Genetic Analysis of a Scytho-Siberian Skeleton and Its Implications for Ancient Central Asian Migrations

Human Biology, Feb 2004 by Ricaut, Francois-X, Keyser-Tracqui, C, Bourgeois, J, Crubezy, E, Ludes, B

Abstract The excavation of a frozen grave on the Kizil site (dated to be 2500 years old) in the Altai Republic (Central Asia) revealed a skeleton belonging to the Scytho-Siberian population. DNA was extracted from a bone sample and analyzed by autosomal STRs (short tandem repeats) and by sequencing the hypervariable region I (HV1) of the mitochondrial DNA. The resulting STR profile, mitochondrial haplotype, and haplogroup were compared with data from modern Eurasian and northern native American populations and were found only in European populations historically influenced by ancient nomadic tribes of Central Asia.

KEY WORDS: ANCIENT DNA, HV1 SEQUENCE, STRS, HAPLOGROUPS, SCYTHIAN POPULATION, CENTRAL ASIA, ALTAI REPUBLIC, KIZIL SKELETON (95-KBI-52), MIGRATION, MOLECULAR ARCHEOLOGY, D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, AMELOGENIN

The Central Asian region has been inhabited since the lower Paleolithic Era and is considered a crossroads of migration routes of ancient nomadic populations. During the Neolithic period, Central Asia was a contact area between nomadic tribes and the neighboring agriculturist populations. The Scythians (700 B.C.-A.D. 200) were one of the most famous nomadic populations living in the Eurasian steppe zone. The Scytho-Siberian group, localized in southern Siberia, composed the eastern extremity of this population.

The history of the Scythians is known only from ancient texts (Achaemenid, Greek, and Chinese sources) and by the excavation of their burial places. The archeological records (Bokovenko 1994; Dvornichenko and Fedorov-Davydov 1994; Francfort et al. 2000) and morphological (Chikisheva 2000a,b) and genetic (Clisson et al. 2002; Voevoda et al. 2000) studies of this ancient population have not led to a conclusive scenario regarding the origin and fate of the Scythians. Only an increased number of ancient nomad grave discoveries, their further study, and biological analysis of the human remains will allow for a better understanding of the origins, migrations, and disappearance of the Scythian populations.

In this study, two types of genetic markers (HVl region of the mtDNA and autosomal STRs) were used to analyze DNA recovered from the remains of a 2500-year-old human skeleton excavated from the Kizil valley (Altai mountains). Autosomal STRs are used in ancient DNA studies to reconstruct genealogies or population history (Izagirre and de la Rua 1999; Keyser-Tracqui et al. 2003) and to detect ancient sample contamination (Hummel et al. 2000). The HVl region of the mtDNA, because of its haploid and maternal mode of inheritance, gives information on the origin and genetic evolution of populations (Richards et al. 2000; Yao, Kong et al. 2002). The aim of this study is to determine the genetic affiliation of the Scytho-Siberian skeleton by analyzing these two genetic markers.

Materials and Methods

Source of Ancient Human DNA. During the summer of 1995 a Belgian and Russian scientific team excavated several Scytho-Siberian kurgans (stone tumuli) at the source of the Kizil River (Altai Republic). This site constitutes a necropolis of about 30 kurgans, at an altitude of 2180 m. The climatic conditions at this altitude allowed the formation of permafrost, which increases the chances of recovering frozen tombs with well-preserved human material (Burger et al. 1999). One of the kurgans (dated to the 4th-2nd century B.C. by carbon-14 dating of samples from beams of the burial chamber) contained a wooden burial chamber with a human adult skeleton associated with the traditional Scytho-Siberian burial gifts, and the remains of a horse (Bourgeois et al. 2000). Anthropomorphic analysis (Orban and Polet 1995) suggested that this skeleton (95-KBI-52) belonged to a mature adult male (35-45 years old) who exhibited characteristic lesions related to horse riding. Only a part of the right femur was used for DNA analyses.

Ancient DNA Extraction. A part of the right femur was abraded (to a depth of 4 mm) with a sanding machine (Dremel) to remove possible contamination from the bone surface. The sample was ground to a fine powder with a drill fitted with a surgical trepan.

Three independent DNA extractions were made according to the method of FiIy et al. (1998). For each extraction, 2 g of bone powder was dissolved in an extraction buffer [5 mM EDTA, 2% SDS, 10 mM Tris HCl (pH 8.0), 0.3 M sodium acetate, l mg/ml proteinase K] and incubated for 16 hr at 56�C. After an organic extraction (phenol/chloroform/isoamyl alcohol, 25:24:1, v/v/v), the aqueous phase was purified using the CleanMix Kit (Talent, France), a method based on the large DNA affinity for the silica in the presence of guanidium thiocyanate, and concentrated to 40 �l using Microcon-30 filters (Millipore, France).

Autosomal STR Analysis. Amplifications were performed with the Amp-FlSTR Profiler Plus Kit (PE Applied Biosystems, France). Nine STR loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) and the amelogenin locus 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, and 1-4 �l of the DNA extract. Cycling parameters were 94�C for 11 min, followed by 37 cycles of 94�C for l min, 59�C for l min, and 72�C for l min, and a final delay of 45 min at 60�C.