Am. J. Hum. Genet. 43:134-143, 1988

Mitochondrial DNA Polymorphism among Five
Asian Populations
S. Harihara,*l' N. Saitou,* M. Hirai,* T. Gojobori,t K. S. Park,4 S. Misawa,§ S. B. Ellepola,1T. Ishida,# and K. Omoto**Department of Anthropology, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo; tNational Institute of Genetics, Mishima, Shizuoka,
Japan; tDepartment of Biology, Sung-Shin Women's University, Seoul; §Department of Legal Medicine, Institute of Community Medicine,
University of Tsukuba, Tsukuba, Ibaraki, Japan; IlDepartment of Medicine, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka;
and #Primate Research Institute, Kyoto University, Inuyama, Aichi, Japan

Summary

Mitochondrial DNA (mtDNA) polymorphisms were detected using 13 restriction enzymes on the total
DNA obtained from blood samples of five Asian populations: Japanese and Ainu of northern Japan, Korean, Negrito (Aeta) of the Philippines, and Vedda of Sri Lanka. Of a total of 28 restriction-enzyme
morphs detected, eight had not been reported previously. By combining the morphs, we were able to classify mtDNAs of 243 individuals into 20 mtDNA types. Phylogenetic analyses using maximum parsimony
and genetic distance methods both showed that the Japanese, Ainu, and Korean populations were closely
related to each other. Aeta was found to show a relatively close relationship to these three populations,confirming the conclusion from previous studies of blood markers. In contrast, Vedda was quite different
from the other four populations.
Introduction

Polymorphism of mitochondrial DNA (mtDNA) detected by restriction enzymes has been analyzed inmost major racial groups, and its usefulness in population genetic studies has been amply demonstrated(see, e.g., Brown 1980; Johnson et al. 1983; Cann etal. 1987). However, relatively little is known about
the mtDNA types of the isolated indigenous groupsof Asia whose racial origins have been in dispute. In
the present paper we deal with three such groups:
Ainu of northern Japan, Negrito of the Philippines,
and Vedda of Sri Lanka (fig. 1).
All of these groups are known to have been huntergatherers until recent times and are considered to betruly indigenous groups of the areas they inhabit.
Received November 25, 1987; revision received March 7, 1988.
1. Present address and address for correspondence and reprints:
Dr. Shinji Harihara, Department of Legal Medicine, Institute ofCommunity Medicine, University of Tsukuba, Tsukuba, Ibaraki
305, Japan.
X 1988 by The American Society of Human Genetics. All rights reserved.
0002-9297/88/4302-0005$02.00

134

Ainu and Negrito show morphological features
markedly different from those of the surrounding
mongoloid groups. Thus, in anthropological literatures they often have been considered to belong to thecaucasoid and to the negroid or australoid groups,
respectively. On the contrary, recent genetic studies
using protein and antigen markers from blood indicated that their genetic affinities were closest to theeast-Asian mongoloid groups (Misawa and Hayashida 1968; Omoto 1972, 1984). Our previous study
on mtDNA polymorphism among Ainu and Japanese (Harihara et al. 1986) also supported the finding
about Ainu.
However, since Ainu and Negrito today show considerable admixture with surrounding mongoloid
populations, there had remained some reserve about
the validity of the close genetic affinities to mongoloids. The origin of Vedda is also unknown, but aprevious genetic study has suggested a link to mongoloids (Kirk et al. 1964).
In the present study the mtDNA types of the Ainu,
Negrito, and Vedda groups are described, together
with those of the Japanese and Korean groups, and135

mtDNA Polymorphism

Japaneseand AinuaFive Asian populations of which sam ples were anaFigure I
lyzed in this study.

used for analyses of genetic affinities of thkese populations. It was hoped that the maternallly inherited
mtDNA (Hutchison et al. 1974) may bse relatively
resistant to gene flow from outside, whiich is more
likely to be due to migrant males than to migrantfemales.
Material and Methods

Blood samples were collected from five Asian populations. Samples of Ainu (48 individuals;) and Japanese (74 individuals) were obtained in the Hidaka
district of Hokkaido, northern Japan. The2se were thesame samples as those used in the previoius study by
Harihara et al. (1986). Samples of 64 in(dividuals of
the Korean population were from Seoul. I lhe Negrito(Aeta) samples (37 individuals) were colle cted on the
Bataan Peninsula of Luzon in the Philippiries. Twenty
blood samples of Vedda were from Hena nagawa, Sri
Lanka.
Total DNA extracted from blood ce lls was digested with the following 13 restrictioln enzymes:
AvaII, BamHI, EcoRI, HincII, HincMI, KpnI,
HpaI, MspI, PstI, PvuII, SstI, XbaI, and1 XhoI. For
the Japanese, only five enzymes (AvalII, BamHI,
HincIl, HpaI, and PvuII) were used. We did not use
the other enzymes, because Horai et al. (1984) andHorai and Matsunaga (1986) had found a very low(if any) degree of polymorphism with thei m. Digested
DNAs were separated on agarose gel (Hladler et al.
1983), and digestion patterns of mtDNjA were detected by a hybridization technique (Sout:hern 1975;
Botchan et al. 1976). The probe used uvas purified

mtDNA from either human placentas or cultured human cells which was nick-translated by the method of
Rigby et al. (1977).
Mitochondrial DNAs of all the individuals wereclassified into mtDNA types by combining the enzyme morphs for each individual. Genetic distancesamong mtDNA types were calculated by the methodof Nei and Li (1979) and Nei and Tajima (1983).
Genetic distances among populations were also calculated by the method of Nei and Li (1979). Phylogenetic trees were constructed by applying the unweighted pair-group method of analysis (UPGMA;
Sokal and Sneath 1963) to the genetic distance data.
The phylogenetic relationship of mtDNA types
was also depicted by the maximum parsimony
method (Camin and Sokal 1965; Eck and Dayhoff1966): an unrooted tree was constructed to connect
all types by setting the total number of changes to aminimum.
Results
mtDNA Morphs for Each Restriction Enzyme

Variations in mtDNA digestion patterns were detected by AvaII, HincII, HpaI, MspI, PstI, PvuII, and
XhoI. A total of 28 enzyme morphs were detected,
20 of which had been reported elsewhere (Denaro etal. 1981; Blanc et al. 1983; Johnson et al. 1983;
Horai et al. 1984; Harihara et al. 1986; Horai andMatsunaga 1986). Eight newly found morphs (fig. 2)
are as follows:
Aval.-Three new morphs were found by using
AvaII in the Korean, Aeta, and Vedda groups, andwe called these AvaII morphs 24Korean, 25Aera, and26Vedda, respectively (fig. 2a). Superscripts denote theorigin of the samples.
AvaII morph 24Korean was a site-gain variant, detected in one individual. In this morph, a 3.0-kb fragment of AvaII morph 1 (Johnson et al. 1983) was
split into two fragments of almost the same size, 1.5
kb, and they could not be distinguished on the autoradiogram. The new site is at or near bp 14900 ofAnderson et al.'s (1981) human mtDNA sequence,and the change is located in the coding region ofcytochrome b.
In AvaII morph 25Aeta, a 9.8-kb fragment of AvaIlmorph 1 became two fragments, one 6.6 kb and theother 3.2 kb. Double digests with SstI and AvaII cut
a 6.6-kb fragment into a 3.6-kb fragment and a
3.0-kb fragment, indicating that the extra site is lo-Ava]I morphs
1

24Korean

9.8

1

1

25Aeta

9.8

6.6

1

26Vedda

9.8

6.0

2.4

2.2 2.1

3.8

3.0

3.2

3.0

3.0

1

0.9
0.8

1.0

0.8
0.7

0.8
0.7

3Ainu

XhoI morphs
XbaI morphs
1
1

14.5

12Aeta0.7

0.5 0.5
0.5

1.0

1.0

PstI morphs
1

.5

1 .2
1.1

1.5

0.8
0.7

.-1,i

MspI morphs

7.5
4.5

2.0
1.8

1

2Ainu

9.5

3Ainu

7.5
5.3

16.5

4.5

2.0

4Korean

*:

1 .8

,'

..

..

,'$.

4.0

-...

4.:

,

rv...

...

P;

2.1*

0.8

1< . 2 . 5

:g:

0.8

..

2

Figure 2
Autoradiogram of eight new mtDNA morphs detected in the present study. Morphs
denoted by numbers at top oflanes, and fragment sizes are designated in kilobases. a, AvaIl morphs 1, 24K rC, 25Aera and 26Ved.a b, Mspl morphs 1 and 12A.CI, PStI
morphs 1 and 3Anu. d, XbaI morphs 1, 2 , and 3Anu. e, XboI morphs 1 and 4Korean*
are

137

mtDNA Polymorphism

cated approximately at bp 6000, in the coding regionof cytochrome c oxidase subunit I.
AvaII morph 26Vedda ic also a site-gain variant, andtwo fragments (6.0 kb and 3.8 kb) were seen insteadof the 9.8-kb fragment of AvaII morph 1. A 6-kb
fragment became a 2.9-kb fragment and a 3.0-kb
fragment after double digests with SstI and Avail,indicating that the extra site is located approximately
at bp 6700, which is in the coding region of cytochrome c oxidase subunit I.
Mspl.-A new morph was found in Aeta in thepresent study and was named MspI morph 12Aeta (fig.
2b). In this morph, a 0.7-kb fragment, which was notdetected in MspI morph 1 (Johnson et al. 1983), wasgenerated without the splitting of any larger fragments. This seems to be the result of fusion of one ofthree 0.5-kb fragments of MspI morph 1 and theadjacent 0.2-kb fragment, which could not be detected on the autoradiogram in the present study; andthe site loss was located at bp 16453 in the noncoding region.
Pstl.-PstI morph 3Ainu was newly found in Ainu
(fig. 2c). This morph is generated by a site gain in thenoncoding region, and the 14.5-kb fragment of PstI
morph 1 (Horai et al. 1984) was split into a 7.3-kb
fragment and a 7.1-kb fragment. Double digests with
EcoRI and PstI generated three fragments (4.5 kb,
1.6 kb, and 1.1 kb) from the 7.3-kb fragment andtwo fragments (3.6 kb and 3.5 kb) from the 7.1-kb
fragment, indicating that the mutation is located approximately at bp 16200 in the noncoding region.
Xbal.-Horai et al. (1984) reported no variant
XbaI morph in Japanese subjects, and all individuals
were XbaI morph 1. In the present study, two newmorphs were detected in Ainu (XbaI morphs 2Ainu
and 3Ainu) (fig. 2d). In XbaI morph 2Ainu, a site loss
occurred at bp 10256 in the coding region of NADH
dehydrogenase subunit 3 (Chomyn et al. 1985), and a
9.5-kb fragment was generated by fusion of a 7.5-kb
fragment and a 2.0-kb fragment of XbaI morph 1.
Xbal morph 3Ainu is also a site-loss variant, the loss
being located at bp 7440 in the coding region of cytochrome c oxidase subunit 1; and a 4.5-kb fragment
and a 0.8-kb fragment fused to form a 5.3-kb fragment.
Xhol.-In XhoI morph 4Korean detected in the Korean group, two fragments, one of 14.0 kb and theother of 2.5 kb, could be seen instead of a 16.5-kb
single fragment of XhoI morph 1 (Horai et al. 1984)(fig. 2e). Sizes of fragments generated by double digests with PstI and XhoI were 8.5 kb, 3.5 kb, 2.5 kb,

and 2.1 kb. The extra site was determined to belocated approximately at bp 12500, in the coding
region of NADH dehydrogenase subunit 5 (Chomyn
et al. 1985).
The frequency distribution of enzyme morphs foreach population is listed in table 1. Except for thedominant ones, the frequencies of most variant
morphs are low in every population, whereas AvaII
morphs 25Aeta and 26Vedda are relatively frequent in
Aeta (10.8%) and Vedda (25.0%), respectively.
mtDNA Types Defined by Eight Enzymes in Five
Asian Populations

When all enzyme morphs for each individual werecombined, mtDNAs of 243 individuals of the five
populations were classified into 20 mtDNA types;
their distribution is listed in table 2. HpaI morphs
were excluded from this analysis, since HpaI morphs
1 and 4 correspond to HincII morphs 1 and 3, respectively, and since HpaI morph 2 is divided into HincII
morphs 2 and 5. The reason for this is that (1) therecognition sequence of Hpal (GTTAAC) is one offour recognition sequences of HincII and (2) there
was no HincIl morph that was divided into two HpaI
morphs in the present study. In other words, HpaI
happened to be not informative in the present study.
In every population, type 1 is the most frequentand the frequencies of other mtDNA types are low,
except for type 19 in Vedda (25.0%).
Phylogeny of mtDNA Types

The phylogeny of 20 mtDNA types is shown as an
unrooted tree obtained by the maximum parsimony
method (fig. 3). All variant mtDNA types can bederived from the most frequent one (type 1) throughone or two changes. A hypothetical type, X, isneeded to connect types 1 and 16. The variant enzyme morph in type X is either AvalI morph 17 or
XhoI morph 4. A total of 20 lines are needed toconnect 21 mtDNA types. The tree in figure 3 is oneof 15 possible ways of connection that are equallyparsimonious. In one alternative way, for example,
types 7 and 8 and types 6 and 20 are connected
whereas the line connecting types 4 and 8 and thatconnecting types 7 and 20 are cut.
Johnson et al. (1983) reported mtDNA polymorphism among Caucasians, Orientals (mainly Chinese), Bantu, Bushman, and American Indian. They
used five restriction enzymes, four of which-AvaII,
BamHI, HpaI, and MspI-were also used in thepresent study. When results obtained in the presentHarihara et al.

138
Table I
Number of Individuals and Percentage of Enzyme Morphs for Each Population
No. (%)MORPH

AvaII:
1....................
2....................
3....................
5 ....................
10 ....................
12 ....................
14 ....................
24Korean24
25sAta.
26tVedda.
26
HincII:
2....................
1....................
3....................
5....................
...............

.....

.......

**....

REFERENCEa

Japanese(N = 74)

Ainu(N = 48)

Korean(N = 64)

Aeta(N = 37)

Vedda(N = 20)

Total(N = 243)

3
3
3
3
3
S
6
7
7
7

66 (89.2)
1 (1.4)00
3 (4.1)1 (1.4)
1 (1.4)00
0

48 (100.0)
0
0
0
0
0
0
0
0
0

64 (96.9)
1 (1.6)00
0
00
1 (1.6)0033 (89.2)
0
0
0
0
0
0
0
4 (10.8)
013 (65.0)0
1 (5.0)1 (5.0)00000
5 (25.0)224 (92.2)
2 (.8)
1 (.4)
1 (.4)
3 (1.2)
1 (.4)
1 (.4)
1 (.4)
4 (1.6)5 (2.1)2
2
2
2

64 (86.5)5 (6.8)3 (4.1)2 (2.7)46 (95.8)
1 (2.1)1 (2.1)056 (87.5)
4 (6.3)
1 (1.6)3 (4.7)37 (100.0)00020 (100.0)000223 (91.8)
10 (4.1)
5 (2.1)5 (2.1)11
1

66 (89.2)
5 (6.8)3 (4.1)46 (95.8)
1 (2.1)1 (2.1)59 (92.2)
4 (6.3)
1 (1.6)37 (100.0)00

20 (100.0)00228 (93.8)
10 (4.1)
5 (2.1)

3
7

48 (100.0)064 (100.0)035 (94.6)
2 (5.4)20 (100.0)
0

4
4
7

47 (97.9)
0
1 (2.1)62
2(96.9)(3.1)
0

37 (100.0)00

20 (100.0)00

48 (100.0)064 (100.0)037 (100.0)020 (100.0)
0

4
7
7

45 (93.8)
2 (4.2)1 (2.1)64 (100.0)0037 (100.0)00

20 (100.0)
0
0

4
7

48 (100.0)063 (98.4)
1 (1.6)37 (100.0)020 (100.0)0

HpaI:
2....................
1....................
4....................

MspI:
1....................
12Ata..............

PstI:
1....................
2....................
3 Ainu.
*in................

PvuII:
1....................
3....................
XbaI:
1....................
2Ainu.
Ain
3 Ainu.
................

XhoI:
1....................
4Korean.

4
6

73 (98.6)
1 (1.4)242 (99.6)
1 (.4)

a 1 = Denaro et al. (1981); 2 = Blanc et al. (1983); 3 = Johnson et al. (1983); 4 = Horai et al. (1984); 5 = Horai and Matsunaga(1986); 6 = Harihara et al. (1986); 7 = present study.

study are combined with their data on variation in
mtDNAs' patterns of digestion by the four restriction
enzymes, a total of 37 mtDNA types (named
mtDNA types 1-37) are produced for the 10 populations. Note that this numbering system is different
from that of table 2.
Genetic distances for these mtDNA types were calculated, and a UPGMA tree was constructed (fig. 4).
In this tree, two main clusters (A and B) are depicted

if three types (16, 19, and 37) that are apart from theothers are excluded. Cluster A consists of mtDNA
types found in non-African populations, except for
type 1 (which is the most frequent) and type 2. On
the other hand, there are four subclusters (1, 2, 3, and
4) in cluster B, and two of them (subclusters 2 and 4)
consist of African mtDNA types exclusively. The
other two subclusters (1 and 3) contain mtDNA
types found in the non-African populations. Al-

mtDNA Polymorphism

139

Table 2
Number of Individuals and Percentage of mtDNA Types for Each Population
No. (%)

Japanese(N = 74)

Ainu(N = 48)

Korean(N = 64)

Aeta(N = 37)

Vedda(N = 20)(N = 243)

1 .................................... 57 (77.0)
2 (HincIl 1) ................................ 4 (5.4)
3 (AvaIl 10) ................................ 3 (4.1)
4 (HincII3) ................................ 2 (2.7)
5 (HincII 5) . .............................. 2 (2.7)
6 (AvaIl) ................................
1 (1.4)7 (AvaII3) ................................
1 (1.4)8 (AvaII 3, HincII 3) .........
..............
1 (1.4)9 (AvaII 12, HincII 1) .........
1 (1.4).............
10 (AvaII 14) . .............................. 1 (1.4)
11 (PvuII 3) ................................
1 (1.4)12 (XbaI 2AinU).
.0
13 (PstI 3AinU).
.0
14 (XbaI3Ainu).
.0
15 (PstI 2) .................................
0
16 (AvaII 24Korean, XhoI 4Korean) ..0
17 (AvaII 25Acta) ............................
0
18 (AvaII 25Aeta, MspI 12Aeta)..0
19 (AvaII 26Vedda) ...........................
0
20 (AvaII5).
.0

42 (87.5)
1 (2.1)0
1 (2.1)
0
0
0
0
0
0
0
2 (4.2)1 (2.1)1 (2.1)
0
0
0
0
0
0

52 (81.3)
4 (6.3)0
1 (1.6)3 (4.1)1 (1.6)
0
0
0
0
0
0
0
0
2 (3.1)1 (1.6)000033 (89.2)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2 (5.4)2 (5.4)00

13 (65.0)000001 (5.0)
0
0
0
0
0
0
0
0
0
0
0
5 (25.0)
1 (5.0)197 (81.7)
9 (3.7)3 (1.2)4 (1.6)5 (2.1)2 (.8)
2 (.8)
1 (.4)
1 (.4)
1 (.4)
1 (.4)
2 (.8)
1 (.4)
1 (.4)
2 (.8)
1 (A4)
2 (.8)
2 (.8)
5 (2.1)1 (.8)

mtDNA TYPE

Total

NoTE.-mtDNA types are classified by combining the enzyme morphs for each individual. Enzyme morphs of the most frequent type,
type 1, are AvaII morph 1, HincII morph 2, MspI morph 1, PstI morph 1, PvuII morph 1, XbaI morph 1, and XhoI morph 1. Variant
enzyme morphs in each type are listed in parentheses. We assumed that no variation could be detected in the Japanese by BamHI, EcoRI,
HindIII, KpnI, MspI, PstI, XbaI, and XhoI.

9J

18N

|

\
16

8

2JAK

4JAK

17

\

14A
1

A

13 I:7~
1

6

2J

J:

Japanese

A:
X:
N :
V:

Ainu
KoreanNegrito (Aeta)
Vedda

/

J

though two main clusters (A and B) are observable infigure 4, the phylogenetic relationships of the populations are not clear.
Therefore, we also applied the maximum parsimony method to the same data set. Figure 5 is oneof the possible maximum parsimony trees. Four hypothetical mtDNA types (X1, X2, X3, and X4) arenecessary to complete the tree. All mtDNA types canbe divided into two main groups separated by adashed line. One of these main groups is for the
mtDNA types detected only in African populations(Bantu and Bushman), and we call this the Africangroup; these results confirm those of Johnson et al.
(1983). The other (non-African) group contains all of
the mtDNA types found in the non-African populaPossible maximum parsimony unrooted tree for
Figure 3
the phylogeny of 20 mtDNA types listed in table 2. Subscripts by
the numbers of each of the mtDNA types indicate the populations
from which the individuals were sampled. Type X is hypothesized
to complete the phylogenetic tree.

Harihara et al.

140
a
3

Genetic Distance (D)

0.5

1 .0

X 0.01
1 .5

2.0

I B' B 0 C

o - 23C - 22C - 17K - 10
V 13ACJ - 3
o - 24C - 18N - 11
N - 12O K A J- 4
J- 7
B O K A J - 2
J- 8
o - 25C K J - 5
C - 21B' B - 28
B' - 36
B - 31
B' - 35
B' B - 29
vJ - 6
C V - 14
C - 1
C - 20J- 9
B' B - 26
B' B - 27
B - 34B' B - 30
B - 32B - 33B' - 37
C - 16

J:
A:
K:
N:
V:
C:

Japanese
Ainu
KoreanNegrito (Aeta)
Vedda
Caucasian
0: Oriental
B: Bantu
B': Bushman
I: American
Indian-C - 19Figure 4
UPGMA phylogenetic tree based on genetic distances for 37 mtDNA types in 10 human populations. Letters at the left sideof the number of each mtDNA type indicate the populations from which the individuals were sampled. Note that this numbering system isdifferent from that of table 2 and fig. 3.
ArRICAN GRouP
36.

250

58

.37

X4

30 BD

xi

Noot-AFRICAN GROUP

8i,

315

298 X3N

X2
34~ /,-

3\u
\R

:%
A

14;O

I

~

1z: Negrito

~

(^t
a,
24

I:
Ae

C can

A

Ainu

KoreanN

Negrito(Aeta)6

V

Vedda

C

Caucasian0

Oriental

5'

BushmanI

AmericanIndian\1\

X

J:Japanesetions, and only types 1 and 2, which are circled infigure 5, contain some African individuals. Types 1
and 2 of the present study correspond to mtDNA
types 1 and 8 of Johnson et al. (1983), respectively.
There are five clusters in this group (boxed by dashedlines; see fig. 5), all radiating from type 1. One cluster
contains two mtDNA types (11 and 12) found only
in Negritos, whereas mtDNA types found only in
Caucasians (16, 18, and 19) make one cluster with ahypothetical type X2. On the other hand, the otherthree clusters contain mtDNA types found in more
Figure 5 Possible maximum parsimony tree for the phylogeny of 37 mtDNA types listed in 10 populations. Subscripts by
the numbers of each of the mtDNA types indicate the populations
from which the individuals were sampled. Four hypothetical types
are needed to complete the tree: types XI (6-1-1-1), X2 (9-3-2-4 or
9-1-2-4), X3 (1-1-3-2 or 1-1-3-3), and X4 (5-1-3-2) (figures in parentheses are enzyme morphs listed in the following order: AvaIl,
BamHI, Hpal, and Mspl). Groupings of mtDNA types are indicated by lines, and six subgroups are indicated by dashed lines.

mtDNA Polymorphism

141

Table 3
Nucleotide Diversities of Five Asian Populations and Genetic Distances among The Five
Populations

Japanese

Ainu

Korean

Aeta

Vedda

Japanese ..........

.00086(.00019)
Korean .000000... .000000.00065(.000127)(.00017)

Ainu .000006. .. .000006.000004.00041(.000122)(.000114)(.00015)

Aeta .000029. .. .000029(.000157)
Vedda .000091... .000091(.000214).000026.000019.00050(.000151)(.000145)(.00022).000100(.000213).000096(.000210)(.000232).00110(.00031).000111

NOTE.-On the diagonal are the nucleotide diversities; and under the diagonal are the genetic
distances. Figures in parentheses are SEs.

than one population, though one of them (cluster oftypes 4 and 7) is for only Asian populations (Japanese, Ainu, and Korean). The remaining two clusters(one for types 5 and 21 and the other for types 6, 9,
14, 15, and 20) contain individuals from the Japanese, Korean, Vedda, and Caucasian groups. These
two clusters correspond to subclusters 1 and 3 ofcluster B of the UPGMA tree (see fig. 4). Interestingly, mtDNA types in these two clusters can be
related to those of the African group in some alternative maximum parsimony trees. Connections between types 5 and 29 and between types 6 and 26 are
possible, and mtDNA types belonging to the twoclusters can be connected to mtDNA types detected
only in the African populations.
Three mtDNA types (16, 19, and 37) that wereoutside clusters A and B in figure 4 seem to fit morereasonably in figure 5. Types 16 and 19 found in
Caucasians belong to the non-African group, and together with type 18 they form a cluster for Caucasians, whereas type 37 found in Bantu belongs to theAfrican group.

6). In this tree, the distance between the Japanese andKorean populations is the smallest, and Ainu is clustered next to them. Aeta are not far away from thesethree mongoloid populations and make one clusterwith the other three populations. On the other hand,
Vedda is located quite apart from the other four populations. The genetic distance between Vedda and theremaining four populations is more than four times
larger than that between Aeta and the other three
Asian populations.
Discussion
In the present study, 12 presumably populationspecific mtDNA morphs were detected in total. Also,
Brega et al. (1986) have reported six distinct morphs
among the Tharu of Nepal, mnorphs that have notbeen found in other populations, including those of
the present study. These population-specific mtDNA

X 10o-

Nucleotide Diversity of Each Population
and Population Phylogeny

Nucleotide diversities for the five populations examined in the present study and genetic distancesamong these populations are presented in table 3.
The largest value (0.00110 + 0.00031) of nucleotide diversity was for the Vedda, and the smallest(0.00041 + 0.00015) was for Ainu.
On the basis of genetic distances, a UPGMA tree
for the five Asian populations was constructed (fig.

u

0
I

5
I

10
I

Japanese
Korean

Ainu
Aeta

Vedda

Figure 6 Phylogenetic tree based on genetic distances (listed
in table 3) for five Asian populations.

Harihara et al.

142

types may be useful for elucidating problems of racial
admixture.
Hpal morph 1 (HincIh morph 1) has been detected
mainly in mongoloid populations: Orientals (mainly
Chinese) (Denaro et al. 1981; Blanc et al. 1983),
American Indians (Wallace et al. 1985), Japaneseand Ainu (Harihara et al. 1986), the Tharu of Nepal(Brega et al. 1986), and Koreans (present study). Although it also has been found in low frequency in
Bantu (Denaro et al. 1981), this morph can be used asa marker for mongoloid populations. HpaI morph 4(HincIl morph 3) has been detected only in the mongoloid populations of east Asia: Orientals (mainly
Chinese) (Denaro et al. 1981; Blanc et al. 1983), Japanese and Ainu (Harihara et al. 1986), and Koreans(present study). This morph can also be used as amongoloid marker.
Among the variant morphs found in the present
study, AvaII morphs 25Aeta and 26Vedda were relatively frequent in Aeta and Vedda, respectively. Thismay be due to the effect of random genetic drift,
which likely has occurred in these small and isolated
populations. A similar finding was reported for theAmerican Indian by Wallace et al. (1985), who attributed the cause to the small number of female
founders.
The maximum parsimony tree (fig. 5) seems to depict phylogeny of mtDNA types more appropriately
than does the UPGMA tree (fig. 4). All mtDNA types
could be divided into two main groups, African and
non-African. However, we should be cautious whena phylogeny of mtDNA types is used for inferring
population phylogeny, because a substantial part ofthe divergence between mtDNA types is thought to
be attributable to ancient polymorphism, which existed well before the divergence of populations(Horai et al. 1986; Saitou and Omoto 1987).
The values of nucleotide diversity are lower by one
order than those obtained by Cann et al. (1987). Thismight be due to differences between our study andtheirs with regard to the restriction enzymes used.
Another probable cause for this discrepancy is that
Cann et al. (1987) may overestimate the values, since
they used polyacrylamide gel for electrophoresis ofdigested mtDNA fragments and detected DNA conformational mutations (Singh et al. 1987).
The Negrito's relatively close affinity to mongoloid
populations in the present study (see fig. 6) is consistent with the results of previous genetic studies(Omoto et al. 1978; Matsumoto et al. 1979; Horai etal. 1981; Omoto 1981, 1984). We computed genetic

distances from Johnson et al.'s (1983) data and thepresent data and found that distances between two
African populations and non-African populations
are much greater than those among non-African populations (data not shown); thus the Negrito's affinity
with the African populations seems to be quite low.
The Vedda's phylogenetic location in the present
study may suggest a long isolation of this population.
A genetic study of Vedda and Sinhalese by means ofblood markers also suggested this tendency (Ellepolaand Wikramanayake 1986). However, since Vedda
individuals today may be considerably mixed withsurrounding populations, and since no mtDNA data
are available for the neighboring Sinhalese andTamils, we reserve any definite conclusions. We also
note that the SE of the genetic distance is much larger
than the genetic distance itself (table 3). Further studies, using other populations and additional restriction
enzymes, will be necessary to clarify the evolution
and phylogeny of the Asian populations.

Acknowledgments
This study was supported by a grant-in-aid for scientificresearch from the Ministry of Education, Science and Culture, Japan.

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