Genetic Structure of a 2,500-Year-Old Human Population in China and Its
Spatiotemporal Changes
Li Wang,*† Hiroki Oota,* Naruya Saitou,‡ Feng Jin,§ Takayuki Matsushita,\ andShintaroh Ueda**Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan; †Department of Medicaland Animal Genetics, Institute of Genetics, Chinese Academy of Sciences, Beijing, China; ‡Laboratory of Evolutionary
Genetics, National Institute of Genetics, Mishima, Japan; §Laboratory of Human Genetics, Institute of Genetics, Chinese
Academy of Sciences, Beijing, China; and \Doigahama Site Anthropological Museum, Houhoku, JapanTo examine temporal changes in population genetic structure, we compared the mitochondrial DNA (mtDNA)
sequences of three populations that lived in the same location, Linzi, China, in different periods: 2,500 years ago(the Spring–Autumn era), 2,000 years ago (the Han era), and the present day. Two indices were used to compare
the genetic differences: the frequency distributions of the radiating haplotype groups and the genetic distancesamong the populations. The results indicate that the genetic backgrounds of the three populations are distinct fromeach other. Inconsistent with the geographical distribution, the 2,500-year-old Linzi population showed greater
genetic similarity to present-day European populations than to present-day east Asian populations. The 2,000-yearold Linzi population had features that were intermediate between the present-day European/2,500-year-old Linzi
populations and the present-day east Asian populations. These relationships suggest the occurrence of drastic spatiotemporal changes in the genetic structure of Chinese people during the past 2,500 years.

Introduction
Molecular studies have identified genetic relationships among the present-day human populations. However, population history is still in controversy; migration
and admixture in the past make it difficult to interpretthese relations in a straightforward manner when examining the origin of modern humans and their expansion across the earth. Genetic research on ancient populations is therefore absolutely requisite for disclosing
past events and investigating population history. Direct
analysis of the Neanderthal DNA has supported the dispersal of modern humans out of Africa, showing its
mtDNA sequence to be outside the variation of presentday human populations (Krings et al. 1997).
Here we investigated temporal changes in genetic
structure of human populations during the past 2,500
years in China using mtDNA sequences. All samples
were collected in Linzi at the lower reaches of the Yellow River in China. In a previous study (Oota et al.
1999b), we examined DNA from the human remains
excavated from the 2,000-year-old site of Linzi. In thepresent study, we extracted DNAs from the human remains excavated from the 2,500-year-old site of Linzi
and the present-day Han Chinese living in Linzi. We
determined nucleotide sequences of their mitochondrial
D-loop regions and compared genetic structures of thethree populations that lived in the same location in different periods by evaluating the frequency distributionsof the radiating haplotype groups and the genetic distances among the populations, including the present-day
Eurasian populations.
Key words: ancient DNA, mitochondrial DNA, Chinese genetic
diversity population structure.
Address for correspondence and reprints: Shintaroh Ueda, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan. E-mail:sueda@biol.s.u-tokyo.ac.jp.
Mol. Biol. Evol. 17(9):1396–1400. 2000
q 2000 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

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Materials and Methods
Sample Collection
All three populations were sampled in Linzi, which
is now part of the city of Zibo in Shandong Province ofChina. In a previous study (Oota et al. 1999b), we examined DNA from human remains found at the 2,000year-old Yixi site of Linzi. In the present study, DNA
was extracted from bone samples of 63 individuals collected at the 2,500-year-old Liangchun site of Linzi, after receiving authorization from the Cultural Relics Bureau of the People’s Republic of China. These sites havebeen dated to the Spring–Autumn (770 B.C.–403 B.C.)
and the Han era (206 B.C.–A.D. 220) in China, respectively, on the basis of the archaeological finds (earthenwares) excavated. To examine the present-day population, we collected blood samples from 50 Han Chinese
individuals living in Linzi whose parents were both born
there.
Ancient DNA Extraction and Amplification
The bone samples were exposed to ultraviolet radiation to destroy possible contaminating DNA on theirouter surfaces. Then, the DNA was extracted from thespongy layer of the bone as previously described (Kurosaki, Matsushita, and Ueda 1993; Oota et al. 1995,
1999a, 1999b). PCR amplification was carried out in 40
ml of a reaction mixture containing 10 mM Tris-HCl (pH8.3), 2 mM MgCl2, 50 mM KCl, 200 mM each of dNTP,
20 pmol of each primer, and 2 U of Taq polymerase(Ampli-Taq Gold, PE Biosystems). PCR began with at
958C for 9 min, followed by 40 cycles of 948C for 30s, 558C for 30 s, and 728C for 1 min. To verify thereliability of the experiments, negative controls containing all of the reagents but without remains/DNA were
included in each DNA extraction/PCR run. PCR products purified on spin columns (S-300, Pharmacia) weredirectly sequenced for both strands using a commercial

Spatiotemporal Changes in Genetic Structure of Chinese Population

kit (FS Taq DyeDeoxy Terminator Cycle Sequencing
Kit, PE Biosystems).
Phylogenetic Analysis
The phylogenetic network of haplotypes and the
population tree were constructed using the network construction (Bandelt et al. 1995) and the neighbor-joining(Saitou and Nei 1987) methods, respectively. In addition
to the nucleotide sequences determined in this study, weused mtDNA sequence data from four central Asian(Comas et al. 1998) and five European (Richards et al.
1996) present-day populations and the data from ourprevious paper (Oota et al. 1999b). Nucleotide diversity(the mean of pairwise nucleotide differences per site)and evolutionary distances (net values of nucleotide
substitutions) between populations (Nei 1987) were calculated using the program dnapopdist, which was newly
developed for this study. The neighbor-joining tree wasdrawn using the program Dendromaker, version 4.1 (Imanishi 1998), and the midpoint rooting method.
Results and Discussion
Ancient DNA samples are usually damaged andfragmented, and sequences of less than a few hundred
nucleotides should be amplified by PCR. Some unknown molecules are difficult to remove and can inhibit
PCR when extremely small amounts of DNA extracted
from ancient remains are being amplified. Overlapping
DNA regions must be sequenced to assess the reliabilityof the results. Therefore, we analyzed hypervariable region I of the mtDNA by PCR using two sets of primersthat amplify the overlapping MT1 (positions 16190–
16422) and MT4 (positions 16135–16366) regions.
We successfully amplified and sequenced both ofthese DNA sequences for 40 of the 63 2,500-year-old
remains from the Liangchun site. The nucleotide sequences of both strands in each DNA region were completely complementary in all of them. However, the nucleotide sequences of the overlapping regions of MT1
and MT4 were not identical in 6 of the 40 samples;
those six samples were excluded from the subsequent
analysis. Since heteroplasmy due to length variation has
been reported for mtDNA sequences (Bendall and Sykes
1995), a cytosine tract (positions 16184–16193) was excluded from the comparison, as it was in our previous
analysis of DNA from 2,000-year-old human remains(Oota et al. 1999b). The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/
GenBank nucleotide sequence databases with the accession numbers AB031108–AB031191.
In ancient DNA analyses, both the sequence lengththat can be amplified and the number of samples available for analysis are limited, problems that are not encountered in studies of present-day DNA samples.
Therefore, we first constructed a phylogenetic reference
network using the 185-bp MT1 and MT4 nucleotide sequences (positions 16194–16378) of 1,298 present-day
Asian and circum-Pacific individuals. Then, we traced
the genealogy of the mtDNA sequences of the ancienthuman remains. Based on the phylogenetic network, we

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identified six radiation groups. These six groups can becharacterized by the five nucleotide sites 16217, 16223,
16304, 16319, and 16362 (see Oota et al. 1999b; moredetails will be published elsewhere). Groups I–IV havesequences TTTGT, TTTGC, TTTAC, TCTGT, CCTGT,and TCCGT, respectively. We would like to note that
haplotype 1 of figure 1 is the same as the Cambridge
reference sequence (CRS) (Anderson et al. 1981). It
should also be noted that haplotype 7 and its offspring
are not clearly classified into group II according to those
five nucleotide sites because of a reticulation (see fig.
1). We did find the same phylogenetic relationship forthe longer sequence data as for the 185-bp sequencedata.
Based on this present-day reference network, weconstructed a network of the mtDNA sequences of thetwo ancient populations and the present-day people ofLinzi (fig. 1). Ten individuals of the 2,500-year-old Linzi
population had mtDNA type with 16274A; this mtDNA
type was not found in either the 2,000-year-old or thepresent-day Linzi populations. Sixty-five percent (22 of34) of the individuals of the 2,500-year-old Linzi population belong to group IV, whereas none of the 2,000year-old population and only 8% of the present-day Linzi population belong to that group. In contrast, 38% (5of 13) of the 2,000-year-old Linzi population belong togroup VI, compared with only 9% and 10% of the
2,500-year-old and the present-day Linzi populations,
respectively. The 2,000-year-old and present-day Linzi
populations showed high frequencies for group I (23%and 30%, respectively) and for group II (31% and 36%,
respectively). Other present-day east Asian populations,
including Mongols, Koreans, and mainland Japanese,
also have high frequencies for groups I and II (fig. 2).
Heterogeneity of substitution rate for the human
mtDNA D-loop region has been known (e.g., Excoffierand Yang 1999; Meyer, Weiss, and von Haeseler 1999),and sites with high rates, such as 16223, 16311, and
16362, in fact caused reticulations, observed in the phylogenetic network of figure 1. Backward mutations occurring at those high-rate sites may cause erroneousclassification of the six groups defined above. However,relative substitution rates of those sites are about fourtimes as high as the average rate (Excoffier and Yang
1999), and the effect of ‘‘contamination’’ caused bybackward mutations may be relatively small. In any
case, frequency estimates of the six groups should beconsidered as a rough measure for comparing different
human populations.
We also estimated the nucleotide diversity withineach population and the genetic distances between the
populations. We used the 172-bp mtDNA sequence datafor comparison due to the lack of 13-bp data (positions
16366–16378) in the present-day European populations.
The nucleotide diversities were 0.020, 0.022, and 0.027
for the 2,500-year-old, the 2,000-year-old, and the present-day Linzi populations, respectively. Nucleotide diversities within the other present-day populations weresimilar (range 0.011–0.027). The smallest genetic distance for the present-day Linzi population was that from
the Mongols, followed by those from mainland Japanese1398

Wang et al.

FIG. 1.—A phylogenetic network. Open circles represent distinct mtDNA types found in present-day Asian and circum-Pacific populations,and full circles represent mtDNA types that are absent from those populations. Shaded circles represent mtDNA types seen in the 2,500-yearold to present-day Linzi people in China. Larger circles represent backbone types 1, 3, 7, 9, 13, 25, 113, 138, and 173. The numbers on the
branches are the nucleotide positions at which substitutions have occurred (add 16,000 to these numbers to obtain the CRS positions). Reticulations in the network indicate the existence of incompatible nucleotide configurations.

and Koreans. Surprisingly, the three smallest genetic
distances for the 2,000-year-old Linzi population werefrom the present-day central Asian populations: the Kirghiz (Sary-Tash), followed by the Kazakh and the Uighurs. Even more surprisingly, the three smallest genetic
distances for the 2,500-year-old Linzi population werefrom the Turkish, Icelander, and Finnish, rather thanfrom the east Asian populations. The results indicate thatthe genetic backgrounds of the three populations in Linzi are distinct from each other. Figure 3 shows the phylogenetic tree based on those genetic distances; presentday populations from east Asia, including the presentday Linzi population, form a cluster, which is consistent
with their geographical distribution. However, the 2,000year-old Linzi population lies outside the present-day
east Asian cluster, and the 2,500-year-old Linzi popu-

FIG. 2.—Geographic distribution of six radiation groups in the east-central Eurasian continent. Radiation groups I–VI are shown from left
to right.

Spatiotemporal Changes in Genetic Structure of Chinese Population

FIG. 3.—A neighbor-joining tree for 19 human populations.
Branch lengths are proportional to genetic distances.

lation clusters with the present-day European
populations.
The multidimensional scaling method was also applied to the genetic distance matrix data (see fig. 4). The
overall constellation of the 19 populations is quite similar to that of the neighbor-joining tree of figure 3. This
two-dimensional scattergram explains 96.2% of the variation, and the significant portion is already explainedby horizontal axis 1. Because the present-day European
populations are on the left side and the present-day east
Asian populations are on the right side, this representation can be equated to the geographical map of populations, except for the two ancient populations in China. This strengthens the very odd location of the 2,500year-old Linzi population.
It should be noted that the population distance analysis is based only on sequence differences within andbetween populations. Therefore, ambiguity of the phylogenetic relationship of mtDNA haplotypes as represented by a network with reticulations (see fig. 1) does
not cause serious problems in the population analyses.
We compared the genetic structures of three populations that lived in the same location during three sep-1399

arate historical periods and found that the genetic structure of the inhabitants of Linzi has changed greatly overtime. The period of Chinese history that dates to 2,500
years ago corresponds to the transition period from theSpring–Autumn era to the Warring States era, and theperiod around 2,000 years ago was in the middle of theHan era. Linzi, our sampling location, was the capitalof the feudal state Qi in the Spring–Autumn and theWarring States eras. Qin, one of the feudal states during
those periods, conquered other states, including Qi, andestablished the first unified nation in China. Subsequently, the Han dynasty followed Qin after great disturbances of war. Therefore, our finding that the population
structure of Linzi changed drastically during those periods can be concordant with these historical events.
The similarity between the genetic structures of the
2,500-year-old Linzi population and the present-day European populations indicates that there was a genetic
shift in the Linzi area from a European-like population
to a population more like those found in present-day east
Asia, probably caused by migration. This is in accord
with the existence of the Eurasiatic superfamily languages, which surround a linguistically unique Sino-Tibetan language, the present-day Chinese language(Ruhlen 1987, 1994; Cavalli-Sforza, Menozzi, and Piazza 1994). Future molecular studies of ancient populations will help us discover the places and times ofhuman diversification and the migration routes of ancient populations.
Acknowledgments
We gratefully acknowledge the Institute of Archaeology, Shandong Province, China, and the Cultural Relics Bureau of the People’s Republic of China. We also
thank Drs. Kangxin Han, Xunzhang Luo, Y. Manabe andJ. Oyamada for encouragement, Dr. O. Kondo for multidimensional scaling analysis, and Drs. J. Bertranpetit
and F. Calafell for mtDNA sequences of the central
Asians. This study was supported by grants from the
Ministry of Education, Science, Sports, and Culture ofJapan and the NIG Cooperative Research Program. L.W.
was supported by The JSPS Postdoctoral Fellowship for
Foreign Researchers in Japan.

FIG. 4.—The multidimensional scaling of 19 human populations. Populations are designated as follows. 1: the 2,500-year-old Linzi population; 2: the 2,000-year-old Linzi population; 3: the present-day Linzi population; 4: mainland Japanese; 5: Koreans; 6: Ainu; 7: Okinawa
Japanese; 8: Mongols; 9: Altai; 10: Kazakh; 11: Kirghiz (Talas); 12: Kirghiz (Sary-Tash); 13: Uighurs; 14: Turkish; 15: Portuguese; 16: Icelander;
17: German; 18: Finnish; 19: Welsh.

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Wang et al.

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MASAMI HASEGAWA, reviewing editor
Accepted May 29, 2000