Genetic history of Southeast Asian populations as revealed by ancient and modern human mitochondrial DNA analysis Genetic History of Southeast Asian Populations as Revealed by Ancient and Modern Human
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 137:425–440 (2008) Genetic History of Southeast Asian Populations as Revealed by Ancient and Modern Human Mitochondrial DNA Analysis Patcharee Lertrit,1* Samerchai Poolsuwan,2 Rachanie Thosarat,3 Thitima Sanpachudayan,1 Hathaichanoke Boonyarit,1 Chatchai Chinpaisal,4 and Bhoom Suktitipat1 Department of Biochemistry, Faculty of Medicine, Mahidol University, Bangkok 10700, Thailand Faculty of Sociology and Anthropology, Thammasat University, Bangkok 10200, Thailand Office of Archaeology, Thai Fine Arts Department, Bangkok 10200, Thailand Department of Pharmacology and Toxicology, Silpakorn University, Nakhonpathom 73000, Thailand KEY WORDS ancient mtDNA; migration history; Southeast Asian ABSTRACT The 360 base-pair fragment in HVS-1 of the mitochondrial genome were determined from ancient human remains excavated at Noen U-loke and Ban LumKhao, two Bronze and Iron Age archaeological sites in Northeastern Thailand, radio-carbon dated to circa 3,500– 1,500 years BP and 3,200–2,400 years BP, respectively These two neighboring populations were parts of early agricultural communities prevailing in northeastern Thailand from the fourth millennium BP onwards The nucleotide sequences of these ancient samples were compared with the sequences of modern samples from various ethnic populations of East and Southeast Asia, encompassing four major linguistic affiliations (Altaic, Sino-Tibetan, TaiKadai, and Austroasiatic), to investigate the genetic relationships and history among them The two ancient samples were most closely related to each other, and next most closely related to the Chao-Bon, an Austroasiatic-speaking group living near the archaeological sites, suggesting that the genetic continuum may have persisted since prehistoric times in situ among the native, perhaps Austroasiaticspeaking population Tai-Kadai groups formed close affinities among themselves, with a tendency to be more closely related to other Southeast Asian populations than to populations from further north The Tai-Kadai groups were relatively distant from all groups that have presumably been in Southeast Asia for longer-that is, the two ancient groups and the Austroasiatic-speaking groups, with the exception of the Khmer group This finding is compatible with the known history of the Thais: their late arrival in Southeast Asia from southern China after the 10th–11th century AD, followed by a period of subjugation under the Khmers Am J Phys Anthropol 137:425–440, 2008 V 2008 Wiley-Liss, Inc The early peopling of Southeast Asia has recently been a shared focus of archaeological, linguistic, and anthropological-genetic investigations (for detailed reviews on this issue see: Jin et al., 2001; Sagart et al., 2005) Several major hypotheses have been advanced concerning the significance of this geographic area One is that it may be an ancestral homeland of almost all present-day East Asian natives (Chu et al., 1998; Jin and Su, 2000; Su and Jin, 2001) Another is that, more recently, expanding populations have moved into the area, in association with specific cultural and linguistic spreads, presumably from central or southern China (Renfrew, 1996; Bellwood, 2001, 2005; Higham, 2003; Blench, 2005; Lu, 2005) However in this area, patterns of cultural diffusion detected archaeologically are not always congruent with current patterns of linguistic and genetic differentiation (Chu et al., 1998; Ding et al., 2000; Yao et al., 2002a; also see reviews in Sagart et al., 2005) This suggests that the formation of ethnic populations in the area was complicated both culturally and genetically A number of causes of such discrepancies have been suggested, including language shifts and cultural diffusion, sometimes without population replacement or major expansion, and substantial population admixture (Ding et al., 2000; Wang, 2001; Lu, 2005) One major demographic question that naturally arises is whether contemporary ethnic populations of this geographic area can be considered as direct descendants, in the genetic sense, of their ethnic and linguistic predecessors, thus validating the use of modern samples to represent and trace the expansion of the presumed long-estab- lished ethnic populations Without a thorough understanding of the dynamics of genetic structures of ethnic populations since the distant past, this problem will remain unsolved Employment of ancient human DNA analysis can partially resolve this serious problem concerning our understanding of recent human evolution in this geographic area In this article, we provide an analysis of ancient human mtDNA drawn from two neighboring prehistoric populations in northeastern Thailand, spanning the period approximately between 3,500 and 1,500 BP, in the context of their spatial and temporal relationships with various contemporary ethnic popula- C V 2008 WILEY-LISS, INC C Additional Supporting Information may be found in the online version of this article Grant sponsor: Siriraj Research and Development Fund, Faculty of Medicine Siriraj Hospital, Mahidol University; Grant number: 002(III)/48 *Correspondence to: Patcharee Lertrit, Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand E-mail: sipwy@mahidol.ac.th and lertrito@yahoo.com Received 10 January 2007; accepted 28 April 2008 DOI 10.1002/ajpa.20884 Published online July 2008 in Wiley InterScience (www.interscience.wiley.com) 426 P LERTRIT ET AL Fig Distribution of Bronze and Iron Age archaeological sites in the Upper Mun River Valley, northeastern Thailand; exact locations of Noen U-Loke and Ban Lum-Khao are labeled (modified from Higham, The origin of the civilization of Angkor, The final report to the National Research Council of Thailand, 2000, pp 1-160, ' Department of Anthropology, University of Otago, reproduced by permission) [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] tions of East and Southeast Asia An attempt was also made to provide more insight into the complex demographic dynamics of Southeast Asian populations, particularly Austroasiatic and Thai speakers MATERIALS AND METHODS Ancient samples Two ancient populations from the Noen U-loke and Ban Lum-Khao archaeological sites were selected for American Journal of Physical Anthropology this study These sites are representative of the ancient settled farming communities prevailing from 3,500 BP up to the late Angkorian period around 600 BP in the Upper Mun valley, Non-Sung District, Nakhorn-Ratchasima Province, northeastern Thailand Figure illustrates the dense distribution of prehistoric sites in the area, including the two aforementioned sites; the cultural similarities and overlapping periods of human occupation observed among these sites (Higham, 2000) would suggest continuous settle- 427 ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES TABLE Details of skeletons sampled in the study of Noen U-Loke No Burial Codea Mortuary Phase Dates (BP) Depth below datum (m) Sex of the skeleton Age at death (year) Position of tooth sampledb 10 11 12 13 14 15 16 17 26 33 48 50 15 31 62 69 74 99 111 113 60 66 68 3 4 4 4 4 5 5 2,400–2,100 2,200–1,800 2,200–1,800 2,200–1,800 2,200–1,800 1,800–1,600 1,800–1,600 1,800–1,600 1,800–1,600 1,800–1,600 1,800–1,600 1,800–1,600 1,700–1,500 1,700–1,500 1,700–1,500 1,700–1,500 1,700–1,500 3.65 2.62 2.81 2.91–3.10 N/A 2.18 1.54 1.82 2.29 2.43 2.57 3.65 0.82 1.27 1.09–1.39 1.52 1.25 # # $ # ? ? $ # # $ # $ # ? # # # mid-adult 25–30 25–30 20–25 N/A $12 35–40 25–30 25–30 [40 25–30 25–30 20–25 25–60 20–25 35–40 25–30 46 38 47 36 38 27 48 38 48 48 38 N/A N/A N/A 28 48 46 a As determined in the excavation report (Higham, 2000) Identified according to International Dental Federation (FDI) nomenclature: first digit is for quadrant (1, maxillary right; 2, maxillary left; 3, mandibular left; 4, mandibular right) and second digit is for tooth position in each quadrant (1, central incisor; 2, lateral incisor; 3, canine, etc.) Loose tooth found in situ in confident association with the corresponding cranial sample was labeled N/A b ment of probably related groups of prehistoric people in the area Excavations at both sites were conducted under collaboration between the Fine Arts Department of Thailand and Department of Anthropology, University of Otago, New Zealand They have yielded numerous human skeletal and cultural remains from the Bronze and Iron Ages, radio-carbon dated to the period between 3,500 and 1,500 BP (Higham, 2000; Higham and Thosarat, 2004) Dental samples (either second or third molars) were collected for mtDNA extraction from 26 individual skeletons found in situ at the Noen U-Loke site and from 16 individual skeletons at the Ban Lum-Khao site This work was undertaken with official permission from the Fine Arts Department of Thailand Individual samples in each site, collected from different mortuary phases, presumably belonged to the same prehistoric population, as indirectly suggested by the continuous usage of the area as a burial ground by the prehistoric people In addition, there is no archaeological evidence of major cultural intrusion that might suggest population replacement Every effort was made to maximize sample sizes from both ancient populations, given the constraints of a paucity of usable specimens and the need to preserve some specimens for usage in further studies Noen U-Loke was a large prehistoric cemetery with mortuary sequences divided into five phases spanning the period between 3,500 and 1,500 BP No dental samples were available for the study from the deepest mortuary phase One, six, ten, and nine dental samples were collected from skeletons in mortuary phases 2, 3, 4, and 5, respectively The available samples from this site thus spanned the period between 2,400 and 1,500 BP Details of the sampled skeletons are shown in Table Ban Lum-Khao was another excavated prehistoric cemetery, some 10 km distant from Noen U-Loke The mortuary sequences at this site spanning the period between 3,200 and 2,400 BP were divided into three phases Three, eight, and five dental samples were collected from skeletons in phases 1, 2, and of the burial site, respectively Details of the sampled skeletons are shown in Table To demonstrate the preservation of ancient DNA in this area, the same DNA extraction procedure was applied to pig remains found in the Ban Lum-Khao excavation site at 1.56 m depth (Fine Arts Department of Thailand, 2004 Modern samples Analysis of mtDNA from present-day natives of Thailand was also conducted Hair roots were collected with informed consent from 74 unrelated healthy individuals whose ancestry could be traced back for at least three generations to confirm their ethnic identification Our samples comprised three groups: 1) Thirty two ThaiKorat individuals from neighboring areas of the archaeological sites (Ban Lum-Khao and Ban Non-Wat, NonSung District, Nakhorn-Ratchasima Province); 2) Twenty individuals of Chao-Bon, a Mon-Khmer speaking group residing in Pak-Thong-Chai District, Nakhorn-Ratchasima Province; and 3) Twenty two Khmer individuals from the Thailand-Cambodia border in Chanthaburi Province Preventive treatment against contamination of ancient DNA All steps of ancient DNA preparation were carried out at Mahidol University under sterile conditions in a laboratory used only for ancient DNA analysis It is physically separated from other laboratories, and was sterilized by Bleach treatment and ultraviolet irradiation at 254 nm nightly Laboratory precautions were taken according to standard protocols (Stoneking, 1995; Handt et al., 1996; Cooper, 1997; Jehaes et al., 1998; Kolman and Tuross, 2000; Eshleman and Smith, 2001; Paăaăbo et al., 2004) to minimize the risk of contamination and to detect any potential contamination during the process of ancient DNA preparation Laboratory coats, face shields, and gloves were used by all personnel at all times Two sets of pipettes were used with filter tips: one for DNA extraction and one for PCR reactions All PCR reagents were aliquoted and used once All glassware American Journal of Physical Anthropology 428 P LERTRIT ET AL TABLE Details of skeletons sampled in the study of Ban Lum Khao No Burial Codea Mortuary Phase Dates (BP) Depth below datum (m) Sex of the skeleton Age at death (year) Position of tooth sampledb 10 11 12 13 64 72 31 42 65 67 109 25 33 39 49 51 1 2 2 2 3 3 3,200–3,000 3,200–3,000 3,000–2,600 3,000–2,600 3,000–2,600 3,000–2,600 3,000–2,600 3,000–2,600 2,600–2,400 2,600–2,400 2,600–2,400 2,600–2,400 2,600–2,400 1.71 1.57 1.22 N/A 1.76 1.26 1.39 1.89 1.56 N/A 1.14 0.79 0.78 $ $ $ # $ # # # # $ $ $ $ 40–44 20–24 Over 40 Over 40 20–29 Over 40 25–29 20–29 17 25–29 25–29 20–24 20–24 18 48 16 18 28 17 38 N/A 17 18 48 N/A 17 a As determined in the excavation report (Higham, 2000) Identified according to International Dental Federation (FDI) nomenclature: first digit is for quadrant (1, maxillary right; 2, maxillary left; 3, mandibular left; 4, mandibular right) and second digit is for tooth position in each quadrant (1, central incisor; 2, lateral incisor; 3, canine, etc.) Loose tooth found in situ in confident association with the corresponding cranial sample was labeled N/A b was treated with N HCl and rinsed with sterile deionized water before use Pretreatment of samples The second or third molar tooth with intact pulp cavity was selected from each sampled skeleton for ancient mitochondrial DNA extraction In the case of an intact jaw, the tooth was extracted using a sterile mini-drill Workers were required to use face masks and disposable hand gloves at all stages of sample preparation in order to prevent contamination with recent DNA Each collected tooth sample was sealed in a plastic bag and kept in a dark and dry environment until use Each tooth sample was prepared for DNA extraction by soaking for 15 in 10% sodium hypochlorite solution to eliminate all sources of external contamination The sample was then washed 2–3 times with sterile distilled water and exposed to UV light for at least 20 on each surface The cleaned and dried tooth was then crushed into powder in an enclosed laminar hood using a sterile mortar and pestle 250 mg of tooth powder was placed into each of five-tubes and kept at 2208C until used for DNA extraction DNA extraction and purification A silica-based protocol (Boom et al., 1990; Hoss and Paăaăbo, 1993) was adopted for ancient DNA extraction and purification Tooth powder was suspended by vortexing in 500-ll of lysis buffer (6M GuSCN, 20 mM EDTA, 10 mM Tris-HCl pH 6.4, 0.65% Triton X-100) and incubated at 608C for 16 h with occasional agitation After centrifuging at 12,000 rpm for 5-min, the supernatant was collected, added to 1-ml of lysis buffer containing mg/ml proteinase K and incubated for h at 608C DNA purification was conducted in the dark at room temperature by adding 40-ll of silica suspension and leaving for 10–15 with occasional agitation DNA-containing silica was sedimented at 6,000 rpm for 3-min, re-suspended in 1-ml of wash buffer (6M GuSCN, 10 mM TrisHCl pH 6.4) and left to stand for a while at room temperature After centrifuging at 6,000 rpm for 3-min, supernatant was removed and the silica pellet was American Journal of Physical Anthropology re-suspended twice with 95% ethanol in order to remove all traces of GuSCN which could inhibit the subsequent PCR The suspended silica was left at 2208C for 15-min and sedimented at 6,000 rpm for 3-min Ancient DNA was then extracted from the silica pellet by the addition of 75-ll of elution buffer (10 mM Tris-HCL pH 8.0) and incubation at 568C for 10-min After sedimenting the silica at 12,000 rpm for min, a 50-ll aliquot of the supernatant containing purified extracted ancient DNA was placed in a new Eppendorf tube and stored either at 2208C for use in PCR amplification within week, or at 2808C for longer storage At least two tubes of tooth powder from each sample were subjected to DNA extraction, which was carried out only on one tube of tooth powder at a time The second extraction of each sample was performed months after the first Blank extraction was also performed simultaneously for all extracted samples and procedures DNA extraction and sequencing from randomly selected ancient samples (NUL 5, NUL 8, NUL 26, NUL 50, BLK 7, BLK 33, BLK 49, and BLK 109) was also performed in the laboratory at the Faculty of Pharmacology, Silpakorn University, located in a different province from the Faculty of Medicine Siriraj Hospital, Mahidol University All of the DNA sequences from these selected ancient samples obtained at Silpakorn University match exactly with the results obtained in our laboratory, supporting the accuracy of the laboratory procedure The ancient pig DNA was also extracted from a third molar using the same protocol Modern DNA from hair follicles was isolated in a different laboratory using the same digestion buffer and proteinase K (Moore, 1995) After incubation at 378C overnight, the digestion mixture was heated at 958C for 10-min and stored at 2208C until used directly for PCR amplification HVS-1 amplification PCR reactions were prepared under a separate laminar flow hood using a dedicated set of pipettes Human mitochondrial DNA hypervariable segment (HVS-1; nt15978-nt16355) was amplified using nested PCR of overlapping fragments (F1 (nt15978-nt16224), F2 (nt15978-nt16112), F3 (nt16064-nt16224) and F4 429 151 61 TGC ATT GGA 182 56 228 58 161 C TG G TC GTC 58 135 58 247 58 151 61 TGC ATT GGA 182 56 233 58 Dog primer Pig primer Human L16190 H16422 Pig L15686 Pig H15867 Dog L15561 Dog H15692 CAT GAT CAT CCA CGT GGT GCT TTC CGC GAT GCA GAT TAC ACG ACA GCC TTA TAA AAG GAG TAT TGT ATG GCC CAA GAT CAT TA GTT CTT G GG GTC – F4 CCC ATT GTA GAA CGT CAT – F3 AGG GTT GAT TGC TGT ACT TG H16224 F2 247 55 Human 58 378 CAC CAT TAG CAC CCA AAG GGG ATT TGA CTG TAA TGT GC CAC CAT TAG CAC CCA AAG F1 L15978 H16224 L15978 H16112 L16064 H16224 L16190 H16417 Pig L15686 Pig H15867 Dog L15561 Dog H15692 CAC AGG CAC GGC TGA AGG CCC TTT GTA GAA CGT CAT CAT GTT CAT TGG CTC GTT CAT CAC CAT CCA CGT GGT TAG GAT TAG CAG ACC GAT GCT GGA CGC GAT GCA GAT CAC TGC CAC TAA CAT TGC TAC GGA ACA GCC TTA TAA CCA TGT CCA TGT CAA TGT AAG TGG TAT TGT ATG GCC AAG ACT AAG ACG CAA ACT CAA TGG CAT TA GTT CTT TG Annealing temperature (8C) Sequence 50 to 30 Name of nested PCR primer No of overlapping fragment Sequence 50 to 30 Annealing temperature (8C) Size of product (bp) L15978 H16355 L15978 All mtDNA segments were aligned using ClustalW Multiple Alignment Software (Hall, 1999) to determine sequence haplotypes Additional sequences from various other East and Southeast Asian populations obtained in other studies were also included in this study for genetic comparison purposes (Table and Fig 2) These populations encompassed four main linguistic families: Austroasiatic (Chao-Bon, Khmer and Chong), Tai-Kadai (ThaiKorat, Thai-Koen kaen, Thai-Chiang Mai, northern Human Data analysis TABLE Oligonuclelotide primers used for primary and nested secondary PCR amplifications of ancient DNA Purified PCR products were sequenced using a BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, USA) with the same primers used for DNA amplification Sequencing analysis was performed using ABI PRISM 377 and ABI PRISM 310 Automated DNA Sequencers (Applied Biosystems, USA) Name of primary PCR primer DNA sequencing No of primary PCR primer (nt16190-nt16417)) Oligonucleotide primers used for primary and nested PCR are shown in Table PCR amplification was carried out in a reaction mixture of 25-ll containing ll of ancient DNA, 13 PCR buffer pH 8.7, 13 Q-solution, 0.2 mM each of dNTP, 20-pmol of each primer and units of Taq DNA polymerase (QIAGEN, USA) for 50 cycles consisting of (948C, 30 s for denaturation, 30 s at the annealing temperature of each primer pair (as detailed in Table for the annealing step) and 728C, 45 s for extension and a final extension step at 728C for Nested PCR was performed as described above but using 1-ll of primary PCR product as a template, unit of Taq DNA polymerase (QIAGEN, USA) and 30 amplification cycles Blank reactions were simultaneously prepared for every PCR reaction in order to verify the reliability of the experiments All ancient DNA amplifications were accompanied by two extraction blanks and one amplification blank Extraction blanks were PCR reactions set up with extracts prepared without any tooth powder (blank extraction) obtained from the extraction step The amplification blank was a PCR reaction containing all of the reagents but with water replacing the ancient DNA extract After preparation of the PCR reactions in the clean, isolated laboratory, the closed reaction tubes were then transferred to the regular laboratory and subjected to thermocycling Subsequent analyses were carried out in regular laboratories None of the control blanks gave rise to PCR product In one ancient human (BLK 7) and one ancient pig DNA sample, PCR amplification of the mitochondrial control region was performed using pig (Leonard et al., 2007) and dog (Malmstrom et al., 2005) specific primers as well as human primers (L16190 and H16422) Primary and secondary PCR reactions were carried out under similar conditions to those described above The pig and dog specific primers used are also listed in Table For the modern DNA samples, the HVS-1 segment was amplified to produce a 514 bp product using forward (15,904–15,930) and reverse (16,398–16,417) primers PCR products both from ancient and modern DNA were purified using either QIAquick PCR Purification Kit when only a specific band product was obtained or QIAquick Gel Extraction Kit when non-specific bands were also shown, prior to DNA sequencing Size of product (bp) ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES American Journal of Physical Anthropology 430 P LERTRIT ET AL TABLE Populations selected for genetic comparison No Population Sample size Linguistic affiliation Immediate homeland 10 11 12 13 14 15 16 17 18 19 20 21 Noen U-Loke (NUL) Ban Lum-Khao (BLK) Chao-Bon (ChB) Khmer (Khm) Chong (Chg) Thai-Korat (Th-K) Thai-Khon Kaen (Th-KK) Thai-Chiang Mai (Th-Cm) Thai-northern (Th-N) Phu-Thai (PTh) Lao-Song (LSg) Zhuang (Zhg) Lisu (Lsu) Mussur (Msr) Han-Yunnan (H-Yn) Han-Guangdong (H-Gd) Han-Wuhan (H-Wh) Han-Qingdao (H-Qd) Han-Liaoning (H-Ln) Han-Xinjiang (H-Xj) Tibetan-Qinghai (Ti-Qh) 17 13 20 22 25 32 44 30 32 25 25 56 25 21 43 30 42 49 51 47 64 Not known Not known Austroasiatic Austroasiatic Austroasiatic Tai-Kadai Tai-Kadai Tai-Kadai Tai-Kadai Tai-Kadai Tai-Kadai Tai-Kadai Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan Sino-Tibetan NE, Thailand NE, Thailand NE, Thailand Cambodia E, Thailand NE, Thailand NE, Thailand N, Thailand N, Thailand N, Laos NW, Vietnam SE, China SW, China SW, China SW, China SE, China C, China NE, China NE, China NW, China NW, China 22 Tu-Huzu (Tu-Hz) 35 Altaic NW, China Thai, Phuthai, Lao-Song and Zhuang), Sino-Tibetan (Lisu, Mussur, Han-Yunnan, Han-Guangdong, HanWuhan, Han-Qingdao, Han-Liaoning, Han-Xinjiang and Tibetan-Qinghai) and Altaic (Tu-Huzu) The net genetic distance (dA) between two populations X and Y was computed from sequence data using the formula dA ẳ dXY dX ỵ dY Þ=2; where dXY is the mean pairwise difference between individuals in population X and population Y, and dX (dY) is the mean pairwise difference between individuals within population X (or Y) (Kimura, 1980; Nei, 1987; Nei and Miller, 1990) The Mantel statistical test was performed to assess the significance of the correlation between genetic and geographic distances using GENALEX software (Peakall and Smouse, 2006); the significance of the correlation coefficient (r) was assessed by comparison with the distribution of correlation coefficients arising from 1,000 random permutations Principal coordinate analysis (PCA) using GENALEX software (Peakall and Smouse, 2006) and unrooted NJ tree construction using MEGA3 software (Kumar et al., 2004) was applied to net genetic distances (dA) to visualize the structure of genetic relationships between studied populations The genetic structure of the studied populations was further investigated using SAMOVA algorithms (Dupanloup et al., 2002) to identify, for a prespecified number of groups of populations, the geographical groups that are maximally differentiated from one another, and possibly the genetic barriers between these groups RESULTS DNA sequence results obtained in this study fulfilled the following criteria of authenticity suggested by Cooper and Poinar (2000) and Hofreiter et al (2001) 1) the work was carried out in a clean, isolated, dedicated, suitable, and properly equipped laboratory, 2) a large number of extraction controls and PCR amplification American Journal of Physical Anthropology Reference Present study Fucharoen et al., 2001 Present study Fucharoen et al., 2001 Yao et al., 2002b Fucharoen et al., 2001 Yao et al., 2002a Fucharoen et al., 2001 Yao et al., 2002a Yao et al., 2002b and Wen et al., 2004 Yao et al., 2002b controls were performed with no blank controls giving a positive result, 3) an inverse correlation between fragment length and amplification efficiency was found (it was not possible to produce/amplify any long fragments in this study), 4) results from a second, independent extract of a sample confirmed the reliability of the first one, and 5) successful independent replications of the results were performed in different laboratories We also demonstrate that the mitochondrial control region of an ancient pig sample discovered at one of the two archaeological sites can be amplified using pig specific primers but not with human specific primers and vice versa (see Fig 3) Neither ancient human nor ancient pig DNA gave rise to the PCR product when dog specific primers were used (data not shown) The PCR product amplified from ancient pig remains was sequenced and the sequence obtained is shown in Table This is a critical piece of supporting evidence showing the survival of human DNA fragments which have been associated with faunal remains, and it also makes a good negative control for human PCR amplifications Moreover, the genetic profiles of all people involved in sample processing were also determined (data not shown) and the results did not match DNA sequences from ancient samples Mitochondrial DNA data Ancient mitochondrial DNAs were successfully extracted and amplified to generate overlapping HVS-1 sequences of various final lengths (from 233 to 366 nucleotides in size) for 22/26 (85%) and 13/17 (76%) samples from Noen U-Loke (NUL) and Ban Lum-Khao (BLK) archaeological sites, respectively To be sufficiently informative and at the same time compatible with available modern sequences, only ancient DNA samples with complete 336 nucleotide sequences from HVS-1 np16048–16383 were selected for comparative analyses Half of the sequence length (176 bp) was obtained from at least two separate sequencing reactions (np16048– 16224); the other 160 bp (np16224–16383) was generated ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES 431 Fig Geographic locations of the populations included in the study Numbers corresponding to population names are shown in Table [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] from sequencing both strands The numbers of usable ancient sequences were then 17 (65%) and 13 (76%) from the Noen U-Loke and Ban Lum-Khao archaeological sites, respectively Ancient as well as modern DNA sequences obtained are shown in Table Problems with the authenticity and reliability of ancient DNA data can arise postmortem degradation of DNA either through the generation of miscoding lesions or through the physical destruction of the molecule (Gilbert et al., 2005) Although we have satisfied almost all of the nine key criteria of Cooper and Poinar (2000), this does not guarantee the authenticity of the sequencing results if the samples were contaminated before the analysis For human samples such as these which are considered at high risk of contamination, three indicators have been suggested to positively exclude or question the authenticity of ancient mtDNA sequencing results (Bandelt, 2005; Gilbert et al., 2005; Salas et al., 2005; Bandelt et al., 2007) They are 1) phylogeographic paradoxes where the putative ancient mtDNA lineages reflect the haplogroups of the excavation team, curating staff or lab personnel rather than those in the geographic area of the ancient population; 2) mosaic structures of putative ancient mtDNA sequences where one fragment fits into a specific part of the mtDNA phylogeny but the combination of overall sequence does not fit any point in the phylogeny; and 3) an abnormal mutational spectrum Our ancient and modern mtDNA data were then examined for these indicators of nonauthenticity First, we looked at numbers of transition and transversion mutations in the mtDNA sequence generated in our laboratory including the BLK, NUL, ChB, Khm, and Th-K samples (Table 6) Of 83 total mutations found in these population, transversion mutations were found in the ancient data sets (A16182C and A16183C), and additional transversion mutations were detected in modAmerican Journal of Physical Anthropology 432 P LERTRIT ET AL ern data (C16073A, C16111A, A16182C, and A16183C, C16257A, C16266A,G) In mtDNA, transitions are much more frequent than transversions and are estimated to outnumber transversions by ratio of more than 24:1 in coding regions (Macaulay et al., 2005) For our ancient data set, the transition-transversion ratio was more than 40 (83:2) whereas in our modern data set, the ratio was less (83:6) The transversions A16182C, A16183C, C16257A, C16266A,G have been reported previously in several populations included in this study (Fucharoen et al., 2001; Yao et al., 2002a,b; Wen et al., 2004) while the mutations at 16,111 and 16,266 were mentioned in the list of site-specific mutational rates summarized by Salas et al (2005) The C16073A mutation was found only in Fig The PCR product from the amplification of ancient human DNA and ancient pig DNA with both human and pig specific primers Lane 1, Blank PCR control with no DNA; Lanes and 3, Blank extraction of ancient human and ancient pig DNA respectively; Lanes and 7, mtDNA from ancient human and ancient pig respectively with species specific primers; Lane 5, ancient human DNA with pig primer; Lane 6, ancient pig DNA with human primer; and Lane 8, DNA size of 100 bp marker two samples of Chao-Bon (ChB08 and ChB18) These two samples were assigned to mtDNA haplogroups B* and B A phylogenetic tree of 22 populations was constructed (data not shown) and mtDNA haplogroups were determined in these 22 populations (Table 7) according to the site-specific mutations in the HVS-1 (Wallace et al., 1999; Schurr and Wallace, 2002) Only the Asian haplogroups (A, B/B*, M, F, and X) were classified No phylogeographic paradox was detected: while the excavation team included many Caucasians, it also included some local people Six out of seventeen and three out of thirteen mtDNA sequences from NUL and BLK fulfilled the site-specific mutations in HVS-1 criteria for haplogroup assignment (Wallace et al., 1999; Schurr and Wallace, 2002) No basal motif of a specific haplogroup was observed in central and southeast Asia except for the Polynesian motif (Schurr and Wallace, 2002) This is probably due to an insufficient number of HVS-1 sequences from each haplogroup in the region (which should ideally be more than 10,000) It is also difficult to detect basal motifs given that only the short sequence of the HVS-1 region was studied We thus looked at single mutational variants instead In NUL sequences, there were nine sequences that belong to mtDNA haplogroup B/B* according to phylogenetic analysis Omitting T16519C which was not available in our data set, two sequences fulfilled the site-specific mutations in HVS-1 criteria for haplogroup determination The other seven sequences missed either 16189C or 16217C This characteristic has also been reported in other populations The other mutations found in the sequences of NUL haplogroup B/B* were either identified in other populations recruited in this study (Fucharoen et al., 2001; Yao et al., 2002a,b; Wen et al., 2004) or in the list of site-specific mutational rates (Salas et al., 2005) This was similar to the BLK sequences belong to haplogroup B/B*, and both NUL and BLK sequences belong to mtDNA haplogroup M and F The two sequences from NUL also fulfilled the criteria of haplogroup X but lay on a different mtDNA background However, four transition mutations were detected only in our ancient data set (16088C in NUL8; 16175G, 16202G, and 16254G in BLK33, BLK49, and BLK31, respectively) These mutations, however, did not affect mtDNA haplogroup assignment in the phylogenetic tree In summary, we believe that we have not detected any of the three indicators of nonauthenticity in this data set Haplogroups were determined for all the mtDNA sequences; their frequencies are reported in Table All the identifiable haplogroups in our samples were limited TABLE Mitochondrial DNA sequence obtained from ancient pig sample aligned with pig reference sequence (http:/www.ncbi.nlm.nih.gov, accession number AJ002189) Pig reference Ancient pig Pig reference Ancient pig Pig reference Ancient pig American Journal of Physical Anthropology 433 TABLE Polymorphic sites in HVS-1 of mtDNA from two ancient and three modern populations ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES American Journal of Physical Anthropology Full names of each population are shown in Table TABLE (Continued) 434 P LERTRIT ET AL American Journal of Physical Anthropology 435 ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES TABLE Haplogroup frequencies determined according to the standard criteria (Wallace et al., 1999; Schurr and Wallace, 2002) for all 22 populations investigated in this study Mitochondrial haplogroup frequencies (%) Population BLK NUL ChB Khm Chg Th-K PTh LSg Zhg Th-KK Th-Cm Th-N Lsu Msr H-Yn H-Gd H-Wh H-Ln H-Qd H-Xj Ti-Qh Tu-Hz Total number 13 17 20 22 25 32 25 25 56 44 30 32 25 21 43 30 42 51 49 47 64 35 A 2 16 10 20 12 0 0 (3.1%) (12.0%) 0 (2.3%) (6.7%) (6.3%) (12.0%) (16.3%) (6.7%) (14.3%) (5.9%) (32.7%) (21.3%) (31.2%) (34.3%) B/B* 10 11 7 12 12 11 (46.1%) (52.9%) (35.0%) (18.2%) (40.0%) (12.5%) (20.0%) (32.0%) (19.6%) (15.9%) (23.3%) (12.5%) (20.0%) (28.6%) (27.9%) (16.7%) (19.0%) (23.5%) (22.4%) (6.4%) (1.6%) (17.1%) to those found in Asian and Native American populations (http://www.mitomap.org/cgi-bin/tbl5gen.pl, Reidla et al., 2003) Haplogroup A was absent in all the Austroasiatic groups, the two ancient samples, and the two TaiKadai groups, LSg and Zhg, included in this study The frequency of this haplogroup is notably lower among the Tai-Kadai and Sino-Tibetan groups from Southeast Asia and central-southern China than among the Sino-Tibetan and Altaic groups from further north: H-Qd, H-Xj, Ti-Qh, and Tu-Hz The frequencies of the B/B* haplogroups are generally higher among the ancient and Austroasiatic samples (apart from Khm) than among the rest of the populations In addition the B/B* frequencies are markedly lower in the two populations from northwestern China, H-Xj and Ti-Qh, than elsewhere The frequency distributions of the other identifiable haplogroups, F, M, and X, were not clearly associated with the population groupings, or with either geographic or linguistic groupings Table S1 shows the shared haplotypes observed within and between the populations No identical sequences were observed within either ancient sample, either NUL or BLK, in contrast to the living samples Only one of the BLK sequences, BLK64, was identical to a sample from another population, NUL31 from the Noen U-Loke ancient sample On the other hand, there were several sequences from NUL samples that were identical sequences with living Southeast- and EastAsian populations: ChB, Th-K, P-Th, Lsu, and H-Xj Among these populations, the Ch-B and Th-K groups live in the vicinity of the NUL and BLK archaeological sites Among Austroasiatic speakers included in this study, identical sequences existed more within populations than between populations The Thai samples from various localities in Thailand, Th-K, Th-KK, ThCm, Th-N, and P-Th, shared more haplotypes among themselves and with East-Asian populations than with their Austroasiatic neighbors The frequencies of F 3 11 12 11 9 7 3 (23.1%) (5.9%) (15.0%) (50.0%) (8.0%) (28.1%) (32.0%) (16.0%) (21.4%) (25.0%) (30.0%) (28.1%) (24.0%) (38.1%) (16.3%) (20.0%) (16.7%) (3.9%) (10.2%) (14.9%) (4.7%) (8.6%) M 4 15 25 21 11 12 10 17 14 15 31 17 16 33 14 (30.8%) (23.5%) (20.0%) (27.3%) (32.0%) (46.9%) (36.0%) (28.0%) (44.7%) (47.7%) (36.7%) (37.4%) (40.0%) (28.6%) (39.5%) (46.6%) (35.7%) (60.8%) (34.7%) (34.0%) (51.6%) (40.0%) X 2 2 3 (11.8%) (30.0%) (4.5%) (20.0%) (6.3%) (8.0%) 0 (3.3%) (6.3%) (4.0%) 0 0 (3.9%) (6.4%) (4.7%) Unidentified (5.9%) 0 (3.1%) (16.0%) (14.3%) (9.1%) (9.4%) (4.7%) (10.0%) (14.3%) (2.0%) (17.0%) (6.2%) shared haplotypes were higher within and among northern-East Asian populations than between this group of populations and the rest of the populations Genetic distances and their correlation with geographic distances Genetic distances between the studied populations are shown in Table Using the Mantel test, the correlation between genetic distance and geographic minimal distances is not significant (r 0.162; P 0.14), indicating that the observed pattern of genetic differentiation among populations, particularly in the maternal line, could not be simply explained using an isolation-by-distance model From the genetic distance data, the close genetic affinity of NUL with BLK was notable; their intra-population nucleotide diversities (1.7297 and 1.9701 for NUL and BLK, respectively) were slightly smaller than the average of modern populations (2.1593) NUL also formed relatively close association with the Chao-Bon, an Austroasiatic (AA) speaking group inhabiting areas neighboring the archaeological sites Among contemporary Southeast Asian natives, both ancient populations showed the next closest genetic affinities with the ThaiKorat, another group also resident in the vicinity of the archaeological sites NUL was also fairly similar to several Han populations Comparing the various language groups, genetic distances observed among AA speaking groups tended to be greater than among Tai-Kadai (TK) groups or Sino-Tibetan (ST) groups excluding Tibetan and Mussur While the Tibetan group was relatively distant from other ST groups, Altaic (AT) Tu on the other hand seemed to form close association with the majority of ST groups including Han and Tibetan TK groups from central Southeast Asia and southern China formed close genetic affinities among themselves and also with southern Han populaAmerican Journal of Physical Anthropology 436 2.3568 0.0245 0.0162 0.0228 0.1664 0.0224 2.3791 0.014 0.01 0.0161 0.0263 0.1474 0.0151 2.5011 0.001 0.0164 0.0482 0.0506 0.0433 0.2116 0.0452 1.8819 0.1255 0.1002 0.1855 0.1777 0.1814 0.1663 0.2773 0.1821 2.1359 0.0133 0.0742 0.0502 0.0637 0.0558 0.0474 0.0171 0.1055 0.0511 2.2138 0.0581 0.1232 0.0153 0.016 0.0563 0.051 0.0442 0.2195 0.0656 1.9238 0.075 0.0688 0.1887 0.097 0.084 0.0851 0.126 0.1072 0.0915 0.2269 0.1292 2.3247 0.0931 0.052 0.0256 0.0707 0.0545 0.112 0.1035 0.1106 0.0925 0.2125 0.1371 2.2503 0.0489 0.0698 0.0597 0.1005 0.0018 0.0426 0.0335 0.073 0.0843 0.0584 0.2205 0.0793 1.94 0.0114 0.0135 0.0963 0.0461 0.0657 0.0743 0.0365 0.0789 0.1275 0.1305 0.1552 0.1131 0.2535 0.1558 2.1609 0.0134 0.0197 0.0013 0.0577 0.0355 0.0172 0.0888 0.047 0.0515 0.0773 0.0848 0.0881 0.0556 0.1894 0.0999 2.2819 0.0142 0.0511 0.0001 0.0247 0.0631 0.0133 0.0397 0.1062 0.0271 0.0337 0.0479 0.0563 0.0535 0.049 0.1827 0.0733 2.1857 0.1267 0.1571 0.2235 0.155 0.2206 0.0644 0.1609 0.1788 0.3169 0.2075 0.2037 0.19 0.1982 0.1615 0.1582 0.275 0.1937 1.8473 0.2409 0.0284 0.0373 0.0409 0.0311 0.0007 0.1213 0.056 0.0574 0.0691 0.0747 0.0758 0.1184 0.1478 0.1524 0.1122 0.3045 0.1786 1.9701 0.1176 0.1024 0.2026 0.0701 0.0925 0.1277 0.1093 0.0748 0.0957 0.0854 0.1132 0.2505 0.1014 0.1087 0.1227 0.1616 0.1382 0.1441 0.3148 0.1831 NUL BLK ChB Khm Chg Th-K Th-KK Th-Cm Th-N PTh LSg Zhg Lsu Msr H-Yn H-Gd H-Wh H-Qd H-Ln H-Xj Ti-Qh Tu-Hz 1.7297 0.0357 0.0582 0.1358 0.183 0.085 0.1382 0.1909 0.1062 0.1502 0.102 0.0537 0.1183 0.2167 0.0508 0.0339 0.0432 0.1116 0.0689 0.1164 0.3177 0.1114 2.4295 0.1437 0.1479 0.1161 0.1422 0.2006 0.1001 0.1715 0.1326 0.0994 0.1535 0.2645 0.1133 0.0937 0.117 0.1229 0.0965 0.116 0.2541 0.1255 H-Gd H-Yn Msr Lsu Zhg LSg PTh Th-K Th-KK Th-Cm Th-N BLK NUL ChB Khm Chg TABLE Intra-population and net nucleotide diversity among 22 selected populations H-Wh H-Qd 2.1474 0 0.0586 0.0022 H-Ln H-Xj Ti-Qh Tu-Hz 2.366 2.1068 0.0666 0.0556 1.7303 0 0.0507 2.0226 P LERTRIT ET AL American Journal of Physical Anthropology tions from Yunnan and Guangdong Interestingly the Khmer, an AA speaking group, was generally more affiliated with TK groups than with other AA groups Population clustering as revealed by principal coordinate analysis and phylogenetic analysis A principal coordinate (PC) map and a phylogenetic tree, both derived from net genetic distance data, are shown in Figures and 5, respectively Figure shows the distribution of the studied populations according to the first and second PC axes, which accounted for 34.67 and 26.70% of the total variation, respectively On the first PC axis, populations were divided roughly congruently with geography, with populations from Southeast Asia and southern China, (AA, southern ST, TK and our two prehistoric populations) intermingling towards one end, and northern East-Asian populations (northern ST and AT) towards the opposite end Clearer divisions of populations according to their ethnic and linguistic affiliations were visible on the second PC axis: the two AA groups, Chao-Bon and Chong, were clearly separated from all TK groups Interestingly, they clustered closely on this axis with the two prehistoric populations NUL and BLK, suggesting a close genetic affiliation along the maternal line The TK groups clustered closely together among themselves, and were closer to the southern ST populations than the northern ST populations The TK groups were relatively distant from the two prehistoric populations as well as from the majority of AA groups, particularly on the second PC axis One major exception was the Khmer group which formed a much closer affiliation with the TK groups than with other AA groups on the second PC axis The closer genetic affiliation of the Khmer group with TK groups from central Southeast Asia (i.e Thai speaking groups) than with their northerly linguistic relatives (i.e Zhuang from southern China and Lao-Song originally from northwestern Vietnam), might suggest significant genetic exchanges in the maternal line between the Khmers and Thais in particular The two southwestern Sino-Tibetan groups, Lisu and Mussur, showed closer affiliation with Southeast Asian and southern-East Asian natives than with northern ST groups, indicating a predominantly southern origin for their maternal lineages All ST-Han populations and the AT-Tu population clustered together at quite a distance from the ST-Tibetan population Figure illustrates an unrooted NJ-tree constructed for maternal genetic relationships among the studied populations All Southeast Asian populations included in this study, whether prehistoric or contemporary, clustered together within one major branch on the NJ tree, while the majority of northerly populations clustered on the other branch The southeast Asian cluster was closer to natives of southern China, namely to Han Chinese from Yunnan and Guangdong, Zhuang, Lisu and Mussur, than to northern ST and AT groups The two ancient populations, which are phylogenetically closest to each other, formed a sub-branch with the two AA groups, Chao-Bon and Chong, and with one TK group, Lao-Song Among Southeast Asian natives, the Khmer group was closer phylogenetically to the majority of Thai than to other AA groups SAMOVA analysis Table gives the composition of the corresponding groups of populations inferred by the SAMOVA algo- ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES Fig PC map of 22 selected populations based on net genetic distances The first PC accounts for 34.67% of the original variation, while the second accounts for 26.70% 437 rithms, their associated fixation indices, and their significance evaluated by permuting the populations without considering their geographic position The variance components among groups were statistically significant for all grouping criteria except the two-group category Interestingly, as the number of group increases from two-group to eight-group categories, the three Austroasiatic samples (Chg, ChB, and Khm) tend to separate from each other; Khm formed a group with all Tai-Kadai samples except Lsg and Zhg, and ChB with the two ancient samples, NUL and BLK, of unknown linguistic affiliation, while Chg remained isolated throughout Ti-Qh consistently formed an isolated group for all group numbers with significant variance among groups The use of eight groups provided a subdivision most compatible with the interpretation of population relationships provided from the PC and the phylogenetic analyses, though with a clearer subdivision of the populations That is, the Lsu and their linguistic close relative, Msr, formed a distinct group, and all samples from China, except TiQh, were clearly separated in accord with geography into northern and central-southern groups Fig Unrooted NJ tree of 22 selected populations based on net genetic distances American Journal of Physical Anthropology 438 P LERTRIT ET AL TABLE Fixation indices corresponding to the groups of populations inferred by SAMOVA algorithms for the 22 populations tested for mitochondrial HVS-1 sequences No of groups Populations Chg Chg Chg Chg Chg Chg Chg a P \ 0.01 P \ 0.001 b NUL,BLK,ChB,Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg, Zhg,Lsu,Msr,H-Yn,H-Gd,H-Wh,H-Qd,H-Ln, H-Xj,Ti-Qh,Tu-Hz Ti-Qh NUL,BLK,ChB,Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg,Zhg,Lsu,Msr,H-Yn,H-Gd,H-Wh, H-Qd, H-Ln,H-Xj,Tu-Hz Ti-Qh NUL,BLK,Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg,Zhg,Lsu,Msr,H-Yn,H-Gd,H-Wh,H-Qd, H-Ln,H-Xj,Tu-Hz Ti-Qh ChB Msr NUL,BLK,Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg,Zhg,Lsu,H-Yn,H-Gd, H-Wh,H-Qd,H-Ln,H-Xj,Tu-Hz Ti-Qh ChB Msr BLK NUL,Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg,Zhg,Lsu,H-Yn,H-Gd, H-Wh,H-Qd,H-Ln,H-Xj,Tu-Hz Ti-Qh ChB Msr BLK,NUL Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh,LSg,Zhg,Lsu, H-Yn, H-Gd,H-Wh,H-Qd,H-Ln,H-Xj,Tu-Hz Ti-Qh ChB,BLK,NUL Lsu,Msr Khm,Th-K,Th-KK,Th-Cm,Th-N,PTh LSg Zhg,H-Yn,H-Gd,H-Wh H-Qd,H-Ln,H-Xj,Tu-Hz DISCUSSION Present-day natives of mainland Southeast Asia can be classified according to their linguistic affiliations into five major groups: Tai-Kadai (TK), Sino-Tibetan (ST), Austroasiatic (AA), Hmong-Mien (HM) and Austronesian (AN) (LeBar et al., 1964) The HM, who were migrants from southern China during the past few centuries, and AN, found only peripheral to coastal Vietnam and Malaysia indicating their archipelago origin, are not focal to this study Only three groups, TK, ST and AA, are of major interest here Diverse groups of ST have come to populate various parts of the area in successive waves of migration from southern China The AA were probably the oldest natives of the area based on ethnographic and epigraphic evidence (Bernatzik and Bernatzik, 1951; Coedes, 1966; Condominas, 1990), though questions concerning their linguistic origin and spread have not yet been answered (see arguments on these issues in: Diffloth, 2005; Reid, 2005) TK speakers had their linguistic homeland in southeastern China (Benedict, 1942; Ostapirat, 2005) and would have spread from there into Southeast Asia during the early second millennium AD (Coedes, 1966; Eberhard, 1977) Their ancestors among the so-called Pai-Yuei, a term adopted in old Chinese records for diverse groups of natives inhabiting southeastern China around the beginning of the Christian era, have left descendants such as the Zhuang and Dai in parts of modern southern China (Du and Yip, 1993) The two prehistoric populations from NUL and BLK archaeological sites represent early agricultural communities prevailing in the river valleys of northeastern Thailand from the fourth millennium BP onwards (Higham, 1989) The linguistic affiliation of these communities has not been accurately ascertained, but it has been suggested to be Austroasiatic This language family could have been spread together with rice-culture as part of a Neolithic human diaspora from the Yangze River valley in central China into Southeast Asia after the ninth millennium BP (Higham, 2001, 2003) The close genetic affinity between these two neighboring and contemporaneous ancient samples is probably because they were drawn from the same local prehistoric gene pool Their close genetic affinity with Chao-Bon, a relaAmerican Journal of Physical Anthropology FCT 0.04558 0.04655a 0.04554b 0.04521b 0.04435b 0.03975b 0.03961b tively isolated AA speaking group inhabiting the western border of northeastern Thailand in close proximity to the archaeological sites, suggests that the genetic constitution of the Chao-Bon might be derived from a long-established gene pool persisting in the area since prehistoric times This genetic continuum is made more plausible in light of the fact that the Chao-Bon, or ‘‘Nyah-Kur’’ (forest/hill indigenous dwellers) as this ethnic people call themselves, have been proposed, based on a systematic linguistic comparison, as being surviving remnants of the old Dvaravati Mon (AA) who inhabited the area during the second half of the first millennium AD (Diffloth, 1984) If so, the linguistic affiliation of the prehistoric natives may have been AA, or at least their descendants spoke a language of that kind during the late first millennium AD The group next closest genetically to the ancient NUL and BLK populations are the Thai-Korat, a TK group inhabiting an area close to the archaeological sites; this might also reflect the genetic contribution of previous inhabitants of the area to the gene pool of the Thai-Korat However, the Thai-Korat group was clustered more closely with other TK groups than with the two ancient populations on the PC map, indicating their closer relationship with other TK groups Close genetic clustering observed among the majority of Thai groups may reflect a more recent genetic differentiation among them relative to their genetically more scattered AA neighbors It is also worth noting that the Thai populations and the two prehistoric populations are relatively distant from each other Moreover, the Thai cluster tended to associate more with the Zhuang, their linguistic relatives in Southern China, and with Southern-Han populations from Yunnan and Guangdong (all the latter three populations belonging to the geographically confined central-southern China group detected in the SAMOVA analysis) than with the probably older native populations of Southeast Asia, i.e the AA populations and the two prehistoric populations All the data are consistent with the hypothesis, based on historical grounds that ancestors of the Thai people migrated from southern China into Southeast Asia in relatively recent times, probably during the early second millennium AD (Eberhard, 1977) The closer mtDNA affiliation of the Khmer group with the TK cluster (both Thais from central Southeast Asia and the Zhuang from southern ANCIENT AND PRESENT HUMAN mtDNA OF SEA NATIVES China) than with other AA groups may indicate that the genetic exchange between the Thai and Khmer groups was significant and truly reciprocal (at least along the maternal line) This is plausible given that that Thai people were subjugated under the Khmers for a considerable period during the 12th and 13th centuries (Coedes, 1966) The split of a group of populations comprising the Thai and Khmer from a group comprising populations from Central and Southern China, as observed in the SAMOVA analysis, might indicate a genetic differentiation between these groups that developed after the arrival of ancestral Thais into Southeast Asia Different patterns of genetic admixture between TK and AA natives were observed in the case of the LaoSong, another TK group, which was relatively distant from the Khmer group, but close to the Chong, another AA group However, the Lao-Song and Chong were separated as isolated groups in the SAMOVA analysis On the basis of their known history, the Lao-Song inhabiting parts of central Thailand were descendants of Black-Tai migrants forced to move from northwestern Vietnam as prisoners of war during the past few centuries (Fine Arts Department of Thailand, 1961) Ethnographic evidence indicated that Black-Tai in their original homeland have assimilated to a large extent with their AA neighbors both culturally and linguistically, resulting in an adoption of the Black-Tai identity by their AA neighbors (Condominas, 1990) Therefore, the genetic contribution of AA natives to the gene pool of the so-called ‘‘Black-Tai’’ of northwestern Vietnam would not have been trivial To prove this point about AA remnant populations in northwestern Vietnam and their relationship with the Chong, genetic information on these remnant populations would be useful; unfortunately such information is currently lacking The analysis of ancient human DNA from well-dated archaeological sites certainly extends our knowledge about the complex demographic history and genetic relations of the currently known ethnic populations of Southeast Asia Presented here are the first ancient DNA data from Thailand They provide insights into the long-established genetic continuity on the maternal line of certain ethnic populations in the area, particularly among the AA speakers, and the route of later expansion of the ancestral Thai (TK), possibly from Southern China into the area Since this study is based solely on mtDNA sequence data, further studies are required to perform complementary analyses of nuclear DNA data, such as autosomal STRs which can now be genotyped in ancient samples (Ricaut et al., 2005) The field is very promising, though currently at the beginning stages of exploration, given that there is already an enormous body of potentially suitable skeletal materials awaiting study ACKNOWLEDGMENTS We would like to acknowledge the Thailand Department of Fine Arts and the University of Otago for permitting access to the ancient dental samples We are grateful to P Na Nagara, P Kullavanijaya, Xie Yuanzhang, and the three anonymous reviewers for their critical comments of the manuscript and constructive discussions We would like to thank Dr Jim Stankovich for the proofreading of this manuscript and Ms R.M.S Lanka Ranaweera for her excellent job in faunal remains DNA extrac- 439 tion, amplification and sequencing and Ms Bussaraporn Kunhapan for her great help in SAMOVA analysis LITERATURE CITED Bandelt HJ 2005 Mosaics of ancient mitochondrial DNA: positive indicators of nonauthenticity Eur J Hum Genet 13:1106–1112 Bandelt HJ, Yao YG, Salas A, Kivisild T, Bravi CM 2007 High penetrance of sequencing errors and interpretativeshortcomings in mtDNA sequence analysis of LHON patients Biochem Biophys Res Commun 352:283–291 Bellwood P 2001 Early agriculturalist population diasporas? 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Identified according to International Dental Federation (FDI) nomenclature: first digit is for quadrant (1, maxillary right; 2, maxillary left; 3, mandibular left; 4, mandibular right) and second digit... International Dental Federation (FDI) nomenclature: first digit is for quadrant (1, maxillary right; 2, maxillary left; 3, mandibular left; 4, mandibular right) and second digit is for tooth position