Cai X., Winograd-Cort E.R., Wen B., Cheng X.,
and Qin Z. (2007) Mitochondrial DNA diversity andpopulation differentiation in Southern East Asia. American Journal of Physical Anthropology, 134: 481–488.
Matsumura H., Oxenham M.F., Dodo Y., Domett K., Thuy
N.K., Cuong N.L., Dung N.K. Huffer D., and Yamagata
M. (2008) Morphometric affinity of the late Neolithic
human remains from Man Bac, Ninh Binh Province,
Vietnam: Key skeletons with which to debate the “Twolayer” hypothesis. Anthropological Science, 116:
123–148.
Mayer S., Weiss G., and von Haeseler A. (1999). Patternof nucleotide substitution and rate of heterogeneity inthe hypervariable regions I and II of human mtDNA.
Genetics, 152: 1103–1110.
Moodley Y., Linz B., Yamaoka Y., Windsor H.M.,
Breurec B., Wu J.Y., Maady A., Thiberge S.,
Phuanukoonnon S., Jobb G., Siba P., Graham D.Y.,
Marshall B.J., and Achtman M. (2009) The Peopling of
the Pacific from a bacterial perspective. Science, 323:
527–530.
Oota H., Kitano T., Jin F., Yuasa I., Wang L., Ueda S.,
Saitou N., and Stoneking M. (2002) Extreme mtDNA
homogeneity in continental Asian populations. American Journal of Physical Anthropology, 118: 146–153.
Patcharee L., Poolsuwan S., Thosarat R., SanpachudayanT., Boonyarit H., Chinpaisal C., Suktitipat B. (2008)
Genetic history of Southeast Asian populations as revealed by ancient and modern human mitochondrial
DNA analysis. American Journal of Physical Anthropology, 137: 425–440.
Raymond M. and Rousset F. (1995) An exact test for population differentiation. Evolution, 49: 1280–1283.
Soares P., Trejaut J.A., Loo J.H., Hill C., Mormina M.,
Lee C.L., Chen Y.M., Hujashov G., Peter Forster,
Macaulay V., Bulbeck D., Oppenheimer S., Lin M., andRichards M. (2008) Climate Change and postglacial
human dispersals in Southeast Asia. Molecular Biology
and Evolution, 25: 1209–1218.
Sykes B., Leiboff A., Low-Beer J., Tetzner S., andRichards M. (1995) The origins of the Polynesians: aninterpretation from mitochondrial lineage analysis.

American Journal of Human Genetics, 57: 1463–1475.
Tajima A., Hayami M., Tokunaga K., Juji T., Matsuo M.,
Marzuki S., Omoto K., and Horai S. (2004). Genetic
origins of the Ainu inferred from combined DNA
analyses of maternal and paternal lineages. Journal of
Human Genetics, 49: 187–193.
Torroni A., Bandelt H.J., Macaulay V., Richards M., Cruciani F., Rengo C., Martinez-Cabrera V., Villems R.,
Kivisild T., Metspalu E., Parik J., Tolk H.V., Tambets
K., Forster P., Karger B., Francalacci P., Rudan P., Janicijevic B., Rickards O., Savontaus M.L., Huoponen K.,
Laitinen V., Koivumäki S., Sykes B., Hickey E., Novelletto A., Moral P., Sellitto D., Coppa A., Al-Zaheri N.,
Santachiara-Benerecetti A.S., Semino O., and Scozzari
R. (2001) A signal, from human mtDNA, of postglacial recolonization in Europe. American Journal of
Humman Genetics, 69: 844–852.
Trejaut J.A., Kivisild T., Loo J.H., Lee C.L., He C.L., Hsu
C.J., Li Z.Y., and Lin M. (2005) Traces of archaic mitochondrial lineages persist in Austronesian-speaking
Formosan populations. PLoS Biol. DOI: 10.1371/journal.
pbio. 0030247 (http://biology.plosjournals.org/)
Umetsu K., Tanaka M., Yuasa I., Saitou N., Takeyasu T.,
Fuku N., Naito E., Ago K., Nakayashiki N., MiyoshiA., Kashimura S., Watanabe G., and Osawa M. (2001)
Multiplex amplified product-length polymorphism
analysis for rapid detection of human mitochondrial
DNA variations. Electrophoresis, 22: 3533–3538.
Umetsu K., Tanaka M., Yuasa I., Adachi N., Miyoshi A.,
Kashimura S., Park K.S., Wei Y.H., Watanabe G., andOsawa M. (2005) Multiplex amplified product-length
polymorphism analysis of 36 mitochondrial single-nucleotide polymorphisms for haplogrouping of East
Asian populations. Electrophoresis, 26: 91–98.
Wen B., Li H., Gao S., Mao X., Gao Y., Li F., Zhang F.,
He Y., Dong Y., Zhang Y., Huang W., Jin J., Xiao C.,
Lu D., Chakraborty R., Su B., Deka R., Jin L. (2005)
Genetic structure of Hmong-Mien speaking populations in East Asia as revealed by mtDNA lineages. Molecular Biology and Evolution, 22: 725–734.
Yao Y.G., Nie L., Harpending H., Fu Y.X., Yuan Z.G., andZhang Y.P. (2002) Genetic relationship of Chinese ethnic populations revealed by mtDNA sequence diversity.
American Journal of Physical Anthropology, 118:
63–76.
Yao Y.G., and Zhang T.P. (2002b) Phylogeographic analysis of mtDNA variation in four ethnic populations fromYunnan province: new data and reappraisal. Journal of
Human Genetics, 47: 311–318.
Yao Y.G., Kong Q.P., Bandelt H.J., Kivisild T., Zhang Y.P.
(2002c) Phylogenetic differentiation of mitochondrial
DNA in Han Chinese. American Journal of Human Genetics, 70: 635–651.