AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 133:1128–1136 (2007) Tracing the Genetic History of the Chinese People: Mitochondrial DNA Analysis of a Neolithic Population from the Lajia Site Shi-Zhu Gao,1,2 Yi-Dai Yang,1 Yue Xu,3 Quan-Chao Zhang,1 Hong Zhu,1 and Hui Zhou1,3* 1Laboratory of Ancient DNA, Research Center for Chinese Frontier Archaeology of Jilin University, Changchun 130012, China 2College of Pharmacia Sciences, Jilin University, Changchun 130021, China 3College of Life Science, Jilin University, Changchun 130023, China KEY WORDS ancient mtDNA; Lajia Site; Chinese people ABSTRACT Ancient DNA analysis was conducted on Possible maternal familial relationships were investi- the dental remains of specimens from the Lajia site, gated through mitochondrial DNA (mtDNA) sequence dating back 3,800–4,000 years. The Lajia site is located in analysis. Twelve sequences from individuals found in one Minhe county, Qinghai province, in northwestern China. house were assigned to only five haplotypes, consistent Archaeological studies link Lajia to the late period of the with a possible close kinship. Results from analyses of Qijia culture, one of the most important Neolithic civiliza- RFLP typing and HVI motifs suggest that the Lajia peo- tions of the upper Yellow River region, the cradle of Chi- ple belonged to the haplogroups B, C, D, M*, and M10. nese civilization. Excavations at the site revealed that the This study, combined with archaeological and anthropo- inhabitants died in their houses as the result of a sudden logical investigations, provides a better understanding of flood. The Lajia site provides a rare chance to study the the genetic history of the Chinese people. Am J Phys putative families, all of whom died at the same instant. Anthropol 133:1128–1136, 2007. VC 2007 Wiley-Liss, Inc. The Lajia site is located in the Guanting basin of the In 2000, archaeologists working in the northeast part of upper Yellow River, in Minhe county, Qinghai province, the Lajia site discovered 16 human remains in two ruined northwestern China (Fig. 1). According to radiocarbon houses, designated F3 and F4. Excavations at the site dating, the Lajia site is 3,800–4,000 years old (Xia et al., revealed that the cause of the death of the inhabitants was 2003). Archaeological studies have associated the Lajia a sudden earthquake followed by a flood (Xia et al., 2003). site with the late period of the Qijia culture, a major The Lajia civilization ceased to exist after this natural dis- culture that flourished during the late Neolithic Age to aster, so that the Lajia site is sometimes termed \the east- early Bronze Age (Ren et al., 2002) in the Hexi Corridor, ern Pompeii" by archaeologists (Zhang et al., 2004). a major route leading to the central regions of the Yellow Two of the 16 human remains were found in F3. The River. The Qijia culture belonged to the cultures of the skeletons of these two individuals were in close apposi- Di-Qiang, an ancient tribe confederation in northwestern tion, against the eastern wall. Anthropomorphic analyses China (Liu, 2003). According to ethnological studies, the suggested that these remains belonged to an approxi- Di-Qiang population contributed to the development of mately 35-year-old female and 3–4-year-old child. It was the current Han and Tibeto-Burman (TB) speaking pop- supposed that the female was the mother of the child ulations (Yang and Ding, 2003). In the late Neolithic Age and was embracing her child tightly to protect him/her to early Bronze Age period, a branch of the Di-Qiang from danger during the disaster. people migrated eastward and merged with tribes in the The 14 skeletons found in F4 were placed into six middle and lower Yellow River valley, bringing into being groups (Fig. 2). One group (Group III) found at the east- one constituent of the Huaxia ethnic group (Yang and ern wall also consists of a presumed mother–child pair. Ding, 2003). The Huaxia ethnic group later absorbed many other cultures, including some from the Yangtze River region, to form the famous Huaxia civilization. Grant sponsor: Natural Science Foundation of China, NSFC; After a long process of integration and expansion, the Grant number: J0530184. Huaxia civilization developed into the Han, which forms the largest ethnic group in present-day China (Tian, *Correspondence to: Hui Zhou, Laboratory of Ancient DNA, Research Center for Chinese Frontier Archaeology, Jilin University, 2001; Xu, 2003). In addition, a wave of the Di-Qiang Jiefang Road 115, Changchun, 130023 China. tribes migrated to the southwest of China starting E-mail: [email protected] 4,000–5,000 years ago and mixed with several indigenous tribes. The southwest migrants of the Di-Qiang people Received 10 April 2006; accepted 28 February 2007 developed into part of the Tibeto-Burman (TB) speaking populations that are now primarily distributed in the DOI 10.1002/ajpa.20623 Qinghai, Tibet, Sichuan, Yunnan, and Hunan regions of Published online 16 May 2007 in Wiley InterScience China (Yang and Ding, 2003). (www.interscience.wiley.com). VC 2007 WILEY-LISS, INC. ANCIENT DNA ANALYSIS OF THE LAJIA POPULATION 1129 Fig. 1. Geographic location of the Lajia population (red star) and the modern Han populations. Population codes are as reported in table 6. Pie charts show the distribution of the A, B, C, D, and M*/M10 in the Han populations. D# represents the sub-hap- logroups of D except D4. B# represents the sub-haplogroups of B except B5. M* includes some unidentified M10 haplotypes. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] southwestern corner. A youth’s remains (Group I) were found near the fireplace at the center of the house and a juvenile’s skeleton (Group II) was found northeast of the fireplace. All are suspected to be close relatives who died in the flood. The familial relationships within these 16 specimens are of great interest. Unlike most archaeologi- cal burial sites that have hundreds of individuals and were occupied often for thousands of years, the Lajia site provides a rare opportunity to study a group of people that lived together and died suddenly at a single point in time. In the present study, we used mtDNA analysis to in- vestigate the maternal relationships of the ancient inhabitants whose remains were found in F3 and F4 and their relationship to modern populations. The results provide valuable new insights into the familial relation- ships of the Lajia specimens as well as their relationship to modern Han and TB populations. MATERIALS AND METHODS Fig. 2. Spatial distribution of skeletons and assignment into groups for F4. Circles having the same color represent individu- Sample collection als sharing the same mtDNA haplotypes. Greyed-out circles rep- resent samples that could not be amplified. Adult individuals Samples for DNA analysis were collected from the are indicated by larger circles. [Color figure can be viewed in the teeth of the 16 human remains found at the Lajia site. online issue, which is available at www.interscience.wiley.com.] The use of teeth as a source of DNA has the advantage that multiple, independent samples can be obtained per Group V and VI are comprised of five skeletons in the individual (O’Rourke et al., 2000). All teeth were sampled north or northwestern corner, and Group IV is comprised from well-preserved molars without caries and cracks to of five skeletons, mostly children, at the west wall a minimize the possibility of modern DNA contamination. American Journal of Physical Anthropology—DOI 10.1002/ajpa 1130 GAO ET AL. The estimated age and sex of the skeletons is shown in PCR amplification was carried out in 25 ll of a reac- Table 1. tion mixture containing 67 mM Tris–HCl (pH 8.8), 2 mM MgCl2, 50 mM KCl, 500 mM each of dNTPs, 0.5 lM of each primer, 1 U of Taq polymerase (Promega) and 1.3 mg/ml BSA. Cycling parameters were 948C for 10 min, Ancient DNA extraction, amplification, followed by 33 cycles with 948C for 1 min, 45 s at 528C and sequencing for HVIa and HVIb or 45 s at 558C for HVIc and HVId, Teeth were first soaked in 10% bleach for 20 min, elongation for 1 min at 728C, extension 10 min at 728C rinsed with ethanol and distilled water, and then UV- and storage at 48C. To verify the reliability of the experi- irradiated for 30 min on each side (Ovchinnikov et al., ments, negative controls containing all of the reagents 1999). Each tooth was powdered in a 6750 Freezer Mill but without the ancient DNA sample were included in (SPEX, USA). DNA was carefully extracted from 500 mg each DNA extraction/PCR run. Amplification products of the tooth powder using the Geneclean for Ancient were checked on a 2% agarose gel and purified with DNA kit (BIO 101, Vista, CA) according to the manufac- QIAquick Gel Extraction Kit (Qiagen). PCR products turer’s protocol. Amplification of the mtDNA was per- were sequenced using the ABI 310 Terminator Sequenc- formed on four subregions (a, b, c, and d) of the first ing kit (PE Applied Biosystems) according to the manu- hypervariable segment of the control region (HVI) using facturer’s instructions and were analyzed on the ABI the primers listed in Table 2. PRISM 3100 automatic sequencer (PE Applied Biosys- tems). DNA sequences were analyzed using CLUSTAL X 1.83. TABLE 1. Estimated age and sex of the Lajia individuals Specimen numbers Sexa Age RFLP typing House (F3) Because control regions of the mtDNA exhibit a rela- Y3 Female About 35 years old tively high evolutionary rate, unique haplogroups should Y4 ? 3–4 years old not be assigned to specimens using only these regions House (F4) (Richards et al., 1996; Macaulay et al., 1999).