Am. J. Hum. Genet. 64:250–258, 1999

Molecular Genetic Analysis of Remains of a 2,000-Year-Old Human
Population in China—and Its Relevance for the Origin of the Modern
Japanese Population
Hiroki Oota,1 Naruya Saitou,2 Takayuki Matsushita,3 and Shintaroh Ueda1
1
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo; 2Laboratory of Evolutionary Genetics, National
Institute of Genetics, Mishima, Japan; and 3Doigahama Site Anthropological Museum, Houhoku, Japan

Summary
We extracted DNA from the human remains excavatedfrom the Yixi site (∼2,000 years before the present) in
the Shandong peninsula of China and, through PCR amplification, determined nucleotide sequences of their mitochondrial D-loop regions. Nucleotide diversity of theancient Yixi people was similar to those of modern populations. Modern humans in Asia and the circum-Pacificregion are divided into six radiation groups, on the basisof the phylogenetic network constructed by means of
414 mtDNA types from 1,298 individuals. We compared
the ancient Yixi people with the modern Asian and thecircum-Pacific populations, using two indices: frequency
distribution of the radiation groups and genetic distancesamong populations. Both revealed that the closest genetic relatedness is between the ancient Yixi people andthe modern Taiwan Han Chinese. The Yixi people show
closer genetic affinity with Mongolians, mainland Japanese, and Koreans than with Ainu and Ryukyu Japanese and less genetic resemblance with Jomon people
and Yayoi people, their predecessors and contemporaries, respectively, in ancient Japan.

Introduction
The genetic relationship between human populations of
the Japanese archipelago and the Eurasian continent has
been the object of many studies. Archaeological discovery in the Japanese archipelago has revealed evidence ofhabitation as far back as the Lower Paleolithic period(Imamura 1996). The four main islands constituting theReceived August 3, 1998; accepted for publication October 26,
1998; electronically published December 22, 1998.
Address for correspondence and reprints: Dr. Shintaroh Ueda, Department of Biological Sciences, Graduate School of Science, Universityof Tokyo, Tokyo 113-0033, Japan. E-mail: sueda@biol.s.u-tokyo.ac.jp
q 1999 by The American Society of Human Genetics. All rights reserved.
0002-9297/99/6401-0031$02.00

250

Japanese archipelago were connected with each other orwith the Eurasian continent during the coldest period of
the latest glacial epoch, ∼18,000 years before the present(ybp) (UNESCO/IOC Western Pacific Subcommission
1995). The Jomon period, corresponding to the Neolithic period in Europe, started ∼12,000 ybp. Both theUpper Paleolithic period and the Jomon period are characterized by a hunter-gatherer economy, although thetraces of plant cultivation and large-scale settlement haverecently been found in many Jomon sites, including Sannai-Maruyama and Uenohara sites (which are locatedat the northern end of Honshu island and at the southernend of Kyushu island, respectively). The following Yayoi
period is characterized by agriculture, especially wet-rice
cultivation.
In accordance with these archaeological features
showing drastic changes from the Jomon period to the
Yayoi period, anthropological studies have pointed outthat the Yayoi people are morphologically divided into
two types. One of them is characterized by shorter stature, relatively low and wide facial contours, rather
square orbital openings, markedly depressed nasal root,and prominent glabella and superciliary arches; thesefeatures are also known to be characteristic of the indigenous Jomon people. The other type is just the opposite—higher stature, higher and narrower facial contours, higher orbital openings, much shallower nasal
root, and remarkably flat glabella and superciliaryarches. The latter type is observed mainly in the Yayoi
people, whose remains have been excavated from sites
in northern Kyushu (in the Japanese archipelago, Kyushu is the southwestern main island nearest to Korea
and China). Therefore, it has been proposed that the
Yayoi people in northern Kyushu might have been immigrants from the Eurasian continent to the Japanesearchipelago (Kanaseki 1966).
During recent decades, a number of studies have reported the genetic structures of the modern Asian populations and have discussed their phylogenetic relationships (e.g., see Nei 1995; Omoto and Saitou 1997).
However, the population history is still in controversy.

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Table 1

Material and Methods

mtDNA Nucleotide Sequences of 2,000-Year-Old Human
Remains from the Yixi Sitea

SEQUENCE

SOURCE/SAMPLE
CRS
Yixi:
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

MT11MT4

MT3

111111111111111111111111
666666666666666666666666
222222222222222222233333
012233456666777789901126
371324962456048971841972

111111112222
455678991457
602705050935

ATCCCCTCCCACCGCCCCTTTGCTTCTCCGCTAACA

G................T.C....
...T...................C...T..............C...T.
...................C....
.....................A.C...T.......T...........C.C...T..................
...T........T..........C..T................C....
...T....................
...................C....
....A.C............CC...
...T..............C...T.
...T.........A..T......*.....T.............C...*G................T.C...*.............A.......A.*..........C..A.........*...T...................*
G......T.........T.C...*...T...TTT.T...TA.C..A.*...T..........T........*...................C...*************************.........–.G...........GC..TT....–.G
C........–.G......T.G..G.T.........G...........G..........TG..C....C.–.G************************************************.T.........G..C......–.G.........–.G.T.........G.....A.....GC..........G************************************************.T.........Ga
The notation of the CRS is used for numbering of bases. A
dot (.) denotes that the nucleotide is identical to that in the CRS(Anderson et al. 1981), a dash (–) denotes a gap, and an asterisk(*) denotes that the identity of the nucleotide is unknown.

This might be due to lack of direct genetic information
about ancient people, because comparison using only
modern humans is often affected by changes with thetimes, such as migration and random genetic drift. Genetic research on ancient populations is an absolute prerequisite for disclosing of past events and investigationof the population history.
To determine the genetic background of the ancient
Chinese people, we extracted DNA from the 2,000-yearold human remains excavated in the Shandong peninsulaof China and determined the nucleotide sequences of
their mitochondrial D-loop regions. We also examined
phylogenetic relationships among the modern and ancient Asian peoples, by constructing an mtDNA tree andevaluating nucleotide diversity among populations, tounderstand the genetic relationship of the peoples livingin eastern Asia.

Site and Samples
The Yixi site is located in Linzi, Shandong Province,
China. Linzi was the capital of the Qi dynasty during
the Spring-Autumn and Warring States period (770
B.C.–221 B.C.). This site has been dated to the period of
the Han dynasty (206 B.C.–220 A.D.) in China, on thebasis of the archaeological finds excavated. A total of
42 teeth (mainly third molars) and 16 bone samples wereused for DNA analysis, by official permission authorized
by the Cultural Relics Bureau of the People’s Republicof China.
DNA Extraction and Purification
To destroy possible contaminant DNA on the remains’surfaces, bone and teeth samples were exposed to UV
irradiation. After storage in a 0.5 M EDTA solution at
47C for 11 mo, the samples, frozen in liquid nitrogen,were physically crushed by a Cryo-Press CP-100 (Microtech Nichion). For 36 teeth among the total of 58
samples, DNA extraction and purification were performed by the silica-based method (Boom et al. 1990;
Höss and Pääbo 1993). Fine-powdered teeth were suspended in 1 ml of lysis buffer. Lysis buffer was preparedby mixing of 10 ml of 0.1 M Tris-HCl (pH 6.4), 12 gof guanidinium thiocyanate (GuSCN), 2.2 ml of 0.02 M
EDTA, and 0.5 ml of Triton X-100. After being wellshaken, the mixture was incubated at 657C overnight,
with occasional agitation by a touch mixer. After centrifugation for 5 min at 1,500 g, 500 ml of the supernatant was removed to another tube, and 500 ml of thelysis buffer was added to it. A 40-ml aliquot of 3% (w/v) silicon dioxide solution, stored at room temperaturein the dark, was added, and the mixture was incubatedfor 15 min at room temperature. After centrifugation
for a few seconds, the supernatant was removed, andthe silica pellet was washed twice with 500 ml of 0.1 M
Tris-HCl (pH 6.4) washing buffer containing 10 M
GuSCN. Subsequently, the silica pellet was washed twicewith 500 ml of 70% ethanol and once with acetone. Afterthe pellet had been dried at 607C in a heat block, ancientDNAs were eluted at 567C in 65 ml of water (Ultraspecy;
Biotech). A 50-ml portion was recovered and used as atemplate for DNA amplification by PCR. Lysis andwashing buffers were used after preincubation with silica
suspension.
For the remaining 22 samples (6 teeth and 16 bones),the EDTA/phenol method was used. The powdered samples were suspended in 10 ml of 0.5 M EDTA solution
and then were extracted, twice, with 12 vol of watersaturated phenol and chloroform/isoamyl alcohol (24:1). The aqueous phase was removed and precipitated252

Am. J. Hum. Genet. 64:250–258, 1999

Figure 1

mtDNA network. Smaller unblackened circles represent distinct mtDNA types found in modern humans, blackened circles
represent types absent in the mtDNA database, and larger unblackened circles represent backbone types 1, 3, 7, 9, 13, 25, 113, 138, and 173.
The numbers on the branches denote nucleotide positions (according to the CRS; note that the prefix “16” has been omitted) where substitutions
have occurred. Reticulations in the network indicate the existence of incompatible nucleotide configurations.

with 2 vol of cold ethanol at 2207C. The precipitate
was redissolved in 2 ml of water. After 0.5 ml of 10 M
lithium chloride solution was added and the mixture was
kept on ice for 30 min, the supernatant was removedby centrifugation and was precipitated with 2 vol of cold
ethanol. After the precipitate was dissolved in 0.1 ml ofwater, the DNA extracts were further purified by thesilica-based method described above. To verify that nocontamination had occurred during DNA extraction, weused, as blank controls, the extracts that did not haveremains.
PCR Direct Sequencing of mtDNA
We used three primer sets designed within the mitochondrial D-loop region, one of which (MT4) was newly
designed in the present study and the remaining two of
which (MT1 and MT3) were from Kurosaki et al.
(1993). The MT1 and MT4 regions are in hypervariable
region I, whereas MT3 region is in hypervariable region
II. The nucleotide sequences correspond to positions
16190–16208 and 16422–16403, positions 90–109 and
326–307, and positions 16135–16154 and 16366–
16347 of the Cambridge Reference Sequence (CRS) (Anderson et al. 1981) for MT1, MT3, and MT4, respec-tively. Ancient DNAs were amplified by the hot-start
PCR technique using anti-Taq DNA polymerase antibody (TaqStart Antibody; Clontech). DNA amplification
was performed in 20, 40, and 40 ml of reaction mixture
containing 100 mM Tris-HCl (pH 8.3), 2 mM MgCl2,
500 mM KCl, 200 M of each dNTP, 2 units of AmpliTaq
DNA polymerase (Perkin-Elmer), and 5, 10, and 20
pmol of the PCR primers for MT1, MT3, and MT4,
respectively. Forty cycles of PCR were performed as follows: denaturation at 947C for 30 s, annealing at 557C
for 30 s, and extension at 727C for 60 s. Infrequently,
second-round PCR amplification was necessary, inwhich the reaction was performed under ordinary conditions without anti-Taq antibody. To verify the reliability of the results obtained, we repeated the experiments, along with negative control experiments without
template DNAs.
DNAs amplified by PCR were purified by means ofan S-300 spin column (Pharmacia) and were used as
sequencing templates. Nucleotide sequences of bothstrands were determined by means of the FS Taq
DyeDeoxy Terminator Cycle Sequencing kit (Applied
BioSystems), according to the manufacturer’s instructions, by an Applied BioSystems 373S DNA sequencer.

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Oota et al.: Genetic Structure of Ancient Chinese

Table 2Distribution of Radiation Groups in Yixi and Jomon Individuals
Radiation Group andAncient Individual(s)Sharing Populations (No. of Individuals Shared) in DNA Database

I:
Yixi-10

Yixi-03 and Yixi-13

Turk (1), Ainu (4), mainland Japanese (2), Korean (2), Ryukyu Japanese (7), Cantonese (3), Mongolian (1),Siberian Altai (1), Native American (2), Indonesian (2), aboriginal Taiwanese (2), Filipino (1), Melanesian(1), Polynesian (1)Korean (1), Taiwan Han Chinese (2), Cantonese (1), Mongolian (5), Native American (1), Polynesian (1)II:
Yixi-02
Yixi-05
Yixi-06
Yixi-08

Turk (1), Ainu (1), mainland Japanese (15), Koreans (11), Ryukyu Japanese (1), Taiwan Han Chinese (3),Asian (1), Mongolian (6), Native American (1), aboriginal Taiwanese (1), Melanesian (8)(Unique)(Unique)(Unique)

III:(None)IV:(None)
V:
Yixi-07

Indonesian (1)Yixi-04 and Yixi-11

Turk (2), Taiwan Han Chinese (5), Cantonese (1), Mongolian (4), Native American (1), Indonesian (1), Filipino (1), Polynesian (3)(Unique)
Mainland Japanese (1)Korean (1), Mongolian (3)VI:

Yixi-01
Yixi-09
Yixi-12

Some sequences were determined by means of primers
end labeled with [g-32P]-ATP by T4 polynucleotide kinase. Samples with conflicting nucleotide sequences between strands or between regions overlapping with eachother were excluded from the subsequent data analysis(in this study, there were three samples whose nucleotide
sequences of both strands obtained were not completely
complementary).
Phylogenetic Analyses
We compared mtDNA sequences of a total of 1,298
individual samples retrieved from the DDBJ/EMBL/
GenBank international nucleotide-sequence database:
45 Turks (Comas et al. 1996), 5 Australian Aborigines(Vigilant et al. 1991; Betty et al. 1996), 51 Ainu, 64South Koreans, 50 Ryukyu Japanese (Horai et al. 1996),
62 mainland Japanese (Vigilant et al. 1991; Horai et al.
1996), 66 Taiwan Han Chinese (Horai et al. 1996), 20
Cantonese (Betty et al. 1996), 17 Asians (Vigilant et al.
1991), 101 Mongolians (Kolman et al. 1996), 17 Altai
of central Siberia (Shields et al. 1993), 353 New World
Native Americans (Vigilant et al. 1991; Ward et al. 1991,
1993; Shields et al. 1993; Batista et al. 1995; Kolman
et al. 1995), 65 Indonesians (Vigilant et al. 1991; Redd
et al. 1995; Sykes et al. 1995), 35 aboriginal Taiwanese(Sykes et al. 1995), 36 Filipinos (Vigilant et al. 1991;
Sykes et al. 1995), 104 Melanesians (Vigilant et al. 1991;
Sykes et al. 1995; Redd et al. 1995), 5 Micronesians(Sykes et al. 1995), 16 Kapongamrangi (Sykes et al.

1995), and 186 Polynesians (Vigilant et al. 1991; Sykes
et al. 1995; Redd et al. 1995). After identical sequencesfrom these data were combined, 414 distinct mtDNA
sequences (types) were obtained. In addition, sequence
data on 5 Jomon (Horai et al. 1989, 1991), 14 Yayoi(Oota et al. 1995), and 28 Pygmies (Vigilant et al. 1991)
were used. Phylogenetic networks of mtDNA types wereconstructed by means of the network methods of Excoffier and Langaney (1989) and Bandelt et al. (1995).
Nucleotide diversity is the average number of nucleotide
differences, per site, between populations and is equivalent to heterozygosity at the nucleotide level, for a randomly mating population (Nei 1987). Distance values
for calculation of nucleotide diversity were estimated bymeans of the program CLUSTAL W (Thompson et al.
1994).
Results
For the total of 58 samples, we obtained 23 and 16
nucleotide sequences, for hypervariable regions I and II,
respectively. There is a cytosine tract in the hypervariable
region I from CRS positions 16184–16193. Since heteroplasmy due to length variation in this region has beenreported (Bendall and Sykes 1995), nucleotide sequences
excluding this tract were used for phylogenetic analysis.
There were a total of 36 phylogenetically informative
sites, which are presented in table 1.
Figure 1 shows a phylogenetic network for nine back-254

Am. J. Hum. Genet. 64:250–258, 1999

Figure 2

Geographic distribution (in %) of six radiation groups in the eastern Eurasian continent and Oceania

bone mtDNA types (types 1, 3, 7, 9, 13, 25, 113, 138,and 173), which were deduced from the huge phylogenetic tree constructed by use of 185 nucleotides of
MT1 and MT4 regions from 414 mtDNA types of 1,298
modern humans in Asia and the circum-Pacific region(H.O., S.U., and N.S., unpublished data). The backbone
type is defined as a center of a star having 110 descendant types. On the basis of the starlike clusters, we identified six radiation groups. Group I, including type 13,
was characterized by C/T transition at CRS position
16223, without T/C transition at position 16362. GroupII includes types 7 and 25 and was characterized by
T/C transition at CRS position 16362, without G/A transition at position 16319. Group III was characterized bythree nucleotide substitutions, at CRS positions 16223,
16319, and 16362. Group V was characterized by T/Ctransition at CRS position 16217, without substitutionsat positions 16223 and 16362. Group VI was characterized by T/C transition at CRS position 16304, without substitutions at positions 16223 and 16362.
To reveal the genealogy of the mtDNA sequences of

the Yixi people, we attached their sequence data to the
network tree obtained on the basis of modern humandata. Unfortunately, the data on the MT3 region in modern humans are fewer. We were thus obliged to use nucleotide sequences of MT1 and MT4 regions alone as
the longest sequence available. A total of 11 mtDNA
types from 13 Yixi individuals (Yixi-01–Yixi-13) are presented in table 1; 4 types are unique to the Yixi, whereas
the remaining 7 types are shared with modern humanpopulations, and 4 of these 7 are shared with more than
five modern human populations (fig. 1 and table 2).
Thus, 9 of the 13 Yixi individuals share their mtDNA
types with modern humans from populations covering
the entire circum-Pacific region.
Figure 2 shows the frequency distributions of six radiation groups in the ancient Yixi and in 12 modern
human populations. Group I was found in all the 13
populations. Groups II, V, and VI, however, had distributions that were unique to each population. For Mongolians, Siberian Altai, Ainu, Koreans, mainland Japanese, and Taiwan Han Chinese, the frequencies of group

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Oota et al.: Genetic Structure of Ancient Chinese

Table 3
Mean Pairwise Comparisons Within and Between Populations
GENETIC DISTANCE FROMa

POPULATION

Yixi(n 5 13)MainlandJapanese(n 5 62)

Korean(n 5 64)

Ainu(n 5 51)

RyukyuJapanese(n 5 50)

TaiwanHan Chinese(n 5 66)

Mongolian(n 5 101)

Pygmy(n 5 28)

Yixi
Mainland Japanese
KoreanAinu
Ryukyu JapaneseTaiwan Han Chinese
Mongolian
Pygmy

2.035
.066
.097
.154
.129
0
.023
1.44

2.131
0
.075
.029
.031
.014
1.44

2.069
.077
.038
.058
.016
1.39

2.274
.072
.125
.113
1.46

1.844
.102
.077
1.39

2.561
.024
1.38

2.456
1.38

3.028

a

All values have been multiplied by 100. Genetic distances (DA) between populations are below the diagonal and havebeen calculated according to the following formula: DA 5 DXY 2 [(DX1DY)/2], where DXY is the pairwise mean between
populations X and Y and where DX (DY) is the pairwise mean within population X (population Y) and is given on the
diagonal. The values given as “0” contain negative-value estimations.

II were 120%, whereas those for Indonesians, aboriginal
Taiwanese, Melanesians, and Polynesians were !20%.
Mongolians, Koreans, and mainland Japanese showed
particularly high frequencies of group II (34%, 44%,
and 40%, respectively). Group V had the highest frequency in Polynesians (77%). High frequency for group
V was also observed in aboriginal Taiwanese (53%) andIndonesians (39%). Thus, the frequencies of group II
were higher in the northern populations than in thesouthern populations of the western circum-Pacific,
whereas those of group V were just the opposite.
Three, four, and one Yixi individuals belonged togroups I, II, and V, respectively. No individuals wereclassified as belonging to groups III and IV, which werethe most major radiation groups for Native Americans(59%) and Europeans (81%), respectively. The mostcharacteristic feature of the Yixi people is that 5 (38%)
of 13 individuals belonged to group VI. Modern humanpopulations showed relatively low frequency for groupVI, except for modern Taiwan Han Chinese, in which
this frequency was 120%. In Siberian Altai, aboriginal
Taiwanese, and Micronesians, there were no individuals
who belonged to group VI, and the frequency was !10%in Ainu, Koreans, mainland Japanese, Ryukyu Japanese,and Melanesians. Distribution frequencies for group II
suggest similarity between the Yixi people and northern
Asian populations. We also compared the frequency distributions of radiation groups by using the 151-bp MT4
information alone, from 23 Yixi samples. One informative site (CRS position 16362) was lost, and there
was no distinction either among mtDNA types 1 and 7
or among mtDNA types 13 and 25. However, the radiation group characteristic of the Yixi people and Taiwan Han Chinese—that is, group VI—remained distinguishable, and its distribution frequency was high (i.e.,
9 [39%] of 23).

Table 3 presents the values of nucleotide diversity
within and between populations. The highest value of
nucleotide diversity within circum-Pacific populations
was observed in Taiwan Han Chinese (2.6%). The Yixi
people had a value of 2.0%. Among the seven Asian
populations, the values for nucleotide diversity within
populations varied within the range of 1.8%–2.6% (thatof Pygmies was 3.0%). Between the Yixi people and theseven modern human populations, there was no significant within-population difference in nucleotide diversity. Between populations, the lowest value in net nucleotide substitutions, 0, was found between the Yixi
people and Taiwan Han Chinese, suggesting that theclosest genetic relationship is between them; the second
lowest value for difference from the Yixi population wasobserved in Mongolians (0.023), followed by mainland
Japanese (0.066) and Koreans (0.097). The lowest valuesof difference from modern mainland Japanese were in
Koreans (0.00), Mongolians (0.014), Ryukyu Japanese(0.029), and Taiwan Han Chinese (0.031). When the
154-bp MT4-site information alone was used, one informative site (CRS position 16362) was lost, but 10
additional samples (23 samples in total) were available
for comparison. Between the Yixi people and other populations, the lowest value in net nucleotide substitutions
was observed in Taiwan Han Chinese (0.000), as in thecase when the 185-bp MT11MT4 region from 13 Yixi
individuals was used for comparison.
Discussion
We compared the genetic relatedness between the ancient Yixi people and the modern Asian populations by
two methods using mtDNA sequences; one is the frequency-distribution comparison of radiation groups on
the phylogenetic network, and the other is the mean

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Am. J. Hum. Genet. 64:250–258, 1999

Table 4
Distribution of Radiation Groups in Jomon Individuals
Jomon Individual(s)a
URAWA-1a (5,900 ybp)
TODA-1a (6,000 ybp),
TAKA-5b (3,000 ybp),
TAKA-21b (3,000 ybp)
TAKA-13b (3,000 ybp)

Sharing Populations (No. of Individuals Shared) in DNA DatabaseIndonesian (1), aboriginal Taiwanese (1), Filipino (6), Micronesian (2)Yixi-02, Turk (1), Ainu (1), mainland Japanese (15), Korean (11), Ryukyu
Japanese (1), Taiwan Han Chinese (3), Asian (1), Mongolian (6), Native
American (1), aboriginal Taiwanese (1), Melanesian (8)Yixi-10, Turk (1), Ainu (4), mainland Japanese (2),Korean (2), Ryukyu (7),Cantonese (3), Mongolian (1), Siberian Altai (1), Native American (2), Indonesian (2), aboriginal Taiwanese (2), Filipino (1), Melanesian (1), Polynesian (1)Radiation
Group
II
II

I

Lowercase letters appended to numerals denote the geographic source: a 5 Saitama Prefecture in mainland Japan (Horai et al. 1989),
and b 5 Hokkaido Island in Japan (Horai et al. 1991).
apairwise sequence comparison based on the nucleotide
diversity among populations. Both show the closest genetic affinity between the Yixi people and the modern
Taiwan Han Chinese.
We added the sequence data from the Jomon people(Horai et al. 1989, 1991) within the phylogenetic treeof the modern human data. All of the five Jomon individuals belonged to either of group I or group II; 1
Jomon individual shared mtDNA type with 10 individ-

Figure 3

uals from Southeast Asia and Oceania, and the remaining 4 Jomon individuals shared mtDNA type with 7 Yixi
and 90 circum-Pacific individuals (see table 4). Thismeans that the Jomon people shared mtDNA types withindividuals from all of the circum-Pacific populations,
including the 2,000-year-old Chinese population.
Figure 3 shows the phylogenetic network constructedby use of the mtDNA sequences of (a) the Yayoi people
from the Takuta-Nishibun site of northern Kyushu, Ja-

mtDNA network among ancient Japanese and ancient Chinese individuals. The network was constructed by use of the 154-bp
MT-1 nucleotide sequences of 14 individuals from the Takuta-Nishibun Yayoi site, 13 individuals from the Yixi Han site, and 9 backbone
mtDNA types. Completely closed circles represent absent types (circle size is not significant). The numbers on the branches denote nucleotide
positions (according to the CRS; note that the prefix “16” has been omitted) where substitutions have occurred. The positions with and without
an asterisk (*) are sites of substitutions by transversion and by transition, respectively. Reticulations in the network indicate the existence ofincompatible nucleotide configurations.

257

Oota et al.: Genetic Structure of Ancient Chinesepan (Oota et al. 1995), (b) the Yixi people (present
study), and (c) the nine backbone types (H.O., S.U., andN.S., unpublished data). Since only shorter sequences(154-bp MT1) were available for comparison, two informative sites—CRS positions 16217 and 16223—were
lost. Therefore, mtDNA background types 1, 9, and 13
also were indistinguishable, and mtDNA backgroundtypes 7 and 25 were indistinguishable; and the network
therefore was split into only four radiation groups(groups I1IV1V, group II, group III, and group VI). No
individuals belonging to group III were found in either
the Yayoi people or the Yixi people. Of the 14 Yayoi
individuals, 5 were included within groups I1IV1V and
9 were included within group II; however, neither any
Yayoi people from the Takuta-Nishibun site nor any
Jomon people were included within group VI.
On the basis of morphometric data, Hanihara (1991)
claimed that large-scale migration from the continent to
the Japanese archipelago occurred during the transition
period from Jomon to Yayoi—that is, ∼2,000 ybp—and
that, subsequently, gene flow by immigrants significantly
contributed to the genetic structure of the mainland Japanese. Present data seem to show less contribution fromthe 2,000-year-old Yixi population to the genetic structure of the modern mainland Japanese.
Hanihara (1991) also proposed the dual-structure
model, one of the hybridization theories, which supposes
two events of major migration for the genetic constitution of modern Japanese—that of the Jomon ancestors
and that of the Yayoi immigrants. This model claims (1)that the indigenous Jomon people and the Yayoi immigrants admixed with each other gradually, (2) that
Ainu and Ryukyu Japanese are direct descendants of the
Jomon people, and (3) that the Jomon people originatedfrom Southeast Asia, whereas the Yayoi immigrants originated from northern Asia. Horai and his colleagues concluded that the modern Japanese are divided into twogroups and that the mtDNA of the Jomon people andthat of Ainu belong to the same group (Horai and Matsunaga 1986; Horai et al. 1991). These investigators also
estimated the divergence time between the indigenous
and immigrant groups as being 125,000 ybp and, moreover, claimed, as evidence, that the Jomon people andSoutheast Asians share identical mtDNA sequence(Horai et al. 1989, 1991). As Oota et al. (1995) subsequently explained, however, such an extended divergence time merely means that genetic polymorphisms
existed in the ancestral population of the Jomon people,
because the Jomon period started only ∼12,000 ybp.
Furthermore, four of the five Jomon individuals reportedby Horai et al. shared mtDNA types with many individuals from northern Asia. The three mtDNA types
found in the Jomon individuals were included withingroups I and II, which have higher frequencies in eastern
Asia than in Southeast Asia. In addition, no Jomon in-dividuals were found to belong to group V, a group thatis characteristic of Southeast Asian and Oceanic populations; thus, there seems to be no strong connection
between them.
The present study shows the closest genetic relatedness
between the 2,000-year-old population in Shandong
peninsula and the modern Taiwan Han Chinese butshows less genetic resemblance between the 2,000-yearold populations from northern Kyushu (Japan) and the
Shandong peninsula (China). Genetic information on theearlier people of the continent holds the key to solutionof the question of the origin of the Jomon/Yayoi people
and, consequently, of human history in the Japanesearchipelago.

Acknowledgments
We gratefully acknowledge Dr. Kangxin Han (Institute ofArchaeology, Beijing) and Dr. Xun Zhang Luo (Institute ofArchaeology, Shandong Province, China). We also thank Drs.
Y. Manabe and J. Oyamada (Nagasaki University) and Dr. I.
Koizumi (Hokkaido University). This study was supported by
grants from the Ministry of Education, Science, Sports, andCulture of Japan, and by the National Institute of Genetics
Cooperative Research Program.

Electronic-Database Information
Accession numbers and URL for data in this article are as
follows:
DDBJ/EMBL/GenBank, http://www.ddbj.nig.ac.jp/Welcome-e.html (for nucleotide sequences [AB021466–AB021489])

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