Hum Genet (2009) 126:395–410

DOI 10.1007/s00439-009-0683-0

ORIGINAL INVESTIGATION

Ancient DNA provides new insights into the history

of south Siberian Kurgan people
Christine Keyser · Caroline Bouakaze ·
Eric Crubézy · Valery G. Nikolaev · Daniel Montagnon ·
Tatiana Reis · Bertrand Ludes

Received: 6 February 2009 / Accepted: 6 May 2009 / Published online: 16 May 2009
© Springer-Verlag 2009

Abstract To help unravel some of the early Eurasian that at the Bronze and Iron Age timeframe, south Siberians
steppe migration movements, we determined the Y-chro- were blue (or green)-eyed, fair-skinned and light-haired
mosomal and mitochondrial haplotypes and haplogroups of people and that they might have played a role in the early
26 ancient human specimens from the Krasnoyarsk area development of the Tarim Basin civilization. To the best of
dated from between the middle of the second millennium our knowledge, no equivalent molecular analysis has been
BC. to the fourth century AD. In order to go further in the undertaken so far.
search of the geographic origin and physical traits of these
south Siberian specimens, we also typed phenotype-infor-
mative single nucleotide polymorphisms. Our autosomal, Introduction
Y-chromosomal and mitochondrial DNA analyses reveal
that whereas few specimens seem to be related matrilin- Kurgans (Russian word for tumuli) are barrows characteris-
eally or patrilineally, nearly all subjects belong to haplo- tic of a culture arising on the steppes of southern Russia
group R1a1-M17 which is thought to mark the eastward about 5000 BC and later spreading into eastern, central and
migration of the early Indo-Europeans. Our results also northern Europe between 4400 and 2800 BC. The Kurgan
conWrm that at the Bronze and Iron Ages, south Siberia was culture is divided into diVerent sub-cultures on the basis of
a region of overwhelmingly predominant European settle- the diVerent kinds of graves under the barrows: pit-graves
ment, suggesting an eastward migration of Kurgan people (Yamna), catacomb-graves (Katakomnaya) and timber-
across the Russo-Kazakh steppe. Finally, our data indicate graves (Srubna). The westwards diVusion of this culture is
sometimes equated with the appearance in eastern Europe
of the Corded Ware culture and the introduction of Indo-
Electronic supplementary material The online version of this European-speaking peoples (Gimbutas 1970).
article (doi:10.1007/s00439-009-0683-0) contains supplementary In an attempt to reconstruct some of the population
material, which is available to authorized users. movements of ancient Kurgan people from the Eurasian
steppes, the genetic background of 32 ancient human speci-
C. Keyser (&) · C. Bouakaze · D. Montagnon · B. Ludes
Laboratoire d’Anthropologie Moléculaire,
mens from the Krasnoyarsk area in southern central Siberia
Institut de Médecine Légale, Université de Strasbourg, (along the Yenisey River; Fig. 1) was characterized at the
11 rue Humann, 67085 Strasbourg Cedex, France nuclear and mitochondrial DNA levels. Among these speci-
e-mail: Christine.Keyser@iml-ulp.u-strasbg.fr; mens, 10 were attributed to the Andronovo culture, 4 to the
ckeyser@mageos.com
Karasuk culture, 12 to the Tagar culture and 6 to the
E. Crubézy Tachtyk one (Table 1). The Andronovo culture, related to
AMIS, CNRS, Université de Toulouse, the timber-grave group, appeared throughout the south Rus-
37 allées Jules Guesde, 31000 Toulouse, France sian steppe, Kazakhstan and western central Asia during the
V. G. Nikolaev · T. Reis
second millenium BC. (Koryakova and Epimakhov 2007).
State Medical University of Krasnoyarsk, The bearers of this Middle Bronze Age culture were
1 rue Partizana Zheleznyaka, 660022 Krasnoyarsk, Russia strongly associated with the Indo-Iranians and credited with

123
396 Hum Genet (2009) 126:395–410

Fig. 1 Map indicating the

locations of the archaeological
sites studied. Numbers refer to
the burial sites noted in Table 1

the invention of the spoke-wheeled chariot (Lamberg- century AD. Xiongnu were nomadic tribes inhabiting the
Karlovsky 2002). The Karasuk culture is a Late Bronze steppes north of China and controlling an empire stretching
Age culture that succeeded the Andronovo culture in southern beyond the borders of modern-day Mongolia. We also per-
Siberia (late second millenium BC.). Karasuk people were formed Y-SNP typing of one Scytho-Siberian specimen
farmers who practiced metallurgy on a large scale. They from the Sebÿstei site in the Altaï Republic (Central Asia)
produced a realistic animal art, which probably contributed dated from the middle of the Wfth century BC. All these
to the development of the later Scytho-Siberian animal art specimens were previously typed for autosomal and
style. The Karasuk culture was replaced by the early Iron mtDNA polymorphisms (Keyser-Tracqui et al. 2003;
Age Tagar culture (Wrst millenium BC.) which Xourished in Ricaut et al. 2004).
Khakassia (southern part of the Krasnoyarsk Krai) produc-
ing an art of animal motifs related to that of the Scythians
of southern European Russia. On the Yenisey River, the Materials and methods
Tagar culture was replaced by the Tashtyk culture, dating
from the Wrst to fourth century AD. Ancient human samples
To investigate the history and origin of these ancient
Krasnoyarsk specimens, two uniparentally inherited marker DNA was extracted from 32 ancient human skeletons exca-
systems were analyzed. Indeed, apart from giving informa- vated from diVerent kurgan sites of the Krasnoyarsk region
tion about paternal and maternal lineages, both the non- in southern Central Siberia during the years 1964–2000. In
recombining portion of the Y-chromosome (NRY) and the this area, the average temperature is 20°C below zero in
mitochondrial DNA (mtDNA) have proven to be good indi- winter. Even if the graves under the kurgans were not fro-
cators of migration events in human population history zen at the excavation, in summer the temperature at the
(Underhill and Kivisild 2007). Autosomal short tandem graves level is never over a few degrees Celsius above or
repeats (STRs) were also typed to conWrm conventional below zero. After being listed and arranged in cardboard
sexing and to assess possible parentage relationships and/or boxes, skeletal remains were sent to the Krasnoyarsk State
exogenous contamination. Finally, since the specimens Medical Academy of Russia, at Krasnoyarsk University,
under study are thought to have been “Caucasoid” (Kozintsev where they have been stored in a dry and cold environment.
et al. 1999; Lebedynsky 2003; Moiseyev 2006), phenotype- In 2004, the cardboard boxes were opened for sampling by
informative single nucleotide polymorphisms (SNPs) were two members of our team and transferred to Strasbourg,
also tested. France, under appropriate storage conditions. On arrival in
To widen the geographic scale of our study, we deter- the laboratory, samples were frozen until DNA extraction
mined the Y-chromosomal haplogroup of several Xiongnu to ensure their good preservation. The location of the kur-
specimens dated from the third century BC. to the second gans is indicated on Fig. 1; their associated culture, the time

123
Hum Genet (2009) 126:395–410 397

Table 1 Samples from the Krasnoyarsk region considered in the study

Specimen Code Site/region/(map number) Culture Period Dates Sex

Bronze 1 S07 Tatarka cemetery, burial 64 Andronovo Middle Bronze Age 1800–1400 BC M
Charypovsky region (1)
Bronze 2 S08 Tatarka cemetery, burial 55 Andronovo Middle Bronze Age 1800–1400 BC F
Charypovsky region (1)
Bronze 3 S09 Solenoozernaïa IV, kourgane I, burial 3 Andronovo Middle Bronze Age 1800–1400 BC M
Krasnoyarsk region (2)
Bronze 4 S10 Solenoozernaïa IV, kourgane I, burial 4 Andronovo Middle Bronze Age 1800–1400 BC M
Krasnoyarsk region (2)
Bronze 5 S11 Solenoozernaïa I, burial 4 Andronovo Middle Bronze Age 1800–1400 BC F
Krasnoyarsk region (2)
Bronze 6 S12 Solenoozernaïa I, burial 15 Andronovo Middle Bronze Age 1800–1400 BC ?
Krasnoyarsk region (2)
Bronze 7 S13 Solenoozernaïa IV, kurgane I, burial 4 Andronovo Middle Bronze Age 1800–1400 BC ?
Krasnoyarsk region (2)
Bronze 8 S14 Solenoozernaïa I, burial 4 Andronovo Middle Bronze Age 1800–1400 BC F
Krasnoyarsk region (2)
Bronze 9 S15 Solenoozernaïa I, burial 29 Andronovo Middle Bronze Age 1800–1400 BC ?
Krasnoyarsk region (2)
Bronze 10 S16 Oust-Abakansty, chief kurgan, Andronovo Middle Bronze Age 1800–1400 BC M
Khakassia republic (3)
Karasuk 1 S17 Katcha, Drokino II, burial 1 Karasuk Late Bronze Age 1400–800 BC M
Emelyanovsky region (4)
Karasuk 2 S18 Oust-Abakansty, kurgan IV, burial 1 Karasuk Late Bronze Age 1400–800 BC F
Khakassia republic (3)
Karasuk 3 S19 Bogratsky, burial I Karasuk Late Bronze Age 1400–800 BC F
Khakassia republic (5)
Karasuk 4 S20 Minoussinsk, Podgorny, burial 1 (6) Karasuk Late Bronze Age 1400–800 BC M
Tagar 1 S21 Novosselovsky region, Anach village, Tagar Iron Age 800 BC–100 AD ?
kurgan I, burial 3 (7)
Tagar 2 S22 Novosselovsky region, Anach village, Tagar Iron Age 800 BC–100 AD F
kurgan II, burial 4 (7)
Tagar 3 S23 Tchernogorsk, burial 1 Tagar Iron Age 800 BC–100 AD ?
Khakassia republic (8)
Tagar 4 S24 Tchernogorsk, burial 6 Tagar Iron Age 800 BC–100 AD M
Khakassia republic (7)
Tagar 5 S25 Oust-Abakansty, Khakassia republic (9) Tagar Iron Age 800 BC–100 AD M
Tagar 6 S26 Beysky region, burial 3 Tagar Iron Age 800 BC–100 AD M
Khakassia republic (10)
Tagar 7 S27 Bogratsky region, kurgan 133, burial 3 Tagar Iron Age 800 BC–100 AD F
Khakassia republic (11)
Tagar 8 S28 Bogratsky region, kurgan 133, burial 3 Tagar Iron Age 800 BC–100 AD M
Khakassia republic (11)
Tagar 9 S29 Bogratsky region, kurgan 133, burial 3 Tagar Iron Age 800 BC–100 AD M
Khakassia republic (11)
Tagar 10 S30 Bogratsky region, kurgan 133, burial 2 Tagar Iron Age 800 BC–100 AD ?
Khakassia republic (11)
Tagar 11 S31 Bogratsky region, kurgan 133, burial 2 Tagar Iron Age 800 BC–100 AD ?
Khakassia republic (11)
Tagar 12 S32 Bogratsky region, Abakano-Pérévoz II, Tagar Iron Age 800 BC–100 AD M
burial 1, Khakassia republic (5)
Tachtyk 1 S33 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F
burial 5, Khakassia republic (5)

123
398 Hum Genet (2009) 126:395–410

Table 1 continued
Specimen Code Site/region/(map number) Culture Period Dates Sex

Tachtyk 2 S34 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F

burial 4, Khakassia republic (5)
Tachtyk 3 S35 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F
burial 2, Khakassia republic (5)
Tachtyk 4 S36 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F
burial 4, Khakassia republic (5)
Tachtyk 5 S37 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F
burial 4, Khakassia republic (5)
Tachtyk 6 S38 Bogratsky region, Abakano-Pérévoz I, Tachtyk Iron Age 100–400 AD F
burial 4, Khakassia republic (5)
Question marks denote that the morphological sex determination was not possible or ambiguous. Map number, noted in bracket, indicates the
location of the grave on Fig. 1

period, the estimated age and the morphological sex of the D21S11, D18S51, D5S818, D13S317, D7S820) and the
specimens studied are presented in Table 1. Some skeletons sex-determining marker amelogenin were simultaneously
came from the same kurgan (e.g., S27–S31); one of them ampliWed. PCR reactions were performed according to the
was excavated from a chief’s kurgan (S16). Skeletal manufacturer’s protocol, except for the 37 cycles used
remains used for the DNA analyses were all long bone frag- instead of the recommended 28, in a reaction volume of
ments. Moreover, the Y-SNPs typing was carried out on ten 10
l thus reducing the volume of the DNA samples. Capil-
of the male Xiongnu specimens of the Egyin Gol valley lary electrophoresis was run on an ABI Prism 3100 Genetic
necropolis (Keyser-Tracqui et al. 2003) as well as on a Scy- Analyzer (Applied Biosystems, Foster City, CA, USA) and
tho-Siberian skeleton from the Sebÿstei site (SEB 96K2) data analysis was performed with the GeneMapper soft-
found in a kurgan of the Altaï Republic and dated from the ware (Applied Biosystems, Foster City, CA, USA). The
middle of the Wfth century BC. (Ricaut et al. 2004). parentage relationships between individuals were tested by
pairwise comparison of the proWles.
DNA extraction
Y-chromosomal STR and SNP analysis
To eliminate surface contamination, the outer surface of the
bones was removed to almost 2–3 mm of depth with a The DNAs of the male ancient specimens were analyzed at
sanding machine (Dremel®, Breda, The Netherlands). Bone 17 Y-chromosomal STR loci [DYS19, DYS385a/b,
powder was generated using a column drill Wtted with a sur- DYS389I/II, DYS390, DYS391, DYS392, DYS393 (mini-
gical trepan. DNA was extracted from bone powders mal haplotype), DYS437, DYS438, DYS439, DYS448,
according to a previously published protocol (Keyser-Trac- DYS456, DYS458, DYS635 (Y GATA C4) and Y GATA
qui and Ludes 2005). H4] using the AmpFlSTR® Y-Wler™ PCR AmpliWcation
Kit (Applied Biosystems, Foster City, CA, USA). The
Real time PCR quantiWcation experimental conditions were those recommended by the
manufacturer except that 34 cycles were used instead of 30.
Nuclear DNA quantitation was performed on an ABI Prism STR products were analyzed on an ABI Prism 3100
7000 Sequence Detection System (Applied Biosystems, Genetic Analyzer with GeneMapper software. The STR
Foster City, CA, USA) using the QuantiWler® Human DNA haplotypes obtained were individually compared to the Y
QuantiWcation Kit (Applied Biosystems, Foster City, CA, chromosome Haplotype Reference Database (YHRD)
USA) according to the manufacturer’s protocol. In addition (http://www.yhrd.org) (»57,000 9-loci haplotypes as of
to the quantiWcation of the nuclear DNA, the presence of December 2008 database search) as well as to a private
PCR inhibitors was determined thanks to the co-ampliWca- world Y-STR database maintained by us and containing
tion of an internal PCR control included in each reaction. data retrieved from the literature (»38,000 9-loci haplo-
types). A two-step comparison procedure was applied: in a
Autosomal STR analysis Wrst step (since most of the data available for comparison
do not include all the markers ampliWed in our study) com-
Autosomal STRs were ampliWed using the AmpFlSTR® parison was made with the nine loci of the minimal haplo-
ProWler Plus™ Kit (Applied Biosystems, Foster City, CA, type; in a second step it was undertaken on the minimal
USA). Nine STRs (D3S1358, vWA, FGA, D8S1179, haplotype plus other loci. No mismatch was allowed in the

123
Hum Genet (2009) 126:395–410 399

comparative analysis: only exact matches were considered rs1805008, rs7495174, rs6497268/rs4778241, rs11855019/
(even if many of the exact matches are likely “equal-by- rs4778138) or their previously reported allele frequency
state” and not “equal-by-descent”). diVerences between populations of the world (rs1545397,
A set of 13 Y-chromosomal SNPs [M3, M9, M17, M45, rs16891982, rs2031526, rs1426654). These SNPs are
M89, M173, M175, M216, M217, M242, 92R7, RPS4Y711 located in six pigmentation candidate genes: HERC2,
(M130), and Tat (M46)] characterizing Asian and Amerin- OCA2, MC1R, MATP/SLC24A2, DCT and Golden Gene/
dian populations were also tested (references for SNP SLC24A5. The choice of these SNPs as well as detailed
selection are given in Bouakaze et al. 2007). These SNPs protocol for genotyping using the SNaPshot minisequenc-
were ampliWed and analyzed according to a previously pub- ing methodology are described in Bouakaze et al. 2009.
lished SNaPshot® (Applied Biosystems, Foster City, CA,
USA) minisequencing protocol (Bouakaze et al. 2007). The Measures against contaminations and validation of the data
Y-SNP haplogroup nomenclature followed that of the Y
Chromosome Consortium (Y Chromosome Consortium Bearing in mind the critical issues of pre-laboratory con-
2002, Jobling and Tyler-Smith 2003; Karafet et al. 2008). taminations encountered in most of ancient DNA (aDNA)
studies (Sampietro et al. 2006), bone samples were col-
Mitochondrial DNA sequencing and SNP typing lected with extensive precautions (e.g., they were handled
with gloves by a reduced number of people). Moreover, to
A 381 bp sequence of the HVI region (positions 16009– check for possible modern contamination, the DNA
16390 of the Cambridge Reference Sequence (CRS; Ander- extracted from saliva samples of all people handling the
son et al. 1981, revised Andrews et al. 1999) was ampliWed material or working in the laboratory was genetically typed
in two overlapping fragments as previously described and then compared with the proWles or haplotypes of all
(Keyser-Tracqui et al. 2003). The cycle-sequencing reac- ancient samples.
tion was performed with the BigDye Terminator v1.1 Cycle The precautions concerning the facilities, the laboratory
Sequencing Kit (Applied Biosystems, Foster City, CA, ware and the reagents were thoroughly respected (labora-
USA). The products were detected on an ABI Prism 3100 tory dedicated to ancient DNA only, strict separation of
automatic sequencer and analyzed with the Sequence Navi- pre- and post-PCR experimental areas, UV irradiation of
gator Software package (Applied Biosystems, Foster City, the rooms and the laboratory ware between each experi-
CA, USA). Haplotypes were assigned to the diVerent ment plus treatment of equipment and benches with DNA
haplogroups using the “near matching” method (Yao et al. contamination-removal solution (DNA away), wearing
2002). appropriate protective clothing (lab coats, facemasks and
To validate exact mtDNA haplogroup determination and double pairs of gloves), use of pipettes with aerosol resis-
allocate mtDNAs to particular haplogroups not clearly deW- tant tips, systematic use of negative controls. Multiple inde-
ned with the control region alone, we also typed haplo- pendent DNA extractions and PCR ampliWcations were
group-tagging SNPs of the mitochondrial coding region carried out for each sample. Moreover, each new DNA
(Fig. S1). Twenty-six SNPs were selected and combined in extraction was directly followed by an AmpFlSTR® proWler
three multiplex reactions using SNaPshot® assays. Multi- Plus™ DNA ampliWcation to ensure that the new extract
plex 1 and 2 included a selection of SNPs deWning Euro- was not contaminated and that the following ampliWcations
pean and Asian haplogroups (UK, U, U2, U4, U5a1, HV, T, (mtDNA, Y-chromosome, autosomes) will be performed
T1, N1a, N9a, A, F1, X, C, Z, D5, G2, H, H3), whereas on the same individual.
multiplex 3 included nearly exclusively polymorphisms
deWning subclades inside haplogroup H (H1, H2, H2a, H5a,
H6, H7, H8; Fig. S1). Results
The distribution of the diVerent deWned haplotypes
among modern and ancient populations was investigated by Autosomal STR analysis
exact sequence searches performed against »40,400-
mtDNA haplotypes collected from the literature and main- Of the 32 individual remains analyzed by multiplex ampli-
tained in a personal database. Wcation, 6 DNA samples [S12, S17, S20, S30, S31 and S38
(Table 1)] appeared severely degraded since no ampliWable
Human pigmentation gene SNP analysis product was obtained from at least three independent
extracts. No inhibition was detected as indicated by the real
Ten autosomal SNPs were selected because of either their time PCR quantiWcation. The remaining extracted samples
association with normal human pigmentation variation, gave 26 more or less complete allelic proWles. Consensus
namely, eye, hair and skin color (rs12913832, rs1805007, data are reported in Table 2. The loci D13S317, D18S51,

123
400 Hum Genet (2009) 126:395–410

Table 2 Consensus allelic proWles of 26 of the specimens under study

Burial Amel. D13S317 D18S51 D21S11 D3S1358 D5S818 D7S820 D8S1179 FGA VWA

S07 XY 8/14 16/18 30/32.2 15/16 7/11 10/12 13/15 21/21 15/16
S08 XX 9/10 15/17 29/30 16/16 12/13 8/8 13/15 23/24 17/18
S09 XX 11/11 17/17 – 14/15 11/11 11/11 11/14 22/22 16/16
S10 XY 9?/12 13/15 30/31 17/17 10/11 11/11 13/13 21/23 17/17
S11 XX 11/11 14/15 30/31 14/18 11/11 11/11 13/14 24/24 18/18
S13 XX ? ? ? 17/18 11/(12) ? 13/13 ? 16/18
S14 XX ? ? ? 15/16 11/12 ? 8/12 22/25 17/18
S15 XX 11/11 ? 30/30 15/16 9/13 ? 13/14 23/24 ?
S16 XY 9/12 12/16 32.2/32.2 16/17 11/12 10/12 10/10 21/23 16/20
S18 XX 11/11 12/15 32.2/36 16/17 11/12 ? 13/13 22/22 18/18
S19 XX 11/11 12/13 30/31 17/18 11/11 9/11 12/13 21/23 15/17
S21 XX 9/12 17/17 28/31.2 15/19 11/12 9/10 12/12 23/24 17/18
S22 XX 11/12 14/15 30/31 16/17 11/12 9/10 13/15 19/24 19/19
S23 XX 8/11 16/16 30/30 16/18 12/13 10/10 14/16 23/24 17/18
S24 XY 8/11 14/16 29/30 14/16 10/13 10/11 12/14 19/24 14/17
S25 XY ? ? 29/30.2 15/17 12/12 ? 13/13 21/25 18/18
S26 XY 11/12 12/15 30/32.2 14/16 12/12 10/12 10/13 23/24 18/19
S27 XX 8/10 16/17 28/32.2 14/16 9/11 9/9 15/16 19/22 15/19
S28 XY 8/11 17/18 31.2/32.2 16/16 9/14 11/11 13/14 24/25 18/18
S29 XY 9/9 14/? ? 14/18 12/13 9/10 13/16 ? 14/19
S32 XY 8/11 ? 29/32.2 15/18 9/11 11/11 14/15 21/22 16/17
S33 XX 12/12 21/21 30/30.2 16/18 12/13 9/11 14/15 22/23 15/17
S34 XY 10/12 12/16 30/32.2 15/17 7/12 9/11 10/13 21/24 17/19
S35 XX 10/11 13/14 30/31.2 14/18 9/11 9/10 10/13 22/24 14/17
S36 XX 11/12 14/15 28/32.2 14/15 11/11 8/11 14/16 25/25 15/19
S37 XX 9/13 14/16 28/29 15/17 11/12 10/11 15/15 22/22 17/20
Question marks denote that alleles could not be clearly ampliWed for the locus in question. Consensus allelic proWles were built after obtention of
a minimum of three DNA proWles for each specimen

D21S11 and D7S820 were often not ampliWed, probably Y-chromosomal STR and SNP analysis
because they are expressed in the higher molecular weight
range. Such an inverse dependance of the ampliWcation To identify male lineages, an analysis of polymorphic
eYciency on the size of the segment to be ampliWed is typi- markers located on the male-speciWc part of the Y-chromo-
cal of DNA retrieved from ancient remains and results from some was performed. Seventeen Y-speciWc STRs were
damage and degradation of the DNA (Smith et al. 2003). typed and used to construct haplotypes. All of the 10 male
Morphological and molecular typing results for sex Siberian specimens were successfully typed at 14 loci (at
determination were in accordance with each other except least) and 6 diVerent haplotypes were diVerentiated
for two specimens (S09 and S34); nevertheless, since the (Table 3). Three of them were shared between at least two
DNA proWles obtained for these two individuals were specimens suggesting that some individuals could have
almost complete, it is highly probable that molecular results belonged to the same paternal lineage (S10 and S16; S24,
were the correct ones. Furthermore, the amelogenin locus S34 and maybe S25; possibly S28 and S29) although buried
allowed us to deduce the sex of four specimens for which in diVerent kurgan and/or with no evidence of a close kin-
morphological indicators of sex were absent (S13, S15, S21 ship link (cf., autosomal STRs). Pairwise comparisons of
and S23). the haplotypes showed that except for S07, all the ancient
Comparison of the proWles in pairs revealed no Wrst male specimens bore closely related allelic proWles,
degree relatives, even for specimens unearthed from the diVering at most at six loci (on the 17 tested) and always by
same kurgan or even burial. It should be noted, nevertheless, one-step mutation only.
that 35% of the DNA proWles were incomplete which might Haplogroup (hg) assignment, based on the Y-chromo-
have hampered the detection of close familial kinship. somal SNP typing, revealed that except for S07, which was

123
Hum Genet (2009) 126:395–410 401

found to belong to hg C(£C3), all ancient specimens were

Specimen DYS19 DYS385 DYS389I DYS389II DYS390 DYS391 DYS392 DYS393 DYS437 DYS438 DYS439 DYS448 DYS456 DYS458 DYS635 YGATA Haplogroup
aYliated to hg R1a1. This Wnding is in agreement with the

C(£C3)
R1a1
R1a1
R1a1
R1a1
R1a1
R1a1
R1a1
relative similarity of the haplotypes mentioned above.
While hg C has a distribution generally limited to popula-
tions of northern Eurasia, eastern Eurasia, Oceania, and the
Americas, R1a1 is widely spread across Eurasia. It is found

13
12
13

13
12
11

12
–
among western Eurasian, southern Asian, central Asian and
Siberian populations. This haplogroup is thought to trace
the migration patterns of the early Indo-Europeans, perhaps

23
22
23
23
23
23
23
23

– denote that alleles could not be ampliWed for the locus in question, alleles in bold deWne a motif shared by 4 of the 5 haplotypes belonging to R1a1 haplogroup
stemming from the Kurgan culture (Zerjal et al. 1999;
Semino et al. 2000). The additional analysis performed on
Xiongnu specimens revealed that whereas none of the
15
16
15
15
15
15
15
15
specimens from the Egyin Gol valley bore this haplogroup,
the Scytho-Siberian skeleton from the Sebÿstei site exhib-
ited R1a1 haplogroup.
16
15
16
16
16
16
16
16

A search in the YHRD database as well as in our own

databank revealed that none of the Y-STR haplotypes
20
19
20
20
20
20
20

obtained from the south Siberian samples perfectly

–

matched (at 17 loci) those included in the databases.

Nevertheless, when not all loci were scored, matches were
10
11
10
10
10
10
10

found for all samples except two (S07 and S32) for which
–
Table 3 Y-STR haplotypes and haplogroups determined for the ancient male specimens from the Krasnoyarsk region

even the search based on the 9-loci minimal haplotype

was fruitless (Table 4). The S10/S16 haplotype matched
11
10
11
11
11
11
11
11

the most frequent R1a1 haplotype (12 loci) seen in the

south Siberian population of Derenko et al. (2006). This
haplotype is notably found at high frequency in Altaians.
14
14
14

14
14
14
14
14

It carries an allelic stucture 16-14-32-25-11-11-13

(DYS19-DYS389I-DYS389II-DYS390-DYS391-DYS392-
DYS393) which is considered as a founder haplotype rel-
14
13
13
13
13
13
13
13

ative to southern Altaians (Kharkov et al. 2007). The S10/

S16 haplotype is also found in eastern Europe (Hungary,
Slovenia, Poland) as well as in Asia (Central Anatolia).
12
11
11
11
11
11
11
12

The S24/S34 haplotype is mainly found in Poland and

Germany. In Asia it is found in Anatolia, Armenia, Nepal
and India. Haplotype of specimen S26 has a wide distribu-
11
11
11
11
11
11
11
9

tion since it appears in Europe as well as in western Asia,

in Central Asia, in southern Asia and in southern Siberia.
The allelic structure 16-24-11-11-13 (DYS19, DYS390,
22
25
24
24
24
25
25
24

DYS391, DYS392, DYS393) found in this haplotype was

described as the most frequent motif observed in a Ukrai-
nian population by Kravchenko et al. (2002). According
30
32
31
31
31
31
31
31

to these authors, this 5 Y-STR-loci haplotype might be an

ancestral one. Haplotype S28 is the most frequently found
in present-day populations. It is essentially carried by
14
14
13
13
13
14
14
13

eastern and northern Europe individuals, as well as south

Siberians. The S32 haplotype was not found in the
12/13
11/14
11/14
11/14
11/14
11/14
11/14
11/14

databases even though it diVers from the S24/S34 haplo-

type by only one-step mutation at locus DYS392. The S07
haplotype also did not appear in the YHRD database even
15
16
17

16
16

17

when one mismatch was allowed in the minimal

–

haplotype search. The current distribution pattern of all

S10/S16
S24/S34

the Y-STR haplotypes found in our ancient sample is

S07

S25
S26
S28
S29
S32

reported in the map on Fig. 2.

123
402 Hum Genet (2009) 126:395–410

Table 4 Results of the search against Y-STR databases

Sample Minimal haplotype Minimal haplotype +2 to 7 additional loci

S07 No match No match

S10/S16 1 Hungarian2; 2 Slovenians, 2 Poles (YHRD) 2 Tuvinians, 14 South Altaians6; 1 Turk4;
1 Hungarian7; 1 Pole22; 1 Cretan16
S24/S34 (S25) 5 Poles17,19; 1 German19; 2 Poles, 1 Nepalese, 1 Turk, 1 German20; 1 Pole24; 1 Indian8
1 German, 1 Armenian (YHRD)
S26 1 German10; 2 Poles17,19; 1 Serbian23; 1 Pole, 1 Kazakh, 1 Turk4; 1 Chinese15; 1 Indo-Pakistani1;
1 Nepalese, 2 Germans, 1 Turk (YHRD) 8 South Siberians6;
1 Indian5; 5 Poles24;1 German20
S28 (S29) 2 Lithuanians13,19; 4 Latvians13,19; 6 Poles17,19, 1 German19; 1 Austrian2; 1 Lithunian18; 1 Uigur26;
1 Byelorussian18; 1 Russian14; 2 Swedish9,11; 3 Serbians23; 20 South Siberians6;
1 Estonian12, 1 Lithuanian12; 6 Slovenians, 13 Czechs, 3 Poles22; 2 Malays25; 2 Indians5; 2 Russians21
4 Hungarians, 7 Germans, 3 Slovaks, 3 Ukrainians,
2 Croats, 1 Siberian Tuvan, 3 Poles, 3 Norwegian (YHRD)
S32 No match No match
References are given in Table 1 in supplementary material

Fig. 2 Current distribution pat-

tern of the Y-STR haplotypes
found in the ancient Siberians
under study. Each square repre-
sents a present-day individual
sharing the same Y-haplotype of
an ancient specimen

Mitochondrial DNA analysis assay allowed us to obtain additional information regarding

the phylogenetic reWnement of two samples, S13 and S32,
The mtDNA haplotype of the 26 ancient individuals for found to belong to subhgs H6 and H5a, respectively.
whom genotypes were obtained, was determined by Overall, 23 diVerent haplotypes were distinguished and
sequencing of the HVI region. To avoid ambiguous conclu- assigned to 16 diVerent haplogroups. Twenty samples were
sions and to corroborate haplogroup assignment, all indi- found to belong to west Eurasian haplogroups (U2, U4,
viduals were additionally typed for some speciWc mtDNA U5a1, T1, T3, T4, H5a, H6, HV, K, and I), whereas the 6
coding SNPs. Results are indicated in Table 5. A good remaining samples were attributed to east Eurasian haplo-
agreement was found between coding and control region groups (Z, G2a, C, F1b and N9a).
data except for three samples (S27, S29, S35) sharing a To assess the present distribution of the mtDNA types
CRS HVI haplotype whose SNapShot assay coding SNPs found in the ancient sample, a search of their occurence
allowed classiWcation as hg U. Moreover, the SNapShot among modern populations of Eurasia was carried out in

123
 Table 5 HVI haplotype and haplogroup attribution for each Krasnoyarsk specimen successfully analyzed and current distribution of the haplotypes
Sample HVI haplotype MtHg SNPHg Occurrence of the haplotype in the world

S07/S14 356C U4 – 2 Turks7,38; 6 Russians1,19,33; 1 Ukrainian33; 5 Mansi10; 18 Volga-Ural region individuals2;

2 Germans40,48; 17 Altai–Sayan region individuals12,18,46; 5 Bosnians34; 1 Slovenian34; 2 Uygur54;
1 Uzbek54; 3 Mongols54,26,14; 2 Chechens42; 15 Latvians39,31; 1 Albanian3, 4 Macedonians3,57,
3 Romanians3, 4 Hungarians4,22; 1 Buryat14; 1 Finn20; 3 Poles19; 1 Lithuanian31, 3 Seto31,
3 Karelians31, 3 Swedish31; 4 Slovaks35; 6 Greeks23; 3 Italians49; 1 ancient Hungarian50
S08 129A 185T 223T 224C 260T 298C Z (Z1) Z 2 Mongols28,29; 1 Kazakh8; 8 Koryaks45, 1 Itel’men45; 1 Russe33; 1 Ket11, 2 Nganasans11,52;
Hum Genet (2009) 126:395–410

3 Yukaghirs52; 11 Volga-Ural region individuals2; 1 Buryat14, 1 Altaian14, 2 Teleuts14;

1 Volot19, 2 Karelians31, 1 Swedish31
S09 126C 163G 186T 189C 294T T1 T1 2 Turks8, 12 Italians17,49; 1 Chinese Han55; 1 Indian0; 1 Uzbek54; 5 Latvians31,39; 1 Mongol26;
8 Hungarians4,22,50; 3 Austrians5; 3 Altaians14 1 Chukchi14; 4 Finns20; 3 Germans48; 2 Greeks28;
3 Byelorussians1;3 Estonians31, 4 Lithuanians31, 1 Seto31, 5 Karelians31, 8 Swedish31;
1 ancient Kazakhstan30 and 1 ancient Xinjiang17 specimens
S10 051G 092C 129C 183C 189C 362C U2e U2 1 Estonian31; 1 Uygur54
S11 126C 294T 324C T4 T 2 Roma24; 1 Byelorussian1,1 Hungarian22; 1 Greek23
S13 187T 362C H H6 1 Corsican16
S15 093C 224C 311C 319A K2b UK 1 Hungarian4; 1 Austrian5
S16 192T 256T 270T U5a1 U5a1 1 Russian1; 2 Kets11; 1 Byryat14; 1 Khakassian14; 1 Mongol26; 1 Albanian3; 2 Macedonians3;
3 Romanians3, 4 Greeks23; 3 Hungarians4,22; 2 Austrians5, 2 Italians49; 1 Finn20,
1 Lithuanian31, 2 Swedish31
S18 114A 256T 270T 294T U5a1 U5a1 1 Northwestern European44
S19 356C 362C U4* U4 1 Indian43; 1 Hungarian22; 4 Volga-Ural region individuals2; 2 Altai–Sayan region individuals13;
1 Estonian31, 1 Lithuanian31; 1 Bosnian34, 2 Slovenians34; 1 Austrian5; 1 Greek23; 2 Italians49
S21/S22 126C 189C 292T 294T T3 T 2 Sardinians15
S23 126C 189C 292T 294T 296T T3 T Not found
S24 129A 223T 304C 391A I (I4) – 1 Indian27; 2 Icelanders21; 1 Buryat14; 5 Swedish31; 5 Hungarians22; 1 ancient Scandinavian36
S25 093C 223T 227G 278T 362C G2a G2 1 Uygur54; 3 Koreans14,29,41; 2 Latvians31,39
S26 148T 223T 234T 288C 298C 327T C C 1 Tuvinian14
S27 CRS H U Too frequent
S28 172C 179T 183C 189C 232A 249C 304C 311C F1b F1 2 Mongols14,54
S29 CRS H U Too frequent
S32 304T 319A H (H5) H5a 1 Austrian5
S33 093C 129A 223T 298C 327T C C 2 Kazakhs8,54; 6 Altaians13; 7 Buryats13,46; 15 Tuvinians13,46;1 Uygur54; 2 Chineses53,41;
10 Evenks14,46; 1 Ulchi46; 6 Yakuts47,14; 2 Mongols14,41; 1 Kalmyk14; 2 Oroqen41; 1 Xiongnu25
S34 172C 311C HV HV 1 Indian43; 1 Uzbek54, 1 Mongol54; 1 Evenk41; 1 Kalmyk14; 1 Macedonian57; 2 Italians49,51
S35 093C 209C H U 1 Estonian31
S36 223T 257A 261T N9a N9a 5 Chinese41,53,55; 3 Koreans32,41,56; 2 Vietnameses23
S37 126C 163G 186T 189C 269G 294T 362C T1 T1 Not found

123
403
404 Hum Genet (2009) 126:395–410

the literature and personally compiled database. Exact 2008). The K2 haplotype harboured by specimen S15 was
matches were observed for almost all sequences. Among observed only twice, in European samples (1 Hungarian
those mostly prevalent was the hg U4 motif 356C (S07/ and 1 Austrian).
S14) which was found in northern, eastern and southeastern The eastern Eurasian lineages are represented by
European populations, as well as in Volga–Ural, Altai– sequences belonging to hgs N9a, Z, G2a, F1b and C. The Z
Sayan and peri-Baïkal area populations. Note that this haplotype observed in the S08 ancient specimen belong to
sequence occupies a central position on U4 phylogeny built subhg Z1. It is observed in northeastern Asians, in south
by Malyarchuk (2004). It was also observed in an ancient Siberian populations as well as in Central Asia. It is also
Hungarian specimen from the tenth to eleventh century present among several populations of the Volga–Ural and
(Tömöry et al. 2007). The sub-hg U4 variant 356C–362C Baltic Sea regions. The S25-G2a sequence has been
(S19) is present in northern, eastern and mediterranean observed in few but dispersed individuals (Koreans, Lat-
Europe, in the Volga–Ural and Altai–Sayan regions as well vians and Uygur). The specimen S28 belongs to hg F1b
as in southern India. Another sequence within hg U show- with a motif reported in two Mongols only. Haplogroup C
ing a wide geographic distribution is the S16-U5a1 haplo- is represented by two sequences: one had a HVI motif
type which matches in southern Siberia, in Central Asia, as observed in only one south Siberian individual (S26). The
well as in northern, western, eastern and southeastern other had a HVI motif mainly found in Siberia and Central
Europe. Conversely, the S18-U5a1 haplotype is most likely Asia (S33). Finally, the S36 specimen carries a N9a-haplo-
rare since found only once in a northwestern European, type identical to those described previously in East Asian
likewise the S10-U2 haplotype which has been described in individuals (Chinese, Koreans, Vietnamese). Figure 3 rep-
one eastern European and one central Asian individual resents the current and past distribution of the overall
only. Specimens S27, S29 and S35 bear a CRS HVI mtDNA haplotypes found in our ancient Krasnoyarsk sam-
sequence belonging to hg U. Such results have already been ple (except the CRS sequences). This distribution is similar
described in Russian and Byelorussian populations where to that depicted in Fig. 2 for the Y-chromosome, despite the
hg U CRS sequences were found in a relatively high pro- sparser pattern of the mtDNA counterpart.
portion (»35%; Belyaeva et al. 2003).
Within hg T, the most prevalent sequence type was that Human pigmentation gene SNP analysis
harbored by specimen S09. This sequence was described as
the root sequence of hg T1 (Richards et al. 2000; Pike In order to deepen the search of the geographic origin of the
2006). Its highest frequency is in western Eurasia (mainly Siberian specimens under study, we typed SNPs located in
the Baltic region) with occasional occurrences in eastern human pigmentation genes. Ten SNP markers located in
Eurasia. This founder haplotype was also observed in two genes that have been described as accounting for variation
ancient specimens, one from Kazakhstan (1400–1300 BC, in human hair, eye and skin color but also in ethnogeo-
Bronze Age; Lalueza-Fox et al. 2004), the other from a graphic ancestry were thus selected and a minisequencing-
Xinjiang site in northwestern China (Gao et al. 2008). Sur- based assay was developed on modern samples (Bouakaze
prisingly, the S37-haplotype, diVering from the previous et al. 2009). This assay was subsequently applied on the
one by two additional mutations, was not observed in our ancient Siberian samples so that complementary informa-
database. The T3-haplotypes harbored by specimens S21, tion provided by phenotype-associated SNPs could add to
S22 and S23 are either rare or absent whereas the S11-T4 previous anthropological and genetic Wndings. The pheno-
haplotype, although being described as a main founder type and ancestry of the ancient Siberian specimens under
cluster within haplogroup T (Richards et al. 2000), was not study are indicated in Table 6 (genotype details for each
commonly found. investigated marker is given in Bouakaze et al. 2009). Sur-
The H haplotypes observed in our ancient sample are prisingly, the typing of a SNP associated to eye color
uncommon in present-day populations since both S13-H6 (rs12913832) shows that at least 60% (15/25) of the Sibe-
and S32-H5a sequences were found in only one European rian specimens had blue (or green) eyes (S27 cannot be
individual. HV lineage are represented by one sequence tested because bone sample and DNA extract were used
(S34) found in India, in Central Asia and in southeastern up). Moreover, the pigmentation SNP analysis showed that
Europe. all except three specimens exhibited a European ancestry,
Specimen S24 was found to belong to haplogroup I, even when they bore an Asian mtDNA haplotype as is the
subclade I4. Exact matches to this I4 type were mainly case for samples S25, S26, S28, S33 and S36, demonstrat-
found in northern and eastern Europe individuals. The fact ing the importance of studying both maternal and paternal
that one ancient Scandinavian specimen (0–400 AD) bore lineages. These results also show that two individuals car-
this sequence gives direct evidence of the antique presence rying the same mtDNA haplotype can be classiWed in oppo-
of such sequence in the north of Europe (Melchior et al. site ethnogeographic groups as is the case for samples S07

123
Hum Genet (2009) 126:395–410 405

Fig. 3 Current distribution pat-

tern of the mtDNA haplotypes
found in the ancient Siberians
under study (except the CRS se-
quence). Each square represents
a present-day individual sharing
the same mtDNA haplotype of
an ancient specimen. Cross rep-
resents an ancient specimen
diVerent from those studied in
this work

and S14: note that these two specimens belong to diVerent contributed to the spread of the Indo-European language. In
paternal lineages since S07 is the single specimen carrying an attempt to answer these questions, we analyzed, in paral-
haplogroup C(£C3) but not haplogroup R1a1. Specimen lel, markers of distinct genetic systems.
S32 appears as having a mixed ancestry; curiously, this The choice of autosomal STR markers as a Wrst approach
specimen exhibits Y-chromosome and mtDNA haplotypes for analyzing the Siberian ancient remains was based not
virtually unknown in present-day populations. Most of the only on their high discriminatory power to investigate close
specimens seem to have been light-skinned people with familial relationship, but also on their ability to detect
blond or light brown hair. degraded and/or contaminated DNA. Twenty-six out of the
32 bone samples collected yielded ampliWable DNA and
65% of the genetic proWles were complete (Table 1). This
Discussion high success rate suggested that the nucleic acids were well
preserved and allowed us to envisage other single-copy
With the present study, we aimed to unravel some of the nuclear genes analyses (i.e., pigmentation genes). No close
early Eurasian steppe migration movements by analyzing kinship link was detected between the subjects under study
paternal, maternal and autosomal genetic variation in even for those found in the same kurgan (S27–S31). Never-
Bronze and Iron Age anthropological remains recovered theless, the Y-chromosomal analyses performed on the ten
from the Krasnoyarsk area in southern Central Siberia. male specimens showed that S28 and S29 shared the same
There is virtually no knowledge either about the origins and haplotype. These Y-chromosomal analyses, based on the
the history of these ancient south Siberian inhabitants or combined use of STRs and SNPs, also revealed that, with
about the language(s) they may have spoken. Nevertheless, the exception of one individual, all samples examined fall
many scholars believe that these Kurgan people, and into hg R1a1.
notably the bearers of the Andronovo culture, spoke a Haplogroup R1a1 is deWned by marker M173 plus M17
Proto-Indo-Iranian or a Proto-Iranian language (Lamberg- (Y Chromosome Consortium 2002; Jobling and Tyler-
Karlovsky 2002). Moreover, the south Siberian tribes under Smith 2003; Karafet et al. 2008) and has a widespread dis-
study (Andronovo, Karasuk, Tagar) have been described as tribution area on the Eurasian continent. It is spread among
exhibiting pronounced Europoid features (Kozintsev et al. western Eurasian (mostly eastern European and Volga–
1999; Lebedynsky 2003; Moiseyev 2006). These data Ural populations), southern Asian (mainly India and Paki-
raised questions as to where these people came from, which stan’s populations), central Asian and Siberian populations
routes have been followed and to which extent they have (especially southern Siberians), whereas it is rather rare in

123
406 Hum Genet (2009) 126:395–410

Table 6 Most probable pheno-

Sample Eye color Phenotype according to OCA2 diplotype Ancestry according
type and biogeographical origin
according to rs1545397,
of each Krasnoyarsk specimen
to rs12913832 rs16891982,
deduced from the 10 phenotype
rs2031526
and ancestry autosomal markers
SNPs analysis S07 Brown Blue or brown eye, dark brown hair, As
fair or medium skin
S08 Brown – As
S09 Blue Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
S10 – Blue or brown eye, brown hair Eu
S11 Blue – Eu
S13 – Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
S14 Brown Blue or brown eye, brown hair, fair or medium skin Eu
S15 Blue – Eu
S16 Blue Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
S18 Blue – Eu
S19 Blue – Eu
S21 Blue Blue or brown eye, brown hair, fair or medium skin Eu
S22 Brown – Eu
S23 Blue – Eu
S24 Blue – Eu
S25 Blue – Eu
S26 Brown Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
S28 Brown Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
S29 Blue – Eu
S32 Brown – Mix
S33 Blue Blue or brown eye, blond or light brown hair, Eu
fair or medium skin
Blue eye color phenotype is S34 Blue – Eu
predictive of blue or green-eyed S35 Brown – Eu
individuals (non brown eye
S36 Blue Blue or brown eye, blond or light brown hair, Eu
color)
fair or medium skin
– indicates that the marker could
S37 Blue Blue or brown eye, blond or light brown hair, Eu
not be ampliWed, As Asian,
fair or medium skin
Eu European, Mix mix

East Asian populations. In western Eurasia, a clear north– by the movement of the Kurgan people from the north of
east/south–west cline has been described (Rosser et al. the Caspian Sea in a much more recent timescale (Rosser
2000; Semino et al. 2000; Wells et al. 2001). Indeed, the et al. 2000; Semino et al. 2000). This “Kurgan people”
R1a1 haplogroup frequency reaches a maximum in Poland, expansion would have resulted in the spread of the Indo-
Hungary, and Ukraine and decreases in the direction of cen- European language as postulated by Gimbutas (1970).
tral and northern Europe. The same occurs in the southern Thereby, R1a1 was viewed by some authors as the marker
direction, towards Anatolia and the Caucasus. These clinal of the Indo-European contribution (Zerjal et al. 1999; Khar-
frequency distributions have been associated with ancient kov et al. 2004). According to PericiT et al. (2005), the
population movements in Europe: according to Semino present distribution pattern of the R1a1 haplogroup was
et al. (2000), the geographical distribution of the R1a1 probably also inXuenced by much later migratory events
haplogroup probably reXects the re-population of Europe like massive Slavic migration from Wfth century AD.
after the last glacial maximum (»20–12 kya) from a refu- In our ancient sample, among the nine specimens carry-
gium in eastern Europe, likely in Ukraine (Passarino et al. ing haplogroup R1a1, Wve diVerent Y-chromosomal haplo-
2001). This postglacial spread might have been magniWed types were observed. Similarities were noted between these

123
Hum Genet (2009) 126:395–410 407

haplotypes, particularly the motif 11/14-11-11-13-14-11- mens indicates that numerous populations carrying diVerent
10-20-16-15-23 (in bold in Table 3) which is common to all mtDNA variants were involved in the formation of south-
of them except S32. This motif is typically an eastern Euro- ern Siberian populations, even reXecting long-distant
pean one since currently found in the Russian federation movements. It would not have presented any major diY-
only (YHRD database). Matching haplotypes were found culty for Bronze Age and Early Iron Age peoples to range
for all the R1a1-specimens except S32. Figure 2 shows that from one end of Eurasia to the other within some centuries.
the current distribution pattern of the Y-STR haplotypes Historical records and archaeology attest that nomadic
found in our ancient sample resembles that of R1a1. groups moved across Eurasia from North of the Black sea,
Indeed, they were observed at high frequencies in Slavic through Central and Inner Asia, to northeast Asia in a mat-
and Baltic populations (with peaks among Poland and ter of centuries (Mair 2005). Some of them are described in
Czech Republic) as well as in the indigenous populations of Chinese historiography as horse-riding, Caucasian-looking,
south Siberia. By contrast, they were only sporadically Indo-European-speaking people and are sometimes referred
observed in central and east Asia and were absent in west- as the “Kurgan Culture” (Zerjal et al. 2002). Paleogeo-
ern Europe. graphic studies provide material which suggests that cli-
Regarding the mtDNA analyses, our Wndings indicate mate change, particularly in the eastern regions of the
that the ancient Krasnoyarsk mtDNA pool harbored both steppes, was among the causes of these population move-
western and eastern Eurasian lineages. Nevertheless, most ments (Van Geel et al. 2004).
of the retrieved sequences (n = 20, 77%) belong to western If we consider that there is a correspondence between the
Eurasian mtDNA haplogroups (HV, H, T, I, U and K). The overall distribution of haplotypes and haplogroups and past
eastern Eurasian lineages (23% of the sequences) were rep- human movements, it seems that the European or Cauca-
resented by haplogroups or subhapologroups C, Z, G2a, soid component observed in the ancient Siberian sample
F1b and N9a. The western Eurasian contribution to the may originate from East European populations. Moreover,
ancient mtDNA pool reached 90% for the Bronze Age and it is likely that some mtDNA lineages were carried to
decreased to 67% for the Iron Age. Thus, despite a small southern Siberia from the Volga–Ural region. Incidentally,
sample size, our data suggests a temporal pattern which is in the Wfth century BC, Herodotus mentioned transit trade
in agreement with the view that west Eurasian populations occurring in Central Asia along a route that stretched from
predominated in the Krasnoyarsk region during the Bronze the Urals in the west to the Altai and the Minusinsk Basin
Age, whereas Asian component began to increase from the in the east (Hemphill and Mallory 2004). In Altai, the pres-
Iron Age on. This result is similar to that obtained in the ence of the R1a1 haplogroup in the middle of the Wfth cen-
ancient DNA study of Lalueza-Fox et al. (2004) who tury BC is conWrmed by the sample SEB 96K2 of Ricaut
showed that all Kazakh sample specimens from before thir- et al. (2004) which was found to belong to this Y-haplo-
teenth to seventh centuries BC belonged to European lin- group. The boundary of the eastern European inXuence
eages. After that time, there was an inXux of East Asian seems to be Wxed at the peri-Baikal area since no R1a1
sequences which are thought to have coexisted with the haplogroup was found in the Xiongnu specimens of the
prior west Eurasian genetic substratum. Northern border of Mongolia.
As shown in Table 5, and particularly in Fig. 3, the cur- According to the “Kurgan hypothesis” of Marija Gimbu-
rent distribution of the ancient mtDNA haplotypes can be tas, nomadic peoples of the Volga steppe region, assumed
broadly divided into three diVerent geographic poles. The to speak a Proto-Indo-European language, inWltrated
Wrst is represented roughly by eastern and northern Europe, Europe in three waves between 4400 and 2800 BC. Around
the second by the Volga–Ural region and the third by south- 4400 BC, Kurgan people from the lower Dnieper and lower
ern Siberia. It is interesting to note that the distribution of Volga regions began moving along the Black Sea littoral
the paternal and maternal lineages is close. Indeed, except into the Danube Basin. They migrated in the Central Bal-
for the Volga–Ural region, both maps overlap. This would kans and further into Central Europe. During the middle of
mean that the story of women matches well that of men. In the fourth millennium BC, the Kurgan culture in the North
other words, the migrations in which south Siberian speci- Pontic Region continued to develop. People travelled
mens were involved seemed to be “whole-population across western Ukraine north of the Carpathian Mountains
movements” rather than “war-like movements” involving to Poland and Central Germany. They also moved south-
the men only. The fact that East Asian mtDNA sequences west into eastern Romania. Shortly after 3000 BC, the third
appeared at the Iron Age could signify that once settled, Kurgan wave (Yamna people), originating once more from
migrants of supposed European ancestry began to establish the Volga steppe, spread from Central Europe to Northwest
relationships with groups coming from the east and to take Germany, the east Baltic area, southern Scandinavia, the
Asian women as wives. Moreover, the relative high diver- upper Dnieper basin and Central Russia. These three waves
sity of the mtDNA gene pool observed in the ancient speci- of migrations might explain the distribution of mtDNA and

123
408 Hum Genet (2009) 126:395–410

Y-chromosome lineages observed in the present work An essential aspect of the present work is the conWdence
(Figs. 2, 3). in the validity of our data. Indeed, the Weld of ancient DNA
The Andronovo culture was preceded by the Afanasievo studies is fraught with technical pitfalls and needs stringent
one, which is held to share the closest similarities with the criteria to ensure the reliability of results, particularly when
Yamna culture found in the Pontic-Caspian region (Hemp- human remains are studied (Hofreiter et al., 2001). In this
hill and Mallory 2004). An eastward migration of the study, extensive precautions (described in the “Materials
Yamna-derived Afanasievo populations in the Eastern and methods”) were taken to avoid the ampliWcation of
steppe thus provides a possible explanation for the appear- contaminating contemporary DNA molecules. Despite the
ance of a European component in the gene pool of ancient fact that not all reported criteria of authenticity could be
south Siberians. met (Cooper and Poinar 2000), the possibility that our data
Whereas archaeological records are inconclusive about arose from contaminating DNA was considered highly
the anthropological traits characteristic of ancient Siberi- unlikely. Of course, the reasons as to why some criteria
ans, our data deduced from the analysis of human pigmen- were not adopted have to be explained (Gilbert et al. 2005;
tation gene SNPs seems consistent with the fact that most Bandelt 2005). AmpliWed products were not subjected to
of them had blue (green) eyes. Indeed, among the SNPs cloning and amino-acid racemization for the following rea-
tested was rs12913832, a single DNA variation within a sons: (1) an indirect procedure (DNA proWling) was used to
regulatory element of HERC2 gene which is associated to assess contamination and biochemical preservation of the
blue eye color in humans. This polymorphism, together DNA samples. Indeed, the (partial) allelic proWles obtained
with the diplotypes obtained from variations of the OCA2 in this study were not mixtures of diVerent individuals’
locus (major contributor to the human eye color variation) DNA and none of them matched any of those involved in
showed that at least 60% of the ancient Siberian specimens the handling of the bones or DNA samples. Moreover,
under study had blue (or green) eyes. Such color phenotype these DNA proWles were diVerent between each other and
is, according to Eiberg et al. (2008), caused by a founder testiWed to the good quality of the extracted DNA (preser-
mutation which most likely originated 6-10 kya from a vation of the nuclear DNA) while showing evidence of its
region around the Black sea, near modern-day Ukraine or antiquity (allelic drop out, inverse correlation between
Turkey and then diVused into Northern Europe. Our data ampliWcation eYciency and length of the ampliWcation
also suggest that south Siberian specimens might have had product). Thus, the cold climatic conditions encountered in
blond or light brown hair and fair skin and that they were of Siberia had undoubtedly protected the recovered specimens
European ancestry, a result which appears as evident as against DNA degradation; (2) the mtDNA sequences were
those of uniparental markers. determined based on at least two (often three) independent
Interestingly, the haplotype of specimen S09 matches DNA extracts and PCRs performed on both strands of the
that of an ancient specimen from the Yuansha site (Takla- DNA; this strategy is costly but eYcient in terms of reli-
makan desert, Xinjiang Province, northwestern China) and ability; (3) the quality of the sequences was, in general,
dated back to 2,135 § 50 years (Gao et al. 2008), suggest- comparable to those produced from modern DNA (sharp
ing genetic relationships between Andronovo populations peaks and little to no background); (4) the mtDNA
and those of the Xinjiang. The Bronze Age inhabitants of sequences of poor quality have not been taken into account
the Xinjiang were intrigued at their “Caucasoid” physical in this work (samples S12, S17, S30 and S31). Additional
appearance and putative “European” origins (Mallory and criteria of authenticity were considered in this study: results
Mair 2000). Two hypotheses have been oVered by archae- of both sex typing methods (morphologic and genetic) were
ologists to account for the origins of these Bronze Age peo- in accordance with each other (except twice); there was
ple believed to have spoken an Indo-European language concordance between mtDNA HVI haplotype and haplo-
called Tocharian and depicted as possessing red or blonde group-deWning SNPs along the coding region and also
hair, long noses and blue or green eyes: the “steppe hypoth- between Y-haplotype and haplogroups. The fact that the
esis” and the “Bactrian oasis hypothesis”. Proponents of the mtDNA analysis in our ancient sample revealed the pres-
latter assert that settlement of the Xinjiang came from sed- ence of founder mitochondrial lineages as well as of
entary based population of the Oxus civilisation found in sequences found in other ancient specimens might also be
Uzbekistan, Afghanistan and Turkmenistan, whereas pro- an indication of their phylogenetic antiquity.
ponents of the “steppe hypothesis” maintain that the Tarim To conclude, in this work we demonstrated that some
region experienced a colonization attributed to Afanasievo carriers of the Kurgan culture, believed to be Indo-Euro-
and Andronovo populations who migrated to Xinjiang from pean speakers, were also carriers of the R1a1 haplogroup.
the Altai–Minusinsk regions north of the Tarim Basin These data lend further support to the idea that R1a1 might
(Hemphill and Mallory 2004). Our results corroborate the be a marker to the migration patterns of the early Indo-
“steppe hypothesis”. Europeans, an idea also supported by the recent article of

123
Hum Genet (2009) 126:395–410 409

Haak et al. (2008) in which individuals of the Corded Ware Europeans. University of Pennsylvania Press, Philadelphia,
Culture, a culture commonly associated with Indo-Euro- pp 155–195
Haak W, Brandt G, de Jong HN et al (2008) Ancient DNA, strontium
pean, might bore R1a1 Y-chromosome (as we deduced isotopes, and osteological analyses shed light on social and kin-
from their Y-STR typing results). The modern distribution ship organization of the Later Stone Age. Proc Natl Acad Sci
of lineages is the outcome of many millennia of population USA 47:18226–18231
movements and therefore the assumption of a Proto-Indo- Hemphill BE, Mallory JP (2004) Horse-mounted invaders from the
Russo-Kazakh steppe or agricultural colonists from western Cen-
European speaker’s homeland in Kurgan region should be tral Asia? A craniometric investigation of the Bronze Age settle-
taken with great caution. Nevertheless, our study opens ment of Xinjiang. Am J Phys Anthrop 124:199–222
possibilities for new debates. We also showed for the Wrst Hofreiter M, Serre D, Poinar HN et al (2001) Ancient DNA. Nat Rev
time that Bronze and Iron Ages south Siberian populations Genet 2:353–359
Jobling MA, Tyler-Smith C (2003) The human Y chromosome: an
displayed “European” physical appearance, thus corrobo- evolutionary marker comes of age. Nat Rev Genet 4:598–612
rating physical anthropological records. Another conclu- Karafet TM, Mendez FL, Meilerman MB et al (2008) New binary
sion that can tentatively be inferred from the data presented polymorphisms reshape and increase resolution of the human Y
here is that the Andronovo culture might be the eastern chromosomal haplogroup tree. Genome Res 18:830–838
Keyser-Tracqui C, Ludes B (2005) Methods for the study of ancient
spread of the Kurgan culture and might be related to DNA. Methods Mol Biol 297:253–264
Tocharian speakers in the Tarim Basin. Keyser-Tracqui C, Crubézy E, Ludes B (2003) Nuclear and mitochon-
drial DNA analysis of a 2,000-year-old necropolis in the Egyin
Acknowledgments The authors are grateful to Prisca Blandin-Frap- Gol valley of Mongolia. Am J Hum Genet 73:247–260
pin, Aurélie Marchet and Sarah Romac for their skilled technical assis- Kharkov VN, Stepanov VA, Borinskaya SA et al (2004) Gene pool
tance. They also thank Marie Lacan for pertinent comments during the structure of eastern Ukrainians as inferred from the Y-chromo-
redaction of the paper, Sophie Lienart for her work and the reviewers some haplogroups. Russ J Genet 40:415–421
for constructive suggestions that help improve the manuscript. Kharkov VN, Stepanov VA, Medvedeva OF et al (2007) Gene pool
diVerences between northern and southern Altaians inferred from
the data on Y-chromosomal haplogroups. Russ J Genet 43:551–
562
References Koryakova LN, Epimakhov AV (2007) The Urals and Western Siberia
in the Bronze and Iron Ages. Cambridge University Press
Anderson S, Bankier AT, Barrell BG et al (1981) Sequence and orga- Kozintsev AG, Gromov AV, Moiseyev VG (1999) Collateral relatives
nisation of the human mitochondrial genome. Nature 290:457– of American Indians among the Bronze Age populations of Sibe-
465 ria? Am J Phys Anthropol 108:193–204
Andrews RM, Kubacka I, Chinnery PF et al (1999) Reanalysis and Kravchenko SA, Slominsky PA, Bets LA et al (2002) Polymorphism
revision of the Cambridge reference sequence for human mito- of STR loci of the Y chromosome in three populations of eastern
chondrial DNA. Nat Genet 23:147 slavs from Belarus, Russia, and Ukraine. Russ J Genet 38:80–86
Bandelt HJ (2005) Mosaics of ancient mitochondrial DNA: positive Lalueza-Fox C, Sampietro ML, Gilbert MTP et al (2004) Unravelling
indicators of nonauthenticity. Eur J Hum Genet 13:1106–1112 migrations in the steppe: mitochondrial DNA sequences from an-
Belyaeva O, Bermisheva M, Khrunin A et al (2003) Mitochondrial cient Central Asians. Proc R Soc Lond B 271:941–947
DNA variations in Russian and Belorussian populations. Hum Lamberg-Karlovsky CC (2002) Archaeology and language. The Indo-
Biol 75:647–660 Iranians. Curr Anthropol 43:63–75
Bouakaze C, Keyser C, Amory S et al (2007) First successful assay of Lebedynsky I (2003) Les nomades. Errance, Paris
Y-SNP typing by SnaPshot minisequencing on ancient DNA. Int Mair VH (2005) Gene, geography and glottochronology: The Tarim
J Legal Med 121:493–499 Basin during late prehistory and history. Journal of Indo-Euro-
Bouakaze C, Keyser C, Crubézy E et al (2009) Pigment phenotype and pean Monograph Series, 50. Institute for the study of man, Wash-
biogeographical ancestry from ancient skeletal remains: infer- ington, DC
ences from multiplexed autosomal SNP analysis. Int J Legal Med Mallory JP, Mair VH (2000) The Tarim mummies. Thames and Hud-
(in press) son, London
Cooper A, Poinar HN (2000) Ancient DNA: do it right or not at all. Sci- Malyarchuk BA (2004) DiVerentiation of the mitochondrial subhaplo-
ence 289:1139 group U4 in the populations of Eastern Europe, Ural, and Western
Derenko M, Malyarchuk B, Denisova GA et al (2006) Contrasting pat- Siberia: implication to the genetic history of the uralic popula-
terns of Y-chromosome variation in South Siberian populations tions. Russ J Genet 40:1281–1287
from Baikal and Altai–Sayan regions. Hum Genet 118:591–604 Melchior L, Gilbert MT, Kivisild T et al (2008) Rare mtDNA haplo-
Eiberg H, Troelsen J, Nielsen M et al (2008) Blue eye color in humans groups and genetic diVerences in rich and poor Danish Iron-Age
may be caused by a perfectly associated founder mutation in a villages. Am J Phys Anthropol 135:206–215
regulatory element located within the HERC2 gene inhibiting Moiseyev VG (2006) Nonmetric traits in early iron age cranial series
OCA2 expression. Hum Genet 123:177–187 from western and southern Siberia. Archaeol Ethnol Anthropol
Gao S, Cui Y, Yang Y et al (2008) Mitochondrial DNA analysis of hu- Eurasia 25:145–152
man remains from the Yuansha site in Xinjiang. China Sci China Passarino G, Semino O, Magri C et al (2001) The 49a, f haplotype 11
Ser C Life Sci 51:205–213 is a new marker of the EU19 lineage that traces migrations from
Gilbert TP, Bandelt HJ, Hofreiter M et al (2005) Assessing ancient northern regions of the Black Sea. Hum Immunol 62:922–932
DNA studies. Trends Ecol Evol 20:541–544 PericiT M, Lauc LB, KlariT IM et al (2005) High-resolution phyloge-
Gimbutas M (1970) Proto-Indo-European culture: the Kurgan culture netic analysis of southeastern Europe traces major episodes of
during the Wfth, fourth and third millennia BC. In: Cardona G, paternal gene Xow among Slavic populations. Mol Biol Evol
Hoenigswald HM, Seen AM (eds) Indo-European and Indo- 22:1964–1975

123
410 Hum Genet (2009) 126:395–410

Pike DA (2006) Phylogenetic networks for the human mtDNA haplo- Underhill PA, Kivisild T (2007) Use of Y chromosome and mitochon-
group T. J Genet Geneal 2:1–11 drial DNA population structure in tracing human migrations.
Ricaut FX, Keyser-Tracqui C, Cammaert L et al (2004) Genetic anal- Annu Rev Genet 41:539–564
ysis and ethnic aYnities from two Scytho-Siberian skeletons. Am Van Geel B, Bokovenko NA, Burova ND et al (2004) Climate change
J Phys Anthropol 123:351–360 and the expansion of the Scythian culture after 850 BC: a hypoth-
Richards M, Macaulay V, Hickey E et al (2000) Tracing European esis. J Archaeol Sci 31:1735–1742
founder lineages in the Near Eastern mtDNA pool. Am J Hum Wells RS, Yuldasheva N, Ruzibakiev R et al (2001) The Eurasian
Genet 67:1251–1276 heartland: a continental perspective on Y-chromosome diversity.
Rosser ZH, Zerjal T, Hurles ME et al (2000) Y-chromosomal diversity Proc Natl Acad Sci USA 98:10244–10249
in Europe is clinal and inXuenced primarily by geography rather Y Chromosome Consortium (2002) A nomenclature system for the
than by language. Am J Hum Genet 67:1526–1543 tree of human Y-chromosomal binary haplogroups. Genom Res
Sampietro ML, Gilbert MTP, Lao O et al (2006) Tracking down 12:339–348
human contamination in ancient human teeth. Mol Biol Evol Yao YG, Kong QP, Bandelt HJ et al (2002) Phylogeographic diVeren-
23:1801–1807 tiation of mitochondrial DNA in Han Chinese. Am J Hum Genet
Semino O, Passarino G, Oefner PJ et al (2000) The genetic legacy of 70:635–651
Paleolithic Homo sapiens sapiens in extant Europeans: a Y chro- Zerjal T, Pandya A, Santos FR (1999) The use of Y-chromosomal
mosome perspective. Science 290:1155–1159 DNA variation to investigate population history: recent male
Smith CI, Chamberlain AT, Riley MS et al (2003) The thermal history spread in Asia and Europe. In: Papiha SS, Deka R, Chakraborty R
of human fossils and the likelihood of successful DNA ampliWca- et al (eds) Genomic diversity: applications in human population
tion. J Hum Evol 45:203–217 genetics. Plenum, New York, pp 91–101
Tömöry G, Csányi B, Bogácsi-Szabó E et al (2007) Comparison of Zerjal T, Wells RS, Yuldasheva N et al (2002) A genetic landscape re-
maternal lineage and biogeographic analyses of ancient and mod- shaped by recent events: Y-chromosomal insights into Central
ern Hungarian populations. Am J Phys Anthropol 134:354–368 Asia. Am J Hum Genet 71:466–482

123