Ancient DNA analysis of human neolithic remains found in northeastern Siberia.код для вставкиСкачать
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 126:458 – 462 (2005) Ancient DNA Analysis of Human Neolithic Remains Found in Northeastern Siberia François-Xavier Ricaut,1,2* A. Fedoseeva,3 Christine Keyser-Tracqui,1 Eric Crubézy,2 and Bertrand Ludes1,2 1 Laboratoire d’Anthropologie Moléculaire, Institut de Médecine Légale, 67085 Strasbourg, France Laboratoire d’Anthropobiologie, Université Paul Sabatier, CNRS, UMR 8555, 3100 Toulouse, France 3 Department of Archeology and Ethnography, Yakutsk State University, Yakutsk 677891, Russia 2 KEY WORDS ancient DNA; HV1 sequence; STRs; north Siberia ABSTRACT We successfully extracted DNA from a bone sample of a Neolithic skeleton (dated 3,600 ⫾ 60 years BP) excavated in northeastern Yakutia (east Siberia). Ancient DNA was analyzed by autosomal STRs (short tandem repeats) and by sequencing of the hypervariable region I (HV1) of the mitochondrial DNA (mtDNA) control region. The STR proﬁle, the mitochondrial haplotype, and The cultural, morphological, and genetic similarities observed between Siberian and Native American populations led to the consensus that the ancestral population(s) of Native Americans migrated from Siberia through Beringia during the last glaciation (30,000 –12,000 years BP) (Cavalli-Sforza et al., 1994; Crawford, 1998). Nevertheless, the numerous genetic studies analyzing the relationship between aboriginal Siberian and Native American populations do not agree on the timing, source population(s), and number of waves of migration. The difﬁculties in retracing the initial peopling of the Americas arise from the many historic and demographic events that have occurred since the initial colonization, and which could have obscured the ancestral Asian gene pool of Amerindians (Forster et al., 2001; Malhi et al., 2002). Indeed, searching the genetic traces of ancestral Siberian population(s) of Native Americans among the Siberian modern gene pool is a wager because the Siberian gene pool has been affected by important changes since the ﬁrst migrants left for the Americas. The inhospitable conditions of Siberia, in addition to a low human density, favored a high degree of Siberian population isolation and genetic drift (Santos et al., 1999). The depopulation of northern Asia during the last glacial maximum (around 20,000 years ago) and its repopulation at the end of the glacial phase 11,000 years ago (Forster et al., 2001) could also have played an important role in these changes. More recently, Russian colonization, which began in the 17th century, led to the extinction of several ethnic groups and to a drastic reduction in the size and © 2004 WILEY-LISS, INC. the haplogroup determined were compared with those of modern Eurasian and Native American populations. The results showed the afﬁnity of this ancient skeleton with both east Siberian/Asian and Native American populations. Am J Phys Anthropol 126:458 – 462, 2005. © 2004 Wiley-Liss, Inc. genetic diversity of Siberian populations (Levin and Potatov, 1964; Forsyth, 1996). Only direct access to the gene pool of ancient aboriginal Siberian populations could clarify 1) northern Asian prehistory, 2) the ancestor-descendant relationships between ancient and modern populations, and 3) the colonization of the New World, independent of historical changes affecting the Siberian gene pool. Ancient DNA data from south Siberian populations were previously published (Clisson et al., 2002; Keyser-Tracqui et al., 2003; Ricaut et al., 2003), but to the best of our knowledge, there are no ancient DNA data of northeastern Siberians available. MATERIALS AND METHODS During summer 1980, the “Prilenskoy” Russian expedition discovered in northeastern Siberia (in the Sakha Republic), near the Panteleikhe River (lower Grant sponsor: French Department of Research; Grant sponsor: ACI “Espaces et Territoires: Le complexe spatial Altaı̈-Baı̈kal. Plaque tournante des ﬂux géniques en Haute Asie de la période protohistorique à l’époque moderne.” *Correspondence to: François-Xavier Ricaut, Department of Biological Anthropology, Leverhulme Centre of Human Evolutionary Studies, University of Cambridge, Downing Street, Cambridge CB23DZ, UK. E-mail: firstname.lastname@example.org Received 23 July 2003; accepted 31 October 2003. DOI 10.1002/ajpa.20257 Published online 13 August 2004 in Wiley InterScience (www. interscience.wiley.com). 459 GENETIC ANALYSIS OF A NEOLITHIC SIBERIAN Kolyma River basin), a frozen Neolithic grave. This tomb (radiocarbon-dated at 3,600 ⫾ 60 years BP by classic analysis of bone artifacts associated with the skeleton) contained a very well-preserved human skeleton of a young woman (20 –25 years old) with indications of Asian-ancestry skull traits (Gokhmana and Tomtosovoy, 1983). Based on archaeological data, this woman could not be afﬁliated with known recent or ancient Siberian ethnic groups (Kistenev, 1992). During each step of sample preparation (abrasion, extractions, and ampliﬁcations), standard precautions were taken to minimize the risk of contamination and to detect any potential contamination. The outer surface of the ancient bone fragment was removed to almost 3– 4-mm depth to eliminate possible surface contamination. Extraction and ampliﬁcation were performed in a dedicated ancient DNA laboratory, routinely sterilized by different treatments (DNAse away威, bleach and ultraviolet light irradiation at 254 nm), wearing full body protective clothing, and using dedicated equipment and reagents. Extraction and ampliﬁcation blanks were used as negative controls, and mitotypes and genetic proﬁles of all persons involved in processing samples were determined (data not shown) and compared to results obtained from the ancient bone sample. Moreover, DNA was extracted at least three times, and at least three PCR ampliﬁcations were made from each extract to assess the reproducibility of results. In this study, a femoral bone fragment from this ancient female skeleton was used to extract ancient DNA according to published protocols (Fily et al., 1998). The aqueous phase was puriﬁed, using a CleanMix Kit (Talent, France), and concentrated to 40 l, using Microcon威-30 ﬁlters (Millipore, France). Mitochondrial DNA analyses were performed on hypervariable region 1 of the mtDNA control region (HV1). This region was divided into two subregions (a and b) ampliﬁed with two sets of overlapping primer pairs, L15989/H16239 5⬘-CCCAAAGCTAAGATTCTAAT-3⬘/5⬘-TGGCTTTGGAGTTGCAGTTG-3⬘ and L16190/H16410 5⬘-CCCCATGCTTACAAGCAAGT-3⬘/5⬘-GAGGATGGTGGTCAAGGGAC-3⬘. DNA ampliﬁcations were performed in 50 l of reaction mixture containing 2– 6 l of the ancient DNA extract, 10 mM Tris HCL (pH 8.3), 50 mM KCL, 1.5 mM MgCl2, 1 mg/ml BSA, 200 M of each dNTP, 0.25 M of each primer, and 2.0 unit of Taq Gold Star (Eurogentec). Cycling parameters were 94°C for 10 min, followed by 38 cycles of 94°C for 30 sec, 48°C for 30 sec for HV1a or 30 sec at 51°C for HV1b, 72°C for 45 sec, and 72°C for 5 min. Ampliﬁcation products were checked on a 1% agarose gel and puriﬁed with Microcon威-PCR ﬁlters (Millipore). Sequence reactions were performed on each strand, with the same primers as those used for PCR ampliﬁcation, by means of the ABI Prism BigDye威 Terminator cycle sequencing Ready Reaction Kit (PE Applied Biosystems), according to the manufacturer’s instructions. Sequence reaction TABLE 1. mtDNA sequence (between bases 16015–16391) of ancient Siberian sample Polymorphic positions1 Sample 16223 16298 16327 CRS Ancient Siberian C T T C C T 1 Numbered according to published Cambridge Reference Sequence (Anderson et al., 1981). products were analyzed on the ABI Prism 3100 automatic sequencer (PE Applied Biosystems). Autosomal short tandem repeats (STRs) were ampliﬁed using the AmpFlSTR威 Proﬁler Plus威 Kit (PE Applied Biosystems). Nine STRs (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820) and the amelogenin locus (determining the individual’s sex) were simultaneously ampliﬁed. Each ampliﬁcation was carried out in 10 l of a reaction mixture containing 3.82 l PCR reaction mix, 2 l primer set, 0.182 l AmpliTaq Gold威 (PE Applied Biosystems), and 1– 4 l of the DNA extract. Cycling parameters were 94°C for 11 min, followed by 37 cycles of 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min, and a ﬁnal delay of 45 min at 60°C. Both nuclear and mtDNA ampliﬁcation products were analyzed on an ABI Prism 3100 automatic sequencer (PE Applied Biosystems). RESULTS AND DISCUSSION The mtDNA HV1 region analysis of the Yakut specimen resulted in the recovery of a 377-base pair (bp) fragment (nucleotide positions 16015–16391 of the Cambridge Reference Sequence (CRS); Anderson et al., 1981), which was conﬁrmed on both strands (Table 1). This sequence was compared to the CRS: there was a total of three variable nucleotide positions (np), all corresponding to transitions. These mutations at np 16223 C-to-T, 16298 T-to-C, and 16327 C-to-T correspond to substitutions which are characteristic of the founding HV1 sequence for haplogroup C in Siberian and Asian populations (Torroni et al., 1993). Analysis of the ancient Siberian haplogroup and haplotype distribution in Eurasian populations showed that they are both widespread in modern Asian populations. They were most frequently found in east and south Siberian populations (Tuvinian, Buryat, Yakut, Evens, Koryak, and Chukchi) (Derenko and Shields, 1997; Starikovskaya et al., 1998; Schurr et al., 1999; Pakendorf et al., 2003; Derenko et al., 2003), and then to a lesser extent in east Asian populations (Mongol, Chinese, and Korean) (Kolman et al., 1996; Lee et al., 1997; Yao et al., 2002). The above distribution and the presence in an ancient northeast Siberian skeleton of an HV1 sequence presenting mutations at nucleotide positions 16223, 16298, and 16327 are in accordance with genetic studies of modern populations which assert that this HV1 haplotype is the founding HV1 sequence for haplogroup C in Siberian and Asian populations 460 F.-X. RICAUT ET AL. TABLE 2. Autosomal STR results obtained with Profiler Plus kit from ancient DNA sample1 Extraction A B C Consensus genotype 1 Amelo genine D8S1179 D21S11 D7S820 D3S1358 D13S317 vWA D18S51 D5S818 FGA XX XX XX XX XX XX XX XX XX 14/14 14/14 14/(15) 14/14 (13)/14 14/14 14/14 14/14 14/14 31/(31) 31/32.2 (32.2)/32.2 31/32.2 31/32.2 31/(31) 31/32.2 (29)/31/32.2 31/32.2 8/8 8/8 8/(11) 8/(9) 8/(11) 8/8 8/(11) 15/15 (14)/15 15/(18) 15/15 15/(16) 15/15 15/15 15/15 15/15 8/9 8/9 8/9(11) 8/9 8/9 8/9 8/9 8/9 8/9 18/18 17/(18) 18/18 18/18 17/(18) 18/18 18/18 18/18 18/18 15/(17) 15/15 15/15 15/15 15/(17) 15/15 (14)/15 (14)/15/(17) 15/(?) 11/12 11/12 11/12 11/12 11/12 (10)/11/(13) 11/12 11/12 11/12 22/(23) (20/24) 22/(23) 22/(24) 22/(23/24) (23/25) 22/(24) 22/(22) 22/(?) 8/(?) Alleles which could not be detected in at least ﬁve different ampliﬁcations are in parentheses. (Torroni et al., 1993). Moreover, the fact that all east Siberian mainland populations shared this ancient haplotype with a relatively high frequency, without restriction to any linguistic or ethnic group, 1) could either be explained by a common ancestral population or by the effect of a signiﬁcant genetic drift favored by the high degree of Siberian population isolation, and 2) allows us to postulate that the Neolithic woman studied here might be considered an ancestor of present-day inhabitants of east Siberia. Moreover, haplogroup C (to which the ancient east Siberian individual belongs) is considered one of four major founding Native American haplogroups (Torroni et al., 1993), and was also widespread in Native American populations (Merriwether et al., 1995; Malhi et al., 2002). However, the ancient east Siberian haplotype was only to be found in two southern Native Americans (one modern Chilean individual, Horai et al., 1993; and one ancient Colombian mummy, Monsalve et al., 1996) and eight northern Native Americans (seven ancient Norris Farms Oneota individuals, Stone and Stoneking, 1998; and one modern Ponca individual, Malhi et al., 2002). As suggested by Malhi et al. (2002), the share of this haplotype in east Siberian/Asian and Native American populations could be due to either convergence or common ancestry. The genetic typing by megaplex ampliﬁcations provided, after a combination of different ampliﬁcation results, an incomplete consensus STR proﬁle (Table 2). Indeed, to reduce ancient DNA STR genotyping errors and ensure the reliability of results, only the ampliﬁed products from each locus that were reproducible in at least ﬁve different ampliﬁcation reactions were considered authentic (Schmerer et al., 1999). This strategy allowed identiﬁcation of artifact alleles and false-homozygosity resulting from sporadic contaminations or ampliﬁcation artifacts (such as shadow bands and allelic dropout) which were caused by a lack of DNA quality and/or quantity (Schmerer et al., 1999; Hummel et al., 2000). Of the nine autosomal loci tested, only six (vWA, D21S11, D8S1179, D5S818, D13S317, and D3S1358) and the amelogenin locus gave clearly reproducible results in ﬁve different ampliﬁcation reactions, and can be considered authentic (Table 2). For three longer STR loci (D7S820, 258 –294 bp; D18S51, 273–341 bp; and FGA, 219 –267 bp), the degree of reproducibility of results was not sufﬁcient to provide a reliable proﬁle and to assess with certainty the homozygous or heterozygous nature of the genotype obtained. The results obtained from AmpFlSTR威 Proﬁler Plus威 Kit (PE Applied Biosystems) indicate that our results were not inﬂuenced by contamination. Indeed, 1) the ampliﬁed products were reproducible from multiple extractions and ampliﬁcations, 2) STR autosomal ampliﬁcation success correlated negatively with the length of the amplicons, 3) the morphological and molecular sex determinations of the Siberian skeleton were in accordance with each other, and 4) the ancient Siberian STR proﬁle (even incomplete) was never found to correspond to someone involved in processing samples. The nuclear data obtained in this study attest to the authenticity of the DNA extract and ampliﬁed products, and prove that ancient DNA molecules were preserved in the sample bone. The HV1 sequence obtained, which was fully reproducible from multiple ampliﬁcations and extractions and was different from that of the French and Yakut staff, can thus be considered authentic. An assignment method, based on analysis of allelic frequencies of the six STR loci considered in the consensus genotype, was performed to investigate the population afﬁnities of the ancient skeleton, by determining the populations in which the STR proﬁle from the ancient skeleton was most likely to occur (Cornuet et al., 1999). The probability of observing an individual with the ancient skeleton STR proﬁle was 10 times higher in Native Alaskan (Athabaskan, Inupiat, and Yupik; Budowle et al., 2002), Native West Canadian (Salesman and Saskatchewan; Budowle et al., 2001), and East Asian (Vietnamese, Korean, Japanese, and Chinese; Budowle et al., 2001) populations than in other Native American populations located to the south (Apache, Navajo, Northern Ontario, and Puna of Argentina; Budowle et al., 2001; Albeza et al., 2002) and European populations (Russian, Polish, Austrian, Spanish, and Greek; Kornienko et al., 2002; Pawlowski and GENETIC ANALYSIS OF A NEOLITHIC SIBERIAN Maciejewska, 2000; Neuhuber et al., 1999; Gusmao et al., 2000; Sanchez-Diz et al., 2002). In spite of the absence of publications presenting STR data from Siberian populations usable in this study, the assignment-method results indicated that populations living in regions neighboring northeastern Siberia, i.e., extreme North America and east Asia, presented the highest afﬁnity with the ancient skeleton STR proﬁle. CONCLUSIONS The ancient DNA analysis of a Neolithic Siberian human bone sample allowed us to report on the most ancient nuclear proﬁle and mtDNA sequence of north Siberia. Comparison of the ancient HV1 sequence and autosomal STR proﬁle with modern Eurasian and Native American populations 1) supports the hypothesis that the HV1 sequence harboring the 16223T, 16298C, and 16327T motifs could be the founding lineage for haplogroup C in Asian populations, and 2) indicates that this ancient skeleton was linked by its maternal lineage and nuclear DNA to both east Siberian/Asian and Native American populations. 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