Hum Genet (****) ***:*** ***

DOI **.****/s*****-*07-0407-2

ORIGINAL INVESTIGATION

Y chromosomes of prehistoric people along the Yangtze River

Hui Li Ying Huang Laura F. Mustavich Fan Zhang

Jing-Ze Tan Ling-E Wang Ji Qian Meng-He Gao

Li Jin

Received: 15 June 2007 / Accepted: 16 July 2007 / Published online: 27 July 2007

Springer-Verlag 2007

Abstract The ability to extract mitochondrial and nuclear around the mouth of the Yangtze River, linking this culture

DNA from ancient remains has enabled the study of ancient to modern Austronesian and Daic populations. A rare haplo-

DNA, a legitimate Weld for over 20 years now. Recently, Y group, O3d, was found at the Daxi site in the middle reaches

chromosome genotyping has begun to be applied to ancient of the Yangtze River, indicating that the Daxi people might

DNA. The Y chromosome haplogroup in East Asia has since be the ancestors of modern Hmong-Mien populations, which

caught the attention of molecular anthropologists, as it is one show only small traces of O3d today. Noticeable genetic

of the most ethnic-related genetic markers of the region. In segregation was observed among the prehistoric cultures,

this paper, the Y chromosome haplogroup of DNA from demonstrating the genetic foundation of the multiple origins

ancient East Asians was examined, in order to genetically of the Chinese Civilization.

link them to modern populations. Fifty-six human remains

were sampled from Wve archaeological sites, primarily along

the Yangtze River. Strict criteria were followed to eliminate Introduction

potential contamination. Five SNPs from the Y chromosome

were successfully ampliWed from most of the samples, with Ancient DNA has been studied for over 20 years, since the

at least 62.5% of the samples belonging to the O haplogroup, genetic examination of the Quagga (Higuchi et al. 1984) and

similar to the frequency for modern East Asian populations. an ancient Egyptian mummy (P bo 1985). Though the

A high frequency of O1 was found in Liangzhu Culture sites authenticity of the results from ancient DNA has always

been questioned, its study has been accepted as legitimate.

Strict procedures were instituted to minimize the potential

for extraneous DNA contamination, and careful checks were

H. Li F. Zhang J.-Z. Tan L.-E. Wang J. Qian L. Jin

performed to verify the authenticity of putatively ancient

MOE Key Laboratory of Contemporary Anthropology,

DNA (P bo 1989; Stoneking 1995; P bo et al. 2004). A

School of Life Sciences, Fudan University,

large amount of work on ancient DNA studies has been

Shanghai 200433, China

reported, most of which were performed with mitochondrial

H. Li L. F. Mustavich

DNA (mtDNA), since mtDNA has many more copies

Lab for Human Polymorphism Studies,

within each sample than nuclear DNA, increasing the oppor-

Department of Genetics, School of Medicine,

tunity for extraction. Even data from Neanderthal mtDNA,

Yale University, New Haven, CT 06520, USA

which is tens of thousands of years old, has been ascertained

Y. Huang M.-H. Gao

(Serre et al. 2004; Dalton 2006). Ancient nuclear DNA has

Department of Museum, School of Humanities,

also been studied (Lawler et al. 1991; B raud-Colomb et al.

Fudan University, Shanghai 200433, China

1995), illustrating that it is possible to obtain information

H. Li from all types of DNA from ancient remains, in order to

Department of Genetics, School of Medicine,

answer questions of anthropological interest.

Yale University, 333 Cedar Street, SHM-I-347,

The genetic relationship among populations of diVerent

P.O.Box 208005, New Haven, CT 06520-8005, USA

archaeological cultures, as well as between ancient and

e-mail: abpiza@r.postjobfree.com; abpiza@r.postjobfree.com

123

384 Hum Genet (2007) 122:383 388

modern populations, is of great interest to archaeologists in C. Miaoping Type (2500 2200 BC). Around the mouth of

East Asia (Su 1999). The highly ethnic-related Y chromo- the Yangtze River, the catena (ZIA 1999) consisted of

some diversity is one of the best materials to describe the Kuahuqiao C. (6000 BC ), Majiabang C. (5100 3900 BC),

relationships (Su et al. 1999; Shi et al. 2005). The Y chro- Songze C. (3900 3300 BC), Liangzhu C. (3300 2100 BC),

mosome haplogroup patterns are quite diVerent among and Maqiao C. (1900 1200 BC). While the cultures of the

diVerent ethnic groups. For instance, O1 is primarily in diVerent periods showed continuity in each catena, no evi-

Austronesian and Daic populations (Zhang et al. 2007). Y dence of intercultural communications has yet been found.

chromosome studies on ancient DNA were reported as well In the region between the two previous regions, there was

(Hummel and Herrmann 1991; Schultes et al. 1999), most another desultory culture catena (Peng 2005): Xianrendong C.

of which concentrated on short tandem repeats (STRs). (8000 BC ), Shinianshan C. (4000 2500 BC), Shanbei C.

Two Y STR studies on East Asian ancient remains have (2800 2000 BC), and Wucheng C. (1500 1100 BC). For

been reported, which revealed some information of the comparison, there were also some developed culture cate-

ancient Hun s social structure (Keyser-Tracqui et al. 2003), nae in the drainage area of the Yellow River in the North

and the relationship between Amerindians and ancient China. The most important was the catena of Peiligang C.

Siberians (Ricaut et al. 2005). However, haplogroups can (7000 5000 BC), Yangshao C. (5000 3000 BC), Longshan C.

not be determined by Y STR data alone, but by single (3000 2200 BC), Erlitou C. (2200 1500 BC), and Shang C.

nucleotide polymorphisms (SNPs). The Wrst reported (1500 1100 BC), which was believed to be the major origin

ancient Y SNP data was typed from a Native American of the Chinese Civilization. Some argue that these Neo-

sample of an extinct tribe (Kuch et al. 2007). Another paper lithic cultures along the Yangtze River were also origins of

reported the Y SNP data of 11 ancient South Siberian sam- Chinese Civilization. It will certainly excite the anthropolo-

gists of East Asia to reveal whether the people of the diVer-

ples (Bouakaze et al. 2007). In this paper, we present Y

SNP data of 48 ancient samples from China, mostly along ent Neolithic cultures were ancestors of modern Chinese or

Yangtze River, to provide a survey on the genetic diversity other nations by genetic methods. It will also raise the

of the prehistory populations in this region. professional enthusiasm to know whether the culture diver-

There were several diVerent archaeological culture (C.) siWcation coincided with the genetic diversiWcation.

regions in East Asia in the Neolithic Age from around

9,000 3,000 years before present (Su 1999). The cultural

diVerences among the regions lasted throughout the whole Materials and methods

Neolithic Age and into the Bronze Age. There were two

series of Neolithic cultures quite distinct from each other in Archaeological sites and samples

the drainage area of Yangtze River. In the Three Gorges

The samples collected in this study were from Wve sites:

region in the middle reaches of the Yangtze River, the cul-

ture catena (CSACH 1998) consisted of Nanmuyuan C. Maqiao, Xindili, Wucheng, Daxi, and Taosi. The locations

(6000 5000 BC), Liulinxi C. (5000 4400 BC), Daxi C. were marked in Fig. 1. Most of the samples belonged to

four diVerent cultures: Daxi C. was the earliest, followed by

(4400 3300 BC), Qujialing C. (3300 2500 BC), and Shijiahe

Fig. 1 Locations of the archae-

ological sites, cultures and the

distributions of Y SNP haplo-

groups

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Hum Genet (2007) 122:383 388 385

the Longshan C. and Liangzhu C. around the same period, and sealed carefully before they were carried out of the pre-

and Wnally, by Wucheng C., the latest in the Shang Dynasty PCR rooms. The PCR room is far from the other rooms,

and airXow between the PCR room and the other rooms are

of the Bronze Age. These cultures were most representative

for the prehistory of China. Some samples from the same strictly avoided. The post-PCR productions were therefore

necropolises that belonged to the historical time (later than physically separated from the pre-PCR procedures.

841 BC in China) were also collected for comparison. Most Only female researchers were involved in the pre-PCR

of them belonged to the Han Dynasty. The remains of the procedures, preventing possible contamination from mod-

same culture were sampled from diVerent necropolises, ern Y chromosome DNA. Each skeleton was sampled

from several diVerent parts: mostly teeth, astragalus, cal-

avoiding the bias of relative samples.

The skeletons were buried directly in the loessal soil, caneus, vertebrae, etc. The teeth we chose were all intact

with no apparent chests or coYns. Most of the skeletons (without carious lesions), and still Wxed to the jaw. We

excavated in the area of Yangtze River were rotten, but only used bones with thick cortical, as the density of corti-

cal bone oVers two advantages compared to spongy bone.

some of the necropolises were built in the higher places and

remained in good condition, covered tightly and protected First, the quantity of the mineral crystal of hydroxyapatite

on which the DNA is Wxed is higher than in the spongy

by the loess. We chose the unbroken and hardest skeletons

for our samples. A loess covering of 2 in. was not removed bone. Second, it helps protect against contamination. The

from each skeleton before it was carried into the dedicated outer surface of the bones was removed to almost 2 mm of

depth. The same procedures were performed in two diVer-

ancient DNA laboratory to minimize the risk of contamina-

ent labs on diVerent samples from the same skeletons for

tion during excavation and transportation. DNA of each

sample was extracted and typed in one month after excava- reproduction controls. For each SNP, at least three rounds

of ampliWcations were repeated in each lab. Extraction

tion, ensuring the freshness of the sample, which was best

for ampliWcation (Pruvost et al. 2007). The sex of each controls were also provided. The same procedure was per-

skeleton was established according to the methodology formed for the wood controls as for the human remains.

Extraction and ampliWcation blanks were still used as neg-

developed by Murail et al. (1999). Only male samples were

subjected to the Y SNP genotyping. In total, 56 individual ative controls.

remains were included in the experiments. Some pieces of We did not clone the PCR products or quantify the num-

wood were also collected in the same way from the same ber of starting templates in the reaction following the crite-

tombs for extraction controls. Although animal remains ria of some researchers (Cooper and Poinar 2000; Gilbert

would make better controls, no animal remains were found et al. 2005), as the damage or jumping PCR will not result

associated to the human skeleton. in mistakes in the determination of Y SNP alleles.

DNA extraction and ampliWcation

Measures taken to avoid contamination and ensure

authenticity

DNA was carefully extracted according to a published pro-

We have two isolated laboratories dedicated to work with tocol (Fily et al. 1998); no adaptation was made on the pro-

ancient DNA. As we have mentioned, samples were never tocol. The sample bones were crushed under liquid nitrogen

touched by any person before they were moved into the iso- in a freezer mill and DNA was extracted by the silicone

lated labs, because they were covered by the loess in the method (Gilbert et al. 2003). As the protocol has been

tombs. Even when they were covered, they were handled described by many papers in brief or detail, we need not

with gloves by a reduced number of anthropologists wear- repeat it here. Extraction was performed in a clean bench

ing face masks and caps. During the transportation, the hood with positive air pressure and UV irradiation, which

samples were packaged up in the plastic bags hermetically. was cleaned and sterilized between the extractions of every

Our ancient DNA labs are strictly controlled following two samples. Only one sample was extracted at a time to

all the criteria for ancient DNA studies (P bo et al. 2004), avoid the cross-contamination.

such as routine sterilization by diVerent treatments (DNAse The SNPs that we typed were M119, M95, M122, M7,

M134. They formed Wve haplogroups (O1, O2a, O3*, O3d,

away, positive air pressure, bleach and ultraviolet light irra-

diation), air Wltration, and isolated rooms for diVerent O3e) according to the YCC nomenclature (YCC 2002). The

ampliWcation protocol was the same as that previously pub-

experimental steps (three rooms for sample cleaning, DNA

extraction, and PCR cocktail preparation, respectively). lished (Su et al. 1999; Ke et al. 2001). The annealing circles

Less than three researchers can work in each lab, wearing of the PCR reactions were added to 60. The length of the

full body protective clothing, and using dedicated equip- PCR productions were all around 100 200 bp, shorter than

ments and reagents. Pre-PCR work was performed in the the general size of ancient DNA between 100 and 500 bp

designated rooms of the labs. PCR cocktails were prepared (Hofreiter et al. 2001).

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386 Hum Genet (2007) 122:383 388

Results and discussion In two sites of Liangzhu Culture, only O1 haplogroup

were found, and the frequencies of O1 in two sites were

No ampliWable product could be obtained from any of the almost the same. That indicated the ancient people of these

extraction control samples (wood pieces), or from eight two sites belonged to the same population. The historical

samples from the same sites did not diVer from the prehis-

individual skeletons. DNA of these eight individuals might

have been severely degraded (shown as missing data in toric samples in the haplogroup patterns. Even modern

Table 1). There were no diVerences between the non-work- populations in this area around Shanghai contain a large

ing samples and the working samples. It was not possible proportion of O1 haplogroup (Wen et al. 2004). The consis-

for us to determine if there were wrong morphological sex tency of the Y haplogroup pattern in this area, from the

determinations for the non-working samples, as the sex Neolithic Age to modern times, reveals that the population

determination fragment of amelogenin could not be ampli- might not have been replaced. O1 reaches the highest

Wed for them either. For some of the samples, not all of the frequencies in the Taiwan aborigines, and also in Daic

Wve SNPs could be ampliWed, thus haplogroups could not be speaking populations in the southwest China (Li 2005).

determined if no mutated alleles were found (shown as Therefore, it is also possible that there are some close rela-

undetermined in Table 1). However, most individuals were tionships among Taiwan aborigines, Daic speakers and the

successfully ampliWed, and the haplogroups were deter- ancient Liangzhu Culture populations.

mined for half of them. Therefore, at least 62.5% of the A high frequency of O3d was only found in Daxi Cul-

individual remains (30 out of 48) belong to O haplogroup, ture. O3d is very rare in modern populations; Hmong-Mien

which is still the major haplogroup of today s East Asians. populations have been found to contain a small proportion

These ancient results, consequently, did not diVer from the of O3d (Feng 2007). Among those Hmong-Mien popula-

modern populations. The resulting DNA types thus made tions, She and Bunu were found to have the highest fre-

phylogenetic sense (the Y chromosome haplogroup struc- quency of O3d (Su et al. 1999). Since O3d occurs at low

ture), helping to verify the authenticity of the ancient DNA. frequency in the Hmong-Mien, the ancient people of Daxi

The previously reported results of ancient Y chromosome Culture might be the ancestors of the modern populations

were all obtained from samples preserved in cold environ- of Hmong-Mien. The absence of O3d in the historical sam-

ments (Keyser-Tracqui et al. 2003; Ricaut et al. 2005; Kuch ples from the Daxi site (it might not have been found

et al. 2007; Bouakaze et al. 2007). However, our samples because of the small sample size), and the migration of

were not buried in a really cold environment. Therefore, the modern Hmong-Mien populations to the southwest might

relatively high ampliWcation rate should have resulted from indicate that the prehistoric population in the Three Gorges

the strict selection of the best-preserved remains. area has been replaced.

Table 1 Case counts of Y SNP

Site Culture Sample size O1 O2a O3* O3d O3e Undetermined Missing

haplogroups of the archaeologi-

cal sites Maqiao Liangzhu 6 4 2

Historical 3 2 1

Xindili Liangzhu 9 5 3 1

Historical 4 3 1

Wucheng Wucheng 4 2 1 1

Daxi Daxi 20 1 1 5 9 4

Historical 5 2 3

Taosi Longshan 5 3 1 1

Frequencies in modern populations Family Reference Sample size O1 O2a O3* O3d O3e

Daic Li (2005) 1,465 34.87 26.52 10.05 0.19 9.36

Austronesian Li (2005) 381 24.07 16.25 22.41 2.01 2.32

Sino-Tibetan Su et al. (1999) 281 1.57 8.49 17.24 0.53 32.74

Hmong-Mien Feng (2007) 934 8.49 21.87 17.19 10.45 26.38

Austro-Asiatic Li (2005) 140 1.79 31.40 18.49 6.25 19.73

Altaic Su et al. (1999) 303 1.74 4.11 6.60

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Hum Genet (2007) 122:383 388 387

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DNA sequences from the quagga, an extinct member of the horse

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Zhejiang Institute of Archaeology, Jiangxi Institute of Archaeology Peng M-h (2005) Culture research about Wucheng. Wenwu Press,

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