Report

Shifts in the Genetic Landscape of the Western

Eurasian Steppe Associated with the Beginning and
End of the Scythian Dominance
Highlights Authors
d 31 new ancient genomes help compare the Scythians to pre- €rve, Lehti Saag,
Mari Ja
and postdating cultures Christiana Lyn Scheib, ..., Aivar Kriiska,
Toomas Kivisild, Richard Villems
d Scythian dominance brought along an increase of eastern
ancestry across the steppe Correspondence
d Results imply some demic diffusion in the spread of the mari.jarve@ut.ee
Scythian culture
In Brief
d Genetic makeup agrees with the Gothic source of post- Ja€rve et al. present 31 ancient Scythians
Scythian Chernyakhiv culture and samples from pre- and postdating
cultures from the Eurasian Steppe. An
increase of eastern ancestry in the steppe
during the Scythian period supports
some demic diffusion in the spread of the
Scythian culture. The genetic makeup of
post-Scythian Chernyakhiv individuals
agrees with their Gothic source.

€rve et al., 2019, Current Biology 29, 1–12

Ja
July 22, 2019 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.cub.2019.06.019
 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

Current Biology

Report

Shifts in the Genetic Landscape of the Western

Eurasian Steppe Associated with the Beginning
and End of the Scythian Dominance
€rve,1,28,* Lehti Saag,1,2 Christiana Lyn Scheib,1 Ajai K. Pathak,1,2 Francesco Montinaro,1 Luca Pagani,1,3
Mari Ja
Rodrigo Flores,1 Meriam Guellil,1 Lauri Saag,1 Kristiina Tambets,1 Alena Kushniarevich,1 Anu Solnik,1 Liivi Varul,4
Stanislav Zadnikov,5 Oleg Petrauskas,6 Maryana Avramenko,6 Boris Magomedov,6 Serghii Didenko,7 Gennadi Toshev,8
Igor Bruyako,9 Denys Grechko,6 Vitalii Okatenko,10 Kyrylo Gorbenko,11 Oleksandr Smyrnov,11 Anatolii Heiko,12
Roman Reida,6 Serheii Sapiehin,13 Sergey Sirotin,14 Aleksandr Tairov,15 Arman Beisenov,16 Maksim Starodubtsev,17

(Author list continued on next page)

1Estonian Biocentre, Institute of Genomics, University of Tartu, 23b Riia Street, Tartu 51010, Estonia
2Department of Evolutionary Biology, Institute of Molecular and Cell Biology, University of Tartu, 23b Riia Street, Tartu 51010, Estonia
3Department of Biology, University of Padova, Via U. Bassi 58/B, Padova 35121, Italy
4School of Humanities, Tallinn University, 29 Narva Street, Tallinn 10120, Estonia
5Museum of Archaeology, V.N. Karazin Kharkiv National University, 4 Svobody Square, Kharkiv 61022, Ukraine
6Institute of Archaeology, National Academy of Sciences of Ukraine, 12 Heroyiv Stalinhradu Avenue, Kyiv 04210, Ukraine
7National Museum of History of Ukraine, 2 Volodymyrs’ka Street, Kyiv 02000, Ukraine
8Zaporizhzhya National University, 33A Dniprovska Street, Zaporizhzhya 69061, Ukraine
9Odessa Archaeological Museum, 4 Lanzheronivs’ka Street, Odessa 65000, Ukraine
10SC SRC ‘‘Protective Archeological Service of Ukraine,’’ Institute of Archaeology, National Academy of Sciences of Ukraine, 12 Heroyiv

Stalinhradu Avenue, Kyiv 04210, Ukraine

11Mykolaiv V.O. Sukhomlynskyi National University, 24 Nikolska Street, Mykolaiv 54030, Ukraine
12National Museum of Ukrainian Pottery in Opishne, 102 Partyzanska Street, Opishne 38164, Ukraine
13Anton Makarenko Museum, Poltava Regional Makarenko Scientific Lyceum, 1-2 Makarenko Lane, Kovalivka 38701, Ukraine
14Institute of Archaeology, Russian Academy of Sciences, 19 Dmitri Ulyanov Street, Moscow 117292, Russia
15South Ural State University, 76 Lenin Avenue, Chelyabinsk 454080, Russia
16A. Kh. Margulan Institute of Archaeology, 44 Dostyk Avenue, Almaty 480100, Kazakhstan
17Sterlitamak Museum of Local History, 100 Karl Marx Street, Sterlitamak 453124, Russia
18LoCom Medien Akademie Europa €isches Bildungsinstitut, Bachstraße 4, Bonn 53115, Germany

(Affiliations continued on next page)

SUMMARY the Chernyakhiv culture and support the hypothesis

The Early Iron Age nomadic Scythians have been
described as a confederation of tribes of different or-
igins, based on ancient DNA evidence [1–3]. It is still
unclear how much of the Scythian dominance in the
Eurasian Steppe was due to movements of people
and how much reflected cultural diffusion and elite
dominance. We present new whole-genome se-
quences of 31 ancient Western and Eastern Steppe
individuals, including Scythians as well as samples
pre- and postdating them, allowing us to set the
Scythians in a temporal context (in the Western,
i.e., Ponto-Caspian Steppe). We detect an increase
of eastern (Altaian) affinity along with a decrease in
eastern hunter-gatherer (EHG) ancestry in the Early
Iron Age Ponto-Caspian gene pool at the start of
the Scythian dominance. On the other hand, samples
of the Chernyakhiv culture postdating the Scythians
in Ukraine have a significantly higher proportion of
Near Eastern ancestry than other samples of this
study. Our results agree with the Gothic source of
that the Scythian dominance did involve a demic
component.
RESULTS AND DISCUSSION
The Eurasian Steppe is known to have been the route of many
(pre)historic population movements. After the massive migra-
tions of the Early Bronze Age (Yamnaya culture;
6,000–4,300
before present [BP]) [4, 5], active movements of people in the
Steppe resumed in the Early Iron Age with the formation of the
extensive and influential Scythian culture (
2,800–2,200 BP)
[6]. At its peak, it was spread in the Eurasian Steppe from Central
Asia across Southern Russia and Ukraine to Central Europe.
However, the origins of the Scythians are still obscure.
The question whether the ‘‘classical’’ Scythians of the circum-
Pontic region formed as a conglomerate of local tribes or as a
result of migrations from the east has puzzled historians since
the ancient Greek historian Herodotus [7]. Arguments for their
local origins link the formation of the Scythians in both the steppe
and forest-steppe regions to the Bronze Age Srubnaya culture
[8, 9]. However, since the 1970s, migration-based explanations
have been prevalent, with the region of Scythian origin mostly

Current Biology 29, 1–12, July 22, 2019 ª 2019 Elsevier Ltd. 1
 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

Vitali Vasilev,18 Alexei Nechvaloda,19 Biyaslan Atabiev,20 Sergey Litvinov,21 Natalia Ekomasova,21,22
Murat Dzhaubermezov,21,22 Sergey Voroniatov,23 Olga Utevska,24 Irina Shramko,5 Elza Khusnutdinova,21,22
Mait Metspalu,1 Nikita Savelev,19,27 Aivar Kriiska,25,27 Toomas Kivisild,1,26,27 and Richard Villems1,2,27
19Institute of History, Language and Literature, Ufa Federal Research Centre of the Russian Academy of Sciences, 71 October Avenue, Ufa
450054, Russia
20Institute for Caucasus Archaeology, 30 Katkhanova Street, Nalchik 361401, Russia
21Institute of Biochemistry and Genetics, Ufa Federal Research Centre of the Russian Academy of Sciences, 71 October Avenue, Ufa 450054,

Russia
22Department of Genetics and Fundamental Medicine, Bashkir State University, 32 Zaki Validi Street, Ufa 450076, Russia
23Department of Archaeology of Eastern Europe and Siberia, State Hermitage Museum, 34 Dvortsovaya Embankment, St. Petersburg

190000, Russia
24Department of Genetics and Cytology, V.N. Karazin Kharkiv National University, 4 Svobody Square, Kharkiv 61022, Ukraine
25Department of Archaeology, Institute of History and Archaeology, University of Tartu, 2 Jakobi Street, Tartu 51014, Estonia
26Department of Human Genetics, KU Leuven, O&N IV Herestraat 49, Leuven 3000, Belgium
27Senior author
28Lead Contact

*Correspondence: mari.jarve@ut.ee
https://doi.org/10.1016/j.cub.2019.06.019

being sought in Central Asia [10]. Craniometric evidence sug-
gests a lack of continuity with the Bronze Age population both
in the steppe region Scythians north of the Black Sea [11] and
in carriers of the Scythian culture from the Ural Mountains to Altai
and South Siberia, with migrations from Central Asia cited as a
possible explanation [12–15].
Recently, studies of ancient Scythian genomes have affirmed
the confederate nature of the Scythian tribes, showing them to
be genetically distinct from one another but finding little or no
support for large-scale east-to-west movements, instead gener-
ally suggesting separate local origins of various Scythian groups
[1–3]. Because the impact of the dominance of Scythians and
closely related Sarmatians on the Ponto-Caspian genetic land-
scape is still unclear, we aim to evaluate it by comparing ancient
Scythians and Sarmatians to individuals from cultures preceding
and succeeding them in the region.
We have extracted DNA and performed whole-genome
shotgun sequencing of 31 ancient samples from the Eurasian
Steppe (Tables S1 and S2; Figure 1; STAR Methods).
For a general genetic characterization, we first performed prin-
cipal component and ADMIXTURE analyses, which confirmed
the confederate nature of Scythian tribes shown by previous
studies [1, 3]: all five spatiotemporal groups—Ukrainian Bronze
Age (Ukr_BA; 2 samples), Ukrainian Scythians (Scy_Ukr; 9 sam-
ples), Ukrainian post-Scythian Chernyakhiv culture (Chern; 3
samples), Scythians and Sarmatians from the Southern Urals
(ScySar_SU; 8 samples), and Scythians from Kazakhstan (Scy_
Kaz, 4 samples)—were clearly distinct.
The PC plot of the samples of this study together with pub-
lished Scythian and Sarmatian and related ancient samples (Ta-
ble S3) projected onto a background of modern populations (for
which only the median coordinates are shown; Figure 2) revealed
that ancient samples generally did not overlap with modern vari-
ation. Although the ScySar_SU samples (except one) and the
single Caucasus Sarmatian clustered rather compactly together
with Scythian and Sarmatian samples from several studies [1, 3,
16] and the Ukrainian Bronze Age samples positioned just
outside modern European variation, the Ukrainian Scythians
formed a cline between these two groups. Compared to other
groups, Scy_Kaz were more spread out (likely partially due to
the very low coverage of two of the Scy_Kaz samples) and drawn
toward East Asian populations, although they still positioned
‘‘west’’ of the Central Saka [1]—despite originating from neigh-
boring burial mounds of the same Tasmola culture—and most
of the Eastern Scythians [3], who are themselves a very hetero-
geneous group both culturally and genetically. On the other
hand, the Chernyakhiv samples overlapped with modern Euro-
peans, representing the most ‘‘western’’ range of variation
among the groups of this study (Figure 2).
This study’s Scythian and Sarmatian samples did not overlap
with any published Bronze Age samples from the Eurasian
Steppe, being clearly more ‘‘eastern’’ than a dense cluster of
mostly Middle–Late Bronze Age populations from various
studies [2, 16–18] at the edge of modern European variation
(Figure S1).
At K = 5, ADMIXTURE analysis revealed three major ancestry
components in all five groups considered: (1) one prevalent in
European hunter-gatherer (HG) samples (dark blue); (2) one
prevalent in Near Eastern Natufian and Neolithic samples (light
blue); and (3) one prevalent in modern and ancient South Asians
(green; Figure S2). However, all samples of this study also
possessed at least one additional eastern component, one of
which was nearly at 100% in modern Nganasans (orange) and
the other in modern Han Chinese (yellow; Figure S2). The eastern
components were present in variable proportions in the samples
of this study, mostly at lower frequencies than the three above-
mentioned components but noticeably higher in two of the four
Scy_Kaz samples and in one ScySar_SU sample. The latter sam-
ple, MJ-42 (UT-4-16), had a higher eastern affinity than other
members of its group in all analyses, as well as being dated as
one of the oldest in its group (Table S1), and although it
geographically originates from the Southern Urals, burial cus-
toms link it to the Tasmola culture from Central Kazakhstan
[19, 20]. Such outliers can be observed in this as well as pub-
lished Scythian studies [1–3], suggesting that (1) far-reaching
contacts existed between different Scythian groups and (2) it
may not be scientifically sound to exclude such samples simply
as ‘‘outliers,’’ as they may in fact accurately characterize the
composition of the nomadic groups. This also agrees with
isotope analysis results from Scythian sites in Ukraine, pointing

2 Current Biology 29, 1–12, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019
to only a few individuals engaging in pan-regional mobility,
although localized mobility as part of an agricultural lifestyle
was more common [21].
To further investigate their ancestry, we modeled Scythians
and Sarmatians, as well as samples from cultures pre- and post-
dating them, from this study and published sources (Table S3;
STAR Methods), using qpAdm and the ChromoPainter/NNLS
pipeline. We selected the sources as eastern hunter-gatherer
(EHG) + Natufian + Altaian and used Yoruba, Ust-Ishim, Kos-
tenki, Mal’ta, and Han as outgroups in qpAdm. Modern Altaians
were selected as the proxy for the East Asian component in
Scythians and Sarmatians due to (1) our f4 test of the relative af-
finity of our studied groups to the suggested Siberian Bronze Age
HG (Glazkovo) [1] or to modern Altaians showing no clear affinity
to either (Data S1), (2) our f4 results additionally demonstrating
consistently higher affinity of Scythians and Sarmatians to Al-
taians compared to Han, and in most cases also to Altaians
compared to Nganasans (Data S1), and (3) bearing in mind the
putative Altaian origin of Scythians that has been suggested
numerous times. The same distal model was tested with CP/
NNLS for each individual, and the results were subsequently
averaged across each sample group.
Overall, both methods provided similar (Pearson r2 > 0.9;
p < 0.01) results (Figure 3), highlighting an increase in
eastern—Altaian—ancestry in Scythians and Sarmatians
(except the Hungarian Scythians that behave as outliers)
compared to populations predating them. The presence of
Altaian ancestry was more pronounced in eastern Scythian
groups, the highest proportion among the sample groups of
this study found in Scy_Kaz, surpassed by published groups
like Eastern Scythians [3] and Central Saka [1] (Figure 3). Among
post-Scythian groups, Late Sarmatians from the Southern Urals
Figure 1. Map and Timeline of the Samples
of This Study
The approximate area of the Eurasian Steppe is
denoted in light green. See also Table S1.
[2] were indistinguishable from Scythians
and Sarmatians, and Chern from Ukraine
(this study) represented the only instance
of discordance between the two
methods. Although both methods
showed them having a lower proportion
of Altaian and higher percentage of Natu-
fian ancestry compared to the Scythians
preceding them, the differences were
much more pronounced according to
CP/NNLS that showed the Chernyakhiv
individuals lacking the Altaian component
entirely (Figure 3).
We further evaluated the contribution of
more recent groups to Scythians and Sar-
matians and populations postdating them
in two proximal models with the sources
of Yamnaya/Srubnaya + Central Euro-
pean Middle Neolithic + Altaians (Data
S1). Analysis of CP/NNLS residuals [23]
suggested that Yamnaya is a better proxy for the population
contributing to Scythians and Sarmatians (Data S1). Some tar-
gets required additional sources in qpAdm for the proximal
models (Data S1), and we prefer the distal model for our studied
groups due to consistent fitting of all our targets. However, both
methods again generally registered a higher proportion of the Al-
taian component in Scythians (except the western groups) and
Sarmatians, but not in the post-Scythian Chernyakhiv individuals
(Data S1).
In order to determine to which extent the proportion of the Al-
taian component depended simply on geography, we correlated
the qpAdm and CP/NNLS distal model estimates of ‘‘Altaian-
ness’’ with great circle distance from Altai (Figure 4). For Scyth-
ians and Sarmatians, we did observe a linear relationship of
decreasing proportion of the Altaian component with increasing
distance from Altai for both methods. However, pre-Scythian
Steppe populations had a noticeably lower proportion of the
Altaian component than would be predicted by their distance
from Altai (except Cimmerians who have been shown previously
to already carry eastern genetic ancestry despite predating the
Scythians) [2], and the same was true for the post-Scythian
Chernyakhiv culture samples when considering the results of
CP/NNLS (Figure 4). This is compatible with a moderate west-
ward increase of the Altaian genetic component in the Steppe
during the Scythian period, implying the involvement of at least
some degree of migration (east to west; the more complicated
scenarios that have been proposed [11] are not supported by
our results) in the spread of the Scythian culture. This fits the pre-
vious observation that the Iron Age nomads of the western
Eurasian Steppe were not direct descendants of the Bronze
Age population [2] and suggests that the ‘‘Scythian world’’
cannot be described solely in terms of material culture. However,
Current Biology 29, 1–12, July 22, 2019 3
 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

Figure 2. Principal Component Plot of the Samples of This Study Together with Published Scythian, Sarmatian, and Related Samples on a
Background of Modern Eurasians
Principal component analysis (PCA) obtained by projecting the ancient samples onto the eigenvectors calculated based on 537,802 autosomal SNPs in 1,422
modern Eurasians. To improve readability, the modern populations have been plotted as population medians (after outlier removal). Ancient individuals (in color)
are as follows: circles, samples from [1]; square, sample from [16]; triangles, samples from [3]; rectangles, samples from [2]; and diamonds, samples from this
study. Modern populations (in gray) are as follows: Europe: Bsh, Bashkirs; Blr, Belarusians; Bos, Bosnians; Bul, Bulgarians; Chv, Chuvash; Cyp, Cypriots; Est,
Estonians; Fin, Finns; Fre, French; FrB, French Basques; Gag, Gagauzes; Ger, Germans; Grk, Greeks; Hun, Hungarians; Kar, Karelians; Kom, Komis; Lat,
Latvians; Lit, Lithuanians; Mac, Macedonians; Mar, Maris; Mrd, Mordovians; Nit, North Italians; Orc, Orcadians; Pol, Poles; Rom, Romanians; NRu, Northern
Russians; CRu, Central Russians; SRu, Southern Russians; Saa, Saami; Sar, Sardinians; Srb, Serbians; Slk, Slovaks; Sln, Slovenians; Swe, Swedes; Ttr, Tatars;
Tus, Tuscans; Udm, Udmurts; Ukr, Ukrainians; Vep, Vepsas; Siberia: Alt, Altaians; Bur, Buryats; Chk, Chukchis; Dol, Dolgans; Evk, Evenkis; Evn, Evens; Ket, Kets;
Khk, Khakases; Kha, Khanty; Kor, Koryaks; Mon, Mongolians; Nen, Nenets; Ngn, Nganasans; Sel, Selkups; Shr, Shors; Tuv, Tuvinians; Yak, Yakuts; Ykg, Yu-
kaghirs; East Asia: Dau, Daur; Han, Han; Hzh, Hezhen; Jap, Japanese; Mgl, Mongola; Orq, Oroqens; Tu, Tu; Uyg, Uygurs; Xib, Xibo; Central Asia: Irn, Iranians;
Kaz, Kazakhs; Krd, Kurds; Kyr, Kyrgyzians; Psh, Pashtun; Tjk, Tajiks; Trm, Turkmens; Uzb, Uzbeks; Near East: Jor, Jordanians; Leb, Lebanese; Pal, Palestinians;
Syr, Syrians; Tur, Turks; Caucasus: Abh, Abkhasians; Ady, Adygei; Arm, Armenians; Aze, Azeris; Blk, Balkars; Che, Chechens; Grg, Georgians; Kal, Kalmyks;
Kum, Kumyks; Lzg, Lezgins; Nog, Nogais; NOs, North Ossetians. See also Figure S1 and Table S3.

we see no evidence that visible morphological features that
could flag macro-geographic affiliations played a major role in
introducing or maintaining the eastern component within the
sampled populations through socio-cultural dynamics (Table
S4; STAR Methods).
Outgroup f3 and D statistics’ results (Data S1; Figure S3)
showed that Chern samples shared more drift with modern
Near Eastern populations than either Ukr_BA or Scy_Ukr, but
their affinity to ancient populations with a significant Near
Eastern component only differed from that of Ukr_BA—Chern
and Scy_Ukr were both closer to European early farmers than
Ukr_BA, but Chern did not exhibit any difference from preceding
Scythians. A comparison of Western and Eastern Scythians
revealed them to be mainly influenced by their geographic differ-
ences: Scy_Kaz shared more drift with most of the forest or
steppe region Iron and Middle Age populations and Scy_Ukr
had closer affinity to almost all ancient European populations
included in the analyses. Furthermore, Scy_Ukr were closer to
modern Near Eastern populations and Scy_Kaz to modern pop-
ulations from Siberia and East Asia.
The higher proportion of Near Eastern and (according to CP/
NNLS) lower proportion of eastern ancestry in the Chernyakhiv
culture samples were mirrored by f4 analyses, where Chern
showed lower affinity to Han compared to Scy_Ukr (Z score
3.097) and to EHG compared to Ukr_BA (Z score 3.643), as
well as higher Near Eastern (Levant_N and Anatolia_N) affinity
than Scy_Ukr (Z scores 4.696 and 3.933, respectively; Data
S1). It is plausible to assume that this excess Near Eastern
ancestry in Chern is related to European populations whose
Near Eastern proportion has exceeded that in the steppe since
the Neolithic expansion of early farmers. This is further confirmed
by the qpAdm and CP/NNLS proximal models, in which the

4 Current Biology 29, 1–12, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

A B C

Figure 3. Inferred Ancestry Proportions in Scythian and Sarmatian Groups and Groups Pre- and Postdating Them
(A and B) Proportions of eastern hunter-gatherer (EHG) (blue), Natufian (red), and Altaian (green) ancestries inferred using the (A) qpAdm and (B) ChromoPainter/
NNLS method.
(C–E) Correlation of qpAdm and CP/NNLS proportions for the three putative sources—EHG (C), Natufian (D) and Altaian (E)—evaluated. Steppe populations
predating the Scythians are as follows: Yamnaya [22]; Yamnaya_Samara; Yamnaya_Kalmykia; Srubnaya [16]; Yamnaya_Ukraine [17]; Srubnaya-Alakulskaya;
Cimmerian [2]; and Ukr_BA (this study). Scythians and Sarmatians are as follows: Nomad_IA [16]; Scythian_East; Sarmatian_SU [3]; HungarianScythian; Sar-
matian; CentralSaka; TianShanSaka; Tagar [1]; Scythian_PC [2]; Scy_Ukr; ScySar_SU; and Scy_Kaz (this study). Populations postdating the Scythians are as
follows: LateSarmatian [2] and Chern (this study).
See also Data S1 and Table S3.

Chern samples were characterized by a high proportion (18%–
45%) of Central European Middle Neolithic (Data S1). Although
the Chernyakhiv culture was likely ethnically heterogeneous
[24–27], the three samples in our Chern group appear to repre-
sent its Gothic component.
Several aspects regarding the Scythians are evident from
the various analyses employed here. First, the ‘‘Scythian-Sar-
matian world’’ consisted of a confederation of local groups
whose genetic differences can largely be explained by geogra-
phy. Second, although distance from Altai is the main contrib-
utor to the proportion of Altaian ancestry in Scythians and
Sarmatians, ancient populations preceding the Scythians
(e.g., Yamnaya) exhibit less Altaian ancestry than would be
predicted by this geographic correlation, indicating that Scyth-
ians have a higher eastern affinity than preceding steppe pop-
ulations. This lends support to at least some degree of demic
diffusion being involved in the spread of the Scythian cultural
and military dominance. Third, after the end of the Scythian
period in the western Eurasian Steppe, the Chernyakhiv cul-
ture samples have higher Near Eastern affinity compared to
the Scythians preceding them, agreeing with the Gothic
component in the multi-ethnic mix of the Chernyakhiv culture
[24–27], although no such post-Scythian genetic shift is de-
tected further east, in Late Sarmatians [2] from the Southern
Urals.
Although the genetic composition of the Scythian world has
been described from its easternmost to westernmost reaches
[1, 3] and advances have been made in clarifying the transitions
to and from the Scythian culture, further characterization of the
groups preceding and succeeding the Scythians across their
wide distribution area is needed to better understand the impact
of this well-known culture in a temporal context. Ongoing syn-
thesis of archaeology, historical linguistics, and aDNA research
promises further progress in understanding the switch from
Indo-Iranian to largely Turkic-speaking pastoral nomadism in
the Eurasian Steppe as well as the interactions of the steppe
and northern forest zone populations.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Ancient samples and their grouping
B Archaeological background of the samples
d METHOD DETAILS
B DNA extraction

Current Biology 29, 1–12, July 22, 2019 5

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

Figure 4. Correlation of the Altaian Ancestry Proportion and Great Circle Distance from Altai
Proportion of Altaian ancestry as estimated by qpAdm (dashed line) and ChromoPainter/NNLS (CP, solid line). preScy, steppe populations predating the
Scythians; Scy, Scythians and Sarmatians; postScy, populations postdating the Scythians—see Figure 3. See also Data S1 and Table S3.

B Library preparation 2020 grant no. 810645. We thank the University of Tartu Development Fund
B DNA sequencing for support to the Collegium for Transdisciplinary Studies in Archaeology, Ge-
d QUANTIFICATION AND STATISTICAL ANALYSIS netics, and Linguistics. O.U. was supported by the Ministry of Education and
Science of Ukraine grant 0117U004836. K.G. and O.S. were supported by
B Mapping
the grant no. 0118U003394 ‘‘Comprehensive study of the archaeological sites
B aDNA authentication of the lower reaches of the Bug River 3rd millennium BC – 15th century (conser-
B Calculating general statistics and determining genetic vation aspect).’’ N.E. was supported by grant no. 19-04-01195 of the Russian
sex Foundation for Basic Research, and E.K. was supported by the Program for
B Variant calling Supporting Bioresource Collections grant no. 007-030164/2. The study was
B Determining mtDNA haplogroups performed as part of the assignment of the Ministry of Science and Higher Ed-
ucation of the Russian Federation (no. AAAA-A16-116020350032-1).
B Y chromosome variant calling and haplotyping
B Preparing the datasets for autosomal analyses
AUTHOR CONTRIBUTIONS
B Principal component analysis
B ADMIXTURE Conceptualization, M.J., S.Z., I.S., N.S., A. Kriiska, T.K., and R.V.; Software,
B Population modeling and geographic correlation Lehti Saag, C.L.S., L.P., R.F., and Lauri Saag; Validation, M.J., Lehti Saag,
B Outgroup f3 and D statistics and C.L.S.; Formal Analysis, M.J., Lehti Saag, C.L.S., A.K.P., F.M., L.P.,
B f4 statistics R.F., M.G., and T.K.; Investigation, M.J., Lehti Saag, C.L.S., K.T., A. Kushniar-
B Inspecting sites highly differentiated between modern evich, A.S., and L.V.; Resources, S.Z., O.P., M.A., B.M., S.D., G.T., I.B., D.G.,
V.O., K.G., O.S., A.H., R.R., S. Sapiehin, S. Sirotin, A.T., A.B., M.S., V.V., A.N.,
Europeans and East Asians
B.A., S.L., N.E., M.D., O.U., I.S., and E.K.; Data Curation, M.J., Lehti Saag,
B Metagenomic screening for pathogen reads C.L.S., and Lauri Saag; Writing – Original Draft, M.J., Lehti Saag, A.K.P.,
d DATA AND CODE AVAILABILITY F.M., L.P., M.G., S.V., N.S., A. Kriiska, and T.K.; Writing – Review & Editing,
M.J., Lehti Saag, C.L.S., F.M., L.P., R.F., K.T., O.P., M.A., A.H., R.R., S. Sapi-
SUPPLEMENTAL INFORMATION ehin, S.L., M.M., A. Kriiska, T.K., and R.V.; Visualization, M.J., Lehti Saag,
A.K.P., F.M., L.P., and R.F.; Supervision, M.M., N.S., A. Kriiska, T.K., and
Supplemental Information can be found online at https://doi.org/10.1016/j. R.V.; Project Administration, M.J. and A. Kriiska; Funding Acquisition, K.T.,
cub.2019.06.019. A. Kushniarevich, K.G., O.S., N.E., O.U., E.K., M.M., A. Kriiska, T.K., and R.V.

ACKNOWLEDGMENTS DECLARATION OF INTERESTS

This work was supported by institutional research funding (IUT24-1 and IUT20- The authors declare no competing interests.
7) from the Estonian Ministry of Education and Research; by the Estonian
Research Council grants PUT1217, PRG243, and PUT1339; by the EU Received: February 23, 2019
European Regional Development Fund research projects 2014-2020. Revised: May 3, 2019
4.01.16-0030, 2014-2020.4.01.16-0125, 2014-2020.4.01.15-0012, 2014- Accepted: June 7, 2019
2020.4.01.16-0771, and 2014-2020.4.01.16-0024; and by the EU Horizon Published: July 11, 2019

6 Current Biology 29, 1–12, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019
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12 Current Biology 29, 1–12, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Biological Samples
Human archaeological remains This paper N/A
Critical Commercial Assays
MinElute PCR Purification Kit QIAGEN Cat No./ID: 28006
NEBNext DNA Library Prep Master New England Biolabs E6070
Mix Set for 454
Deposited Data
Human reference genome NCBI build 37, Genome Reference http://www.ncbi.nlm.nih.gov/projects/genome/
GRCh37 Consortium assembly/grc/human/
Modern comparison dataset [28] http://evolbio.ut.ee/khazar/
Modern comparison dataset [29] https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE21478
Modern comparison dataset [30] http://evolbio.ut.ee/afghan/
Modern comparison dataset [31] https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE46828
Modern comparison dataset [32] http://evolbio.ut.ee/balkan/
Modern comparison dataset [33] http://evolbio.ut.ee/slavic/
Modern comparison dataset [34] http://hagsc.org/hgdp/files.html
Modern comparison dataset [35] https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE50727
Modern comparison dataset [36] https://www.ncbi.nlm.nih.gov/sra/?term=SRA010102
Modern comparison dataset [37] https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?
acc=GSE108646
Modern comparison dataset [38] http://evolbio.ut.ee/caucasus/
Modern comparison dataset [39] http://evolbio.ut.ee/turkic/
Ancient comparison dataset [1] https://www.ebi.ac.uk/ena/data/search?query=PRJEB20658
Ancient comparison dataset [2] https://www.ebi.ac.uk/ena/data/view/PRJEB27628
Ancient comparison dataset [3] https://www.ebi.ac.uk/ena/data/view/PRJEB18686
Ancient comparison dataset [16] https://reich.hms.harvard.edu/datasets
Ancient comparison dataset [18] https://reich.hms.harvard.edu/datasets
Ancient comparison dataset [22] https://www.ebi.ac.uk/ena/data/view/PRJEB26349;https://
www.ebi.ac.uk/ena/data/view/PRJEB25389
Ancient comparison dataset [17] https://reich.hms.harvard.edu/datasets
Ancient comparison dataset [40] erj35@cam.ac.uk
Ancient comparison dataset [41] mittnik@shh.mpg.de
Ancient comparison dataset [42] http://www.ebi.ac.uk/ena/data/view/PRJEB21037
European Steppe aDNA data This paper http://evolbio.ut.ee/; http://www.ebi.ac.uk/ena/data/view/
PRJEB32764
Oligonucleotides
NEBNext Multiplex Oligos for Illumina New England Biolabs E7335
Software and Algorithms
cutadapt [43] https://cutadapt.readthedocs.io/en/stable/#
Burrows-Wheeler Aligner (BWA) [44] http://bio-bwa.sourceforge.net/
samtools [45] http://samtools.sourceforge.net/
picard N/A http://broadinstitute.github.io/picard/index.html
GATK [46] https://software.broadinstitute.org/gatk/
(Continued on next page)

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 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
mapDamage [47] https://ginolhac.github.io/mapDamage/
mtDNA contamination estimation [40] N/A
ANGSD [48] http://www.popgen.dk/angsd/index.php/ANGSD
sex identification algorithm [49] http://www.sciencedirect.com/science/article/pii/
S0305440313002495
HaploGrep2 [50, 51] http://haplogrep.uibk.ac.at
Haplofind [52] http://www.haplofind.unibo.it
BEDTools [53] http://bedtools.readthedocs.io/en/latest/
PLINK [54] http://pngu.mgh.harvard.edu/purcell/plink/
EIGENSOFT [55] https://github.com/DReichLab/EIG
ADMIXTURE [56] https://www.genetics.ucla.edu/software/admixture/
ADMIXTOOLS [57] https://github.com/DReichLab/AdmixTools
ChromoPainter/NNLS pipeline [58–60] N/A
ParDre [61] https://sourceforge.net/projects/pardre/
Kraken2 [62] https://ccb.jhu.edu/software/kraken2/
Metaphlan2 [63] http://huttenhower.sph.harvard.edu/metaphlan2

LEAD CONTACT AND MATERIALS AVAILABILITY

€rve
Requests for further information, resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mari Ja
(mari.jarve@ut.ee).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Ancient samples and their grouping

We extracted DNA and performed whole genome shotgun sequencing of 31 ancient samples from the Eurasian Steppe (Tables S1
and S2; Figure 1). The samples were radiocarbon dated at the Poznan  Radiocarbon Laboratory in Poland, the dates were calibrated
with OxCal v4.3.2 using the IntCal13 atmospheric curve [64–66]. The oldest sample (Yamnaya) was dated to 4,800–4,400 cal BP and
the youngest (Chernyakhiv) to 1,700–1,400 cal BP, but most to 2,700–2,100 cal BP (Table S1). Based on both geographic and (in case
of Ukrainian samples) temporal affiliation, we have divided the samples into 6 groups for the purpose of downstream analyses: Ukrai-
nian Bronze Age (Ukr_BA, 2 samples), Ukrainian Scythians (Scy_Ukr, 9 samples), Ukrainian post-Scythian Chernyakhiv culture
(Chern, 3 samples), Scythians and Sarmatians from the Southern Urals (ScySar_SU, 8 samples), and Scythians from Kazakhstan
(Scy_Kaz, 4 samples). Five samples are not included in group-based analyses, but rather presented individually: the single Sarmatian
from North Caucasus (Sar_Cau), a Late Srubnaya sample from Ukraine whose dating does not allow grouping with the Bronze Age
samples, and three Cimmerians from Ukraine that are genetically too heterogeneous to form a group.

Archaeological background of the samples

Ukrainian Bronze Age (group Ukr_BA)
Early Bronze Age was a period of active population movements and interactions in the Eurasian Steppe. It is classified as the Yam-
naya (Pit-Grave) historical-cultural community, dated to ca. 4000-2300 BC, with the classical period distinguished between 3300-
2600 BC [4]. The Yamnaya phenomenon was spread across a huge territory in the Eurasian Steppe and in some places forest-steppe
zone. Its core area stretched from the Ural Mountains to the Dnieper River region, but its periphery extended from Western Siberia in
the east to Central Europe in the west [67–69]. The origin of the cultural complex has been linked to the region between the Urals and
the Dnieper River, with the Eneolithic Khvalynsk and Srednestogovskaya cultures proposed as its basis; from there it spread likely as
a result of migrations [69]. Regional differences can be observed in material culture [69]. The mostly nomadic Yamnaya culture people
largely engaged in animal husbandry, in some regions also crop farming, with additional food provided by fishing [70]. For moving
about, they also used wagons, parts of which have been found at burial sites [71]. The main type of sites from this period are burial
sites – deep rectangular graves where the dead were buried in a crouched position and that were then covered with a mound –, rela-
tively few settlement sites are known [69]. There were age- and sex-related differences in burial customs [72]. This study includes one
sample from the Kumy burial site of the Bronze Age Yamnaya culture, from the territory of modern Ukraine.
In Middle Bronze Age, the so-called Catacomb historical-cultural community with a number of local versions, dated to ca
2600-2000 cal BC, is distinguished in the European steppe and forest-steppe zone from the vicinity of the Ural Mountains to the
mouth of the Donau River (only the Middle Don cultural group extended significantly into the forest-steppe) [73]. Also from this period,

e2 Current Biology 29, 1–12.e1–e10, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

mainly burial sites are known, settlement sites and hoards less often [74]. The people mostly led a nomadic, in some places also
(partly) settled life. Besides animal husbandry and, in the forest-steppe regions, also crop farming, some of the food was obtained
by fishing and hunting [73]. The dead were buried in a crouched position into burial chambers under mounds. The burial chambers are
called catacombs, which has given the name to this cultural phenomenon. This study includes one sample from the Mamai-Gora
burial site of the Catacomb culture.
Ukrainian Iron Age (group Scy_Ukr)
At the beginning of the Iron Age, large-scale population movements took place in the territory of present-day Ukraine, resulting in a
number of transitions in the settlement pattern of both the steppe and forest-steppe regions. At the end of the 9th century BC the
peoples inhabiting the left bank of the Dnieper River (Late Srubna and Bondarikhinska culture) came under pressure from likely
nomadic groups (probably Cimmerians) and left the area. For a time the region seems to have been inhabited only by a small nomadic
population [75]. At the same time the Chernoles culture continued in the forest-steppe region of the right bank of the Dnieper River,
but likely it also took in immigrants from the southeast [76]. In the second half of the 8th century BC some people who had so far in-
habited the forest-steppe of the right bank of the Dnieper River relocated to the middle reaches of the Vorskla River in the left bank of
the Dnieper River region [77–80]. From the last quarter of the 8th century to the middle of the 6th century BC both regions mentioned
above were stably inhabited by agriculturalists, confirmed around the middle reaches of the Vorskla River by discovered settlement
sites [80–82]. Elsewhere in the Dnieper-Donetsk forest-steppe, only the mounds of nomadic people are known. Settlement sites and
hill forts are absent there until the middle or the last quarter of the 6th century BC [80, 82, 83].
In the second half of the 7th century BC, the right bank of the Dnieper River, the Posulʹe and Povorskl’ya region, and the western
part of the Severskiy Donets drainage basin were invaded by nomads (Cimmerians and probably Scythians), some of whom had likely
participated in military campaigns to the Near East. There is reason to suppose that a military-political alliance was formed between
the local settled population and the nomads, at the behest of the latter [84], with the Trakhtemirovskoye hill fort built in the region of
the right bank of the Dnieper River [85–88] and the Nemirovskoye hill fort built by the Bug River [89]. Meanwhile in the Dnieper-Do-
netsk forest-steppe, there is no undisputed evidence of hill forts from this period, likely indicating the absence of danger from neigh-
bors, primarily from the steppe.
Profound changes occurred in the middle of the 6th century BC, when the region experienced a notable influx of agricultural people
who populated almost the entire Dnieper-Donetsk forest-steppe. At the same time, many hill forts were built in the forest-steppe Scy-
thia, and in the Vorskla River drainage basin, the Belskoye hill fort with a number of separate fortifications was constructed [77–79, 90,
91], which flourished in the following decades. This hill fort became the economic, political and religious center of the forest-steppe
Scythia [90], the place where contacts with the western forest-steppe, the Thrace and Illyria regions, the North Mediterranean centers
of the antiquity, but also with nomads from the steppe region were established [91–95]. The populace of the hill fort was characterized
by a complex social structure [96]. Burial customs reveal stratification of the society: the differentiation of a wealthy aristocratic elite
(nobility) already in the early Scythian period [93]. According to a number of researchers, the Belskoye hill fort may be the city of Gelon
mentioned in the ‘‘Histories’’ by Herodotus – e.g [90, 97].
The development of the agricultural settlement of the forest-steppe was influenced in the 6th century BC by the appearance of new
eastern nomadic groups through the regions of Volga, Predkavkaz’ye and the northern coast of the Black Sea. The settlement of the
region flourished from the middle of the 6th to the beginning of the 5th century BC [98].
In the second half of the 6th century BC, many settlements, both fortified and open (East Belskoye, Kolomakskoye, Lyubotinskoye,
Tsirkunovskoye, etc.), together with associated burial sites, were built in the region of the left bank of the Dnieper River. Many hill forts
became centers of craftsmanship. Meanwhile many earlier settlements founded in the early Scythian period were abandoned. The
lack of danger from the steppe favored bartering between the local population and the towns of the antiquity on the northern coast of
the Black Sea.
At the end of the first third of the 5th century BC the peaceful development of the forest-steppe region was interrupted by the bellig-
erence of the Scythians who had risen to dominance in the southern part of East Europe already at the end of the previous century.
The spread of agricultural settlement was somewhat diminished and nomadic burial sites appeared in the forest-steppe region [84,
99–101]. Possibly the local military elite acknowledged the Scythian dominance and the inhabitants of the region were incorporated
into the confederation of tribes often called Great (Classic) Scythia. Likely the forest-steppe warriors participated in the military ac-
tions of the steppe peoples and received part of the spoils. Changes in agriculture are also visible, the role of animal husbandry
increased significantly, as shown among other things by settlements being short-term and located on river banks.
In the steppe region north of the Black Sea the Scythian culture is represented from the second half of the 7th century BC by burial
grounds of mounds [102]. By the 5th century BC, the number of burials has more than doubled, but most burials date to the 4th century
BC [102]. The ‘‘tsarskiye kurgany’’ (royal mounds) and burials of Scythian nobility in the steppe region north of the Black Sea are dated
to the end of the 5th century and to the 4th century BC [102–104], the Mamai-Gora burial site of common Scythians (more than 350
burials) dates to the range of the end of the 5th century to the beginning of the 3rd century BC [105]. From the end of the 4th century to
the beginning of the 3rd century BC the Ukrainian forest-steppe region was a part of Scythia.
Chernyakhiv culture (group Chern)
The Chernyakhiv culture was one of the greatest archaeological cultures of the European Barbaricum in late Roman times. The cul-
ture is dated from the middle of the 3rd century to the first half of the 5th century AD [106, 107]. It was spread across a wide area
covering the southeastern Europe forest-steppe zone from the lower reaches of the Danube to the upper reaches of the Siverski Do-
nets. The northern border of its distribution area coincided with the boundary between the forest and the forest-steppe zone; in the

Current Biology 29, 1–12.e1–e10, July 22, 2019 e3

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

south it bordered with the Black Sea (encompassing the territories of modern Ukraine, Romania, Moldova and a part of Russia)
[26, 108].
The Chernyakhiv culture was likely an ethnically heterogeneous mix based on Goths (Germanic tribes) but also including
Sarmatians, Alans, Slavs, late Scythians, Dacians and the antique population of the northern coast of the Black Sea [24–27].
Archaeological sites characteristic of the Chernyakhiv culture are mostly open settlements [24] and pit grave burial grounds lacking
grave markers with both cremation and inhumation burials [26, 109–111]. The latter burial custom may have been caused by various
factors, incl. the influence of Christianity. The burials contain a variety of grave goods: sacrificial food and ceramic and glass vessels,
personal items such as horn combs, costume details (fibulae, buckles, beads) [112].
The Chernyakhiv culture is characterized by advanced agriculture, pottery, bone working, metallurgy and textile manufacture [113,
114]. This is evidenced by agricultural and handicraft artifacts, jewelry, horn and metal combs, household items such as loom
weights, etc [26, 112, 115, 116]. Extensive contacts with the Roman Empire are evidenced by the many Roman imports (coins,
ceramic, glass and metal vessels, etc.) that have been found at Chernyakhiv sites [117–120] as well as by the borrowing of techno-
logical achievements such as wheel made pottery, glass processing and, possibly, agriculture.
Contacts with neighboring regions were active, and the Chernyakhiv culture is associated with a number of historical events that
took place in Europe at that time. In particular, during the Scythian or Gothic wars of the 230 s and 270 s, barbarians living in the
territory of the Chernyakhiv culture (Goths, Ferules, Carps, Bastarns, etc.) carried out regular raids across the Danube Limes of
the Roman Empire. However, from the end of the 3rd century the relations of the barbarians with the Roman Empire gained a certain
stability. From the reign of Constantine I the Goths, who were part of the Chernyakhiv culture, became federates (military allies) of the
Empire [121].
The Goths also interacted with the inhabitants of the East European forest zone. The Roman historian Jordanes described the mil-
itary campaigns of the Gothic king Ermanaric against northern peoples (the ancestors of Vends, Slavs, etc., and the inhabitants of the
northern Volga region) [122].
Jordanes also wrote about the ‘‘State of Ermanaric’’ which may associated with the Chernyakhiv culture. The existence of an early
barbarian state can be assumed since the time of the Scythian or Gothic wars (230-270) [123]. However, the state soon disappeared
in the midst of the complex ethnopolitical processes of the Migration Period (mainly, a Hunnic invasion) [122].
The Chernyakhiv culture dissolved due to a number of factors: climate change [124], a Hunnic invasion [125, 126], internal political
crisis, and participation in migration processes. Part of the population fled from the Huns into the Roman Empire, while some
participated in the Hunnic invasion of the Empire and returned home afterward [122, 127].
Scythians and Sarmatians of the Southern Urals (group ScySar_SU)
Due to its geographic position, the Southern Urals region became a contact zone of different ethnic groups at the beginning of the
Early Iron Age [20]. The prominent natural feature of the region is the mountain range of the Urals that divides the Eurasian steppe
zone into its European and Asian parts. The relative proximity to the nations of Central Asia and the Near East, the abundance of
natural resources and the existence of large centers of metallurgy made the region attractive for nomads, which culminated in the
formation of a large-scale union of nomads in the 5th–4th century BC.
Due to ecological and demographic factors, the Southern Urals steppe was practically uninhabited at the end of the Bronze Age,
only a handful of burials are known from that period [128]. More numerous burial sites reappear only in the 7th century BC, suggesting
the population of the region by nomads [19]. Burial rites specifics enable us to distinguish a number of ethno-cultural components
among the so-called Early Saka cultural period nomads from the Southern Urals, tracing back to Central Asia, the Altai-Sayan region,
Central and Northern Kazakhstan and elsewhere [129].
The northernmost sites of the Early Saka culture are located at the boundary of the foothills of the Urals. The Itkul culture is distin-
guished there, based around a center of metallurgy that more and more began to fulfil the needs of the nomads wandering in the
region [19, 130, 131]. This cultural group is dated to the 7th–5th century BC. One of the samples analyzed in this study (MJ-42 /
UT-4-16) originates from the Nikolaevka II burial site, one of the earliest sites of this culture [132]. The population of the Itkul culture
was likely gradually assimilated by the early Prokhorovka culture (‘‘Early Sarmatian’’) people [133].
At the end of the 7th century and beginning of the 6th century BC the areas west of the Mugodzhary Range and south of the Urals
around the upper and middle reaches of the Ilek and Or Rivers also began to be inhabited by nomads [134]. This group originated from
the area of the Tasmola culture in Central Kazakhstan and as such was close to one of the ethnic components of the Saka culture
described above.
Common burial features enable the distinction of a group of burial sites in the western part of the Southern Urals steppe at least until
the 4th century BC [135], characterized by stone and earth mounds (kurgans), kerbs encircling the burials, wide oval grave pits, en-
circling inlay of the graves, the orientation of the bodies to cardinal directions, the burning of wooden covers, and a frequent
occurrence of multi-layered burials [136]. Analogous mounds were also erected in the steppes and mountain steppes east of the
Urals, and the tradition also extended to the forest-steppe foothills and later during the Prokhorovka culture period to the forest-
steppe west of the Urals [137]. One sample (MJ-40 / UT-87-15) from burial 1 of the Manhar-2 mound in the region east of the Urals
originates from one of the younger burial sites of this group [138].
Another group of nomadic burial sites in the steppes west of the Urals formed in the northern part of the steppe zone in the second
half and at the end of the 6th century BC, and its origins are connected to the nomads who lived in the periphery of Ancient Khorezm
[139]. Sites of this type are located around the lower and middle reaches of the Ilek River, but also in the steppes east of the Urals, and
the westernmost around the middle reaches of the Volga River (Samarskaya Luka) [140]. The youngest ‘‘pure’’ sites are dated to the

e4 Current Biology 29, 1–12.e1–e10, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

end of the 5th century BC, in the 4th century BC the carriers of this group were assimilated by the abovementioned ‘‘Early Sarmatians’’
[141]. The main group of sites are burial structures built on the ancient surface. These are often collective graves, with various struc-
tures of wooden logs or posts (often burnt), in some cases they have had entrances on the ground (dromos’es). Some of the sites of
this group are mausoleums built of raw bricks and later burnt.
At the end of the 6th century BC yet a third group of sites emerged in the steppe west of the Urals, characterized by mounds of earth,
under which wide standard rectangular grave pits were dug, covered by flat wooden covers. These are often collective burial sites
where the dead have been placed with their heads oriented to the west [142]. Such mounds have been found around the lower rea-
ches of the Ilek River and the middle reaches of the Ural River, the more eastern ones from the region where the Or River flows into the
Ural River. Due to similarity to the Povolga Region complexes this can be called the Blumenfeld group. The Povolga group is identified
as the ‘‘Herodotos Sauromatians’’ [143].
Throughout the 5th century BC, active assimilation took place in the Southern Urals steppe, evidenced by the different burial sites of
various nomadic groups [141]. In the first half of the 4th century BC, the ethno-cultural integration and levelling came to an end, re-
sulting in the establishment of relatively standard burial customs. Simple rectangular grave pits were dug, but also very large and
complex collective graves that were round, rectangular or ‘‘cross-shaped,’’ with dromos’es, deep shafts and catacombs, where
the dead were placed with their heads oriented to the south [144]. The center of the formation of this complex (with notable ‘‘foreign
elements’’ that clearly influenced the elite of the society) may have been the region around the lower and middle reaches of the Ilek
River (sites of the ‘‘royal’’ Filippovka-1 cemetery type) [145]. After the Prokhorovka burial site studied over 100 years ago by S.I. Ru-
denko (published by M.I. Rostovtsev) [146] this can be named the Early Prokhorovka complex.
During the 4th century BC, this tradition spread across the entire Southern Urals region inhabited by nomads. In the Southern Urals
(burial site Perevolochan-1), the deposition of the Early Prokhorovka complex on top of the preceding Eastern Priaralian complex and
the absorption of the latter are clearly documented [141]. The area of this tradition (a practically uniform ethno-cultural formation)
measures at least 800 3 800 km. In the steppe zone, three contemporary ‘‘tribal centres’’ formed, first around the middle and lower
reaches of the Ilek River and then at the mouth of the Kumak River flowing into the Ural River and in Prisakmre [147]. The Southern
Urals forest-steppe was probably claimed as a summer territory. The region east of the Urals is characterized by strong militarisation,
probably related to the need to control the Itkul metallurgy center and gold mines south of the modern city of Chelyabinsk [148].
Five of the samples analyzed relate to the Early Prokhorovka period. Two of them (MJ-56 / UT-94-15 from Perevolochan and MJ-39
/ UT-95-15 from Ivanovka-1) originate from the center of the Prokhorovka area, two (MJ-41 / UT-88-15 from Avlasovo and MJ-43 /
UT-26-16 from Sibai-1) from its northern periphery and one (LS-13 / UT-99-15 from Novo-Muraptalovo-7) from the northern boundary
of the steppe west of the Urals.
The disappearance of this union of nomads and the entire regional system of interaction, the ‘‘Filippovka circle’’ sites, in the first
decades of the 3rd century BC was likely related to deteriorating climatic conditions. The cultural dominance of the Southern Urals
‘‘nomadic steppe’’ passed to new migrants related to Central Asia and the southern part of Western Siberia [149].
One of the analyzed samples (MJ-44 / UT-40-16 from Chumarovo-1) originates from the forest-steppe west of the Urals. It is from
an individual buried diagonally into a rectangular grave pit, indicating the arrival of the Middle Sarmatian culture (Alans?) to the region
relatively early, in the 3rd–2nd centuries BC.
Tasmola culture in Central Kazakhstan (group Scy_Kaz)
From Kazakhstan, this study includes individuals from the burial sites of the Tasmola culture (group Scy_Kaz). Based on archaeo-
logical material, this cultural stage is often demarcated as the extensive Tasmola historical-cultural union. Tribes of this union in-
habited the central and northern part of modern Kazakhstan and the steppes east of the Urals in modern Russia, the Tasmola culture
in Central Kazakhstan being a typical example [19, 150–152]. The similarities of these regions are not only apparent in the elements
and objects of the burial ritual, but also in the locations of the settlement sites [153–155].
The archaeological sites of the Tasmola culture were first discovered and studied in Central Kazakhstan over half a century ago by
M.K. Kadyrbaev. Studies carried out in the 21st century have significantly expanded knowledge of the period [150, 156, 157]. By now
more than 220 funerary structures have been examined and more than 50 settlement sites discovered, in 12 of which archaeological
excavations have been carried out [158]. Most of the sites studied are located in the eastern part of Central Kazakhstan.
Recent research, incl. numerous radiocarbon datings, allows us to date the Tasmola culture to 8th–5th century BC [158, 159]; the
samples included in this study match that age.
Anthropological studies indicate that the formation of the Tasmola culture was linked to the Bronze Age population of Kazakhstan
and Siberia and that the Central Kazakhstan Tasmola culture people were in contact with the inhabitants of regions further east [160].
Archaeological material clearly shows contacts of the Tasmola culture with the Altai-Sayan region, the southern part of the region east
of the Urals, and other parts of Kazakhstan. These connections are also visible in the finds of the Taldy-2 burial site where one of the
samples analyzed in this study originates. Among the artifacts found from the Taldy-2 mounds similarities with finds from both the
Eastern Kazakhstan Shilikty and the Russian Kichigino (east of the Urals) and Arzhan-2 (Tuva) burial sites can be detected. These
directions of communication characterize the entire Tasmola culture in Central Kazakhstan.

METHOD DETAILS

All of the laboratory work was performed in dedicated ancient DNA laboratories of the Department of Archaeology and Anthropology,
University of Cambridge, and of the Institute of Ecology and Earth Sciences, University of Tartu. The library quantification and

Current Biology 29, 1–12.e1–e10, July 22, 2019 e5

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

sequencing were performed at the Cambridge Biochemistry DNA Sequencing Facility and the Estonian Biocenter Core Laboratory.
The main steps of the laboratory work are detailed below.

DNA extraction
Sections of tooth roots were broken off using a hammer and used for extraction. To remove contaminants from the surface of tooth
root pieces, they were soaked in 6% bleach for 15 minutes, rinsed twice with double distilled water (ddH2O) and then soaked in 70%
ethanol for 2 minutes. Finally, the samples were left to dry under a UV light for 30 minutes on both sides.
The samples were weighed and per mg of sample 100ml of EDTA and 50ml of proteinase K (10 mg/ml) was added and the left to
digest for 72 hours on a slow shaker at 20 C.
The DNA solution was concentrated to 250 ml (Amicon Ultra-15 30 kDa, Merck Millipore) and purified in large volume columns (High
Pure Viral Nucleic Acid Large Volume Kit, Roche) using 2.5 mL of PB buffer, 1 mL of PE buffer and 50 mL of EB buffer (MinElute PCR
Purification Kit, QIAGEN).

Library preparation
Sequencing libraries were built using NEBNext DNA Library Prep Master Mix Set for 454 (E6070, New England Biolabs) and Illumina-
specific adaptors [161] following established protocols [161–163]. The end repair module was implemented using 18.75 mL of water,
7.5 mL of buffer and 3.75 mL of enzyme mix, incubating at 20 C for 30 minutes. The samples were purified using 500 mL PB and 650 mL
of PE buffer and eluted in 30 mL EB buffer (MinElute PCR Purification Kit, QIAGEN). The adaptor ligation module was implemented
using 10 mL of buffer, 5 mL of T4 ligase and 5 mL of adaptor mix [161], incubating at 20 C for 15 minutes. The samples were purified as
in the previous step and eluted in 30 mL of EB buffer (MinElute PCR Purification Kit, QIAGEN). The adaptor fill-in module was imple-
mented using 13 mL of water, 5 mL of buffer and 2 mL of Bst DNA polymerase, incubating at 37 C for 30 and at 80 C for 20 minutes. The
libraries were amplified and both the indexed and universal primer (NEBNext Multiplex Oligos for Illumina, New England Biolabs) were
added by PCR using HGS Diamond Taq DNA polymerase (Eurogentec). The samples were purified and eluted in 35 mL of EB buffer
(MinElute PCR Purification Kit, QIAGEN). Three verification steps were implemented to make sure library preparation was successful
and to measure the concentration of dsDNA/sequencing libraries – fluorometric quantitation (Qubit, Thermo Fisher Scientific), parallel
capillary electrophoresis (Fragment Analyzer, Advanced Analytical) and qPCR.

DNA sequencing
DNA was sequenced using the Illumina NextSeq-500 platform with the 75 basepair single-end method.

QUANTIFICATION AND STATISTICAL ANALYSIS

Mapping
Before mapping, the sequences of adaptors and indexes and poly-G tales occurring due to the specifics of the NextSeq 500 tech-
nology were cut from the ends of DNA sequences using cutadapt 1.11 [43]. Sequences shorter than 30 bp were also removed with the
same program to avoid random mapping of sequences from other species.
The sequences were mapped to reference sequence GRCh37 (hs37d5) using Burrows-Wheeler Aligner (BWA 0.7.12) [44] and
command mem with re-seeding disabled.
After mapping, the sequences were converted to BAM format and only sequences that mapped to the human genome were kept
with SAMtools 1.3 [45]. Next, data from different flow cell lanes was merged and duplicates were removed with picard 2.12 (http://
broadinstitute.github.io/picard/index.html). Indels were realigned with GATK 3.5 [46] and lastly, reads with mapping quality under 10
were filtered out with SAMtools 1.3 [45].
Endogenous DNA content (proportion of reads mapping to the human genome) was variable as is common in aDNA studies,
ranging from < 5% to > 95%, with the average for the 31 samples being 40.1% (Table S2).

aDNA authentication
As a result of degrading over time, aDNA can be distinguished from modern DNA by certain characteristics: short fragments and a
high frequency of C > T substitutions at the 50 ends of sequences due to cytosine deamination. The program mapDamage2.0 [47] was
used to estimate the frequency of 50 C > T transitions.
mtDNA contamination was estimated using the method from [40], which aligns the raw mtDNA reads to the RSRS [164], determines
the haplotype using GATK pileup [46], counts the number of het sites on haplotype-defining sites as well as adjacent sites and cal-
culates a ratio that takes into account ancient DNA damage by excluding positions where the major allele is C or G and the minor is T
or A respectively. For the male individuals with average X chromosome coverage > 0.1x, contamination was also estimated based on
the X chromosome using the two contamination estimation methods first described in [165] and incorporated in the ANGSD software
[48] in the script contamination.R.
On average, the samples showed 12.81% C > T substitutions at the 50 ends (Table S2). The mtDNA contamination estimate for
samples with > 10x mitochondrial coverage ranged from 0 to 1.80% with an average of 0.92%; the estimates for two samples
with < 10x mtDNA coverage are given in brackets and not taken into account calculating the average (Table S2). One of the low mito-
chondrial coverage samples (MJ-53) has a contamination estimate that falls within the range of higher coverage samples, while the

e6 Current Biology 29, 1–12.e1–e10, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

other (MJ-47) shows a higher level of mtDNA contamination at 4.55%. However, this sample did not show any abnormalities in auto-
somal analyses, grouping with other Ukrainian Scythians, and the contamination estimate may be related to its low mtDNA coverage,
as its average genomic coverage (0.073x), while on the low side, is not extremely low compared to other samples – three samples
(MJ-36, MJ-40, MJ-54) have lower average genomic coverage, but higher average mtDNA coverage, and lower mtDNA contamina-
tion estimates (Table S2). Either the mtDNA-based estimate for MJ-47 is inaccurate due to coverage, damage or a biological reason,
or there is a possibility that this sample has mtDNA contamination, but not nuclear. As the individual is female, it is not possible to do
an X chromosome-based estimate.
The average of the two X chromosome contamination methods of male individuals with average X chromosome coverage > 0.1x
was between 0.19 and 2.27% with an average of 0.71% (Table S2). For most of the male individuals, average X chromosome
coverage was very low (< 0.1x) and therefore the contamination estimates were not calculated.

Calculating general statistics and determining genetic sex

SAMtools 1.3 [45] option stats was used to determine the number of final reads, average read length, average coverage etc.
Genetic sex was calculated using the script sexing.py from [49], estimating the fraction of reads mapping to Y chromosome out of
all reads mapping to either X or Y chromosome.
The average coverage of the whole genome for the samples was between 0.02 and 0.72 (Table S2). Genetic sexing mostly
confirmed morphological sex estimates, but contradicted them in four instances (MJ-41, MJ-43, MJ-54, MJ-56), in each of which
a sample classified as male was shown to be female. The sex of one of the samples (MJ-53) could not be reliably estimated due
to low coverage, but since its morphological sex estimate was male and it was successfully haplotyped for the Y chromosome
(see section ‘‘Y chromosome variant calling and haplotyping’’), we have tentatively classified it as male.

Variant calling
Variants were called with ANGSD [48] command–doHaploCall, sampling a random base for the positions that are present in the
EBC-chipDB [28–39].

Determining mtDNA haplogroups

Raw reads were mapped to the revised Cambridge Reference Sequence [166] and resulting bam files were indexed for viewing in
Tablet v1.13.04.22 [167]. Variants were called using SAMtools 1.3 mpileup variant-only option and filtered using bcftools 1.1 [45].
Haplogroups were assigned using Phylotree build 17 [168] accessed at http://www.phylotree.org, Haplogrep [50, 51] accessed at
https://haplogrep.uibk.ac.at, and Haplofind [52] accessed at http://www.haplofind.unibo.it.
All of the 31 individuals were successfully haplotyped (Table S2). Despite the small sample size, the 31 samples of this study exhibit
a remarkable heterogeneity of mtDNA haplogroups (hgs) (Table S2). Both the pre-Neolithic West Eurasian U5 lineages [169–171] and
hgs H, J and T associated with the Neolithic expansion [170] are represented. Most of the samples (24/31) belong to mtDNA hgs
nowadays mainly spread in West Eurasia (H, J1, T1, T2, U2, U5), with a few representatives of the rare hgs X, W and C1e (5/31)
and of East Eurasian hgs (A23, F2), the latter expectedly being two Scy_Kaz samples (Table S2). The 7 samples belonging to hg
H are all from the western sample groups (Ukr_BA, Scy_Ukr, Chern, Cimmerians), there are no representatives of this hg in Scy-
Sar_SU or Scy_Kaz. Hg U4, nowadays fairly typical among the Finno-Ugric peoples of West Siberia and the Volga-Uralic region
[37], is absent from the samples of this study. Compared to the present-day distribution of mtDNA hgs [172, 173], the mitochondrial
profile of Scythians and Sarmatians, incl. most of our samples from the Southern Urals and half of them from Kazakhstan, is some-
what more ‘western’, reflecting autosomal results that place most of the samples of this study closer to modern Europeans than Cen-
tral Asian or Western Siberian populations (Figure 2).

Y chromosome variant calling and haplotyping

Y chromosome variants were called from the BAM files of the samples using ANGSD [48]–doHaploCall. The resulting VCF files were
filtered for regions of a total length of 8.8 Mbp of sequence that uniquely maps to human Y chromosome when using short read
sequencing technology [174]. Variants called within this 8.8 Mbp region were further filtered for 113,217 haplogroup informative po-
sitions [174–177] [EGC, unpublished] using the BEDTools 2.19.0 [53] intersect option. Haplogroup assignments of each individual
sample were made by determining the haplogroup with the highest proportion of informative positions called in derived state in
the given sample. SNPs from 1000G, http://www.yfull.com/tree [174], and [175] were used for Y chromosome haplogroup assign-
ment. Y chromosome haplotyping was performed on all samples to check whether any of the samples estimated to be female would
also give a result.
None of the female samples were successfully haplotyped as expected. 16 out of the 16 males were successfully haplotyped,
although for the low coverage sample MJ-53, the haplogroup could only be determined at the level of R1, and MJ-52 was typed
as belonging to haplogroup J1b-P58 based on 3 informative positions out of 10 (Table S2).
Out of the 31 samples of this study, 16 are male, and with sufficient Y chromosome coverage for haplogroup assignment (Table S2).
R1a (43%) and I (27%) are the two most frequent Y chromosome hgs in present-day Ukrainians [33]. R1a is also the predominant
lineage among Cimmerians, Scy_Ukr and ScySar_SU in our data, and present among Scy_Kaz as well. Thus, although acknowl-
edging our small sample size, the individuals sampled from archaeological context associated with Scythian identity do not appear
to stand out from the context of other groups living in the region before and after them. One notable difference from the present is the

Current Biology 29, 1–12.e1–e10, July 22, 2019 e7

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

absence of hg N, nowadays widespread in the Volga-Uralic region and West Siberia as well as among Mongols and Altaians
[178–180]; however, this result is consistent with the absence of hg N among Bronze Age and Eneolithic males from the Steppe
[181]. In context of their claimed Altaian homeland it is interesting to note that one Scy_Ukr and the single Sar_Cau sample belong
to the Q1c-L332 lineage which is a sub-clade of hg Q1c-L330 that today has peak frequency of 68% in Western Mongolians [182] and
occurs at 17% in South Altaians [183] while being very rare (< 1%) in East European populations and absent elsewhere (https://www.
yfull.com/tree/Q-L330/).

Preparing the datasets for autosomal analyses

The EBC-chipDB [28–39] was used as the modern DNA background. Samples from [1–3, 16–18, 22, 40–42] were used as the ancient
DNA background. The inclusion of modern and ancient populations in various analyses is given in Table S3. The full genome
sequencing data of the aDNA background dataset [1, 2, 22, 40, 42] in the form of FASTQ files was called as described in the Variant
calling section. The 1240k capture data of the aDNA background dataset [3, 16–18, 41] was downloaded in EIGENSTRAT format. The
data of the two comparison datasets and of the samples of this study was converted to BED format using PLINK 1.90 (http://pngu.
mgh.harvard.edu/purcell/plink/) [54], the datasets were merged and the 537,802 SNPs of the modern comparison dataset were kept.
As all individuals of this study had > 11,000 SNPs of the 537,802, none were removed from further autosomal analyses.

Principal component analysis

We performed principal component analysis (PCA) using the program smartpca (lsqproject: YES and shrinkmode: YES) from
EIGENSOFT 7.2.1 (https://github.com/DReichLab/EIG) [55], projecting 951 aDNA samples (ancient DNA background and this study)
onto the eigenvectors calculated based on 537,802 autosomal SNPs in 1,422 modern Eurasians from 92 populations from the
EBC-chipDB. To improve readability, we plotted the modern populations as population medians (after outlier removal, resulting in
1,346 individuals from 91 populations). Since the number of published ancient samples has also grown large enough to make plots
difficult to read, we opted to plot several versions of relevant ancient populations on the modern background (Figures 2 and S1).
Given that PCA was run in shrinkmode to account for shrinkage effects that can arise due to projection, the lack of overlap of
most of the ancient samples with modern variation (especially since there were also exceptions to this) was likely not a result of
PCA shrinkage but rather a genuine difference.

ADMIXTURE
1,531 modern individuals from 102 populations from the EBC-chipDB (the modern dataset without outliers used for PCA plus 185
South Asians from 11 populations) and 951 aDNA samples (ancient DNA background and this study) were included in the ADMIX-
TURE analysis. The analysis was carried out using ADMIXTURE 1.30 [56] with the P option, projecting the ancient samples onto the
genetic structure calculated based on the modern dataset. The dataset of modern samples was pruned to decrease linkage
disequilibrium using the indep-pairwise option with parameters 200 25 0.4 in PLINK 1.90 (http://pngu.mgh.harvard.edu/ purcell/
plink/) [54]. This resulted in a set of 206,241 SNPs. We ran ADMIXTURE on this set using K = 3 to K = 15 in 100 replicates, which
enabled us to assess the convergence of the different models. K = 5 was the model with the largest number of inferred genetic clus-
ters for which > 10% of the runs that reached the highest Log Likelihood values yielded very similar results. We use this as a proxy to
assume that the global Likelihood maximum for this particular model was reached. We then used the inferred genetic cluster pro-
portions and allele frequencies of the best run at K = 5 to run ADMIXTURE, projecting the aDNA samples on the inferred clusters (Fig-
ure S2). The resulting membership proportions to K genetic clusters are sometimes called ‘ancestry components’ which can lead to
over-interpretation of results. The clustering itself is, however, an objective description of genetic structure and as such a valuable
tool in population comparisons.

Population modeling and geographic correlation

Sample groups used in population modeling:

d Pre-Scythians: Yamnaya [22], Yamnaya_Samara, Yamnaya_Kalmykia, Srubnaya [16], Yamnaya_Ukraine [17], Srubnaya-Ala-
kulskaya, Cimmerian [2], Ukr_BA (this study)
d Scythians and Sarmatians: Nomad_IA [16], Scythian_East, Sarmatian_SU [3], Hungarian Scythian, Sarmatian, Central Saka,
Tian Shan Saka, Tagar [1], Scythian_PC [2], Scy_Ukr, ScySar_SU, Scy_Kaz (this study)
d Post-Scythians: Late Sarmatian [2], Chern (this study)

1. qpAdm
We constructed various population admixture models using the qpWave [184, 185] and qpAdm [5] setup. The method first models a
‘target’ population group of interest, exploiting the genetic drift shared between a set of reference ancient sources and a set of out-
groups, and subsequently extracts the ancestry proportions of the ‘target’ population stemming from the blend of these reference
ancient sources. We intended to differentiate between ancestral source components of relevance, based on previous results and
prior information [1], to answer the questions we address in the present study.

e8 Current Biology 29, 1–12.e1–e10, July 22, 2019

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

We tested separate distal and proximal models for estimating ancient contributions to our studied target groups from temporally
different sources.
The distal (temporally distant) model we tested for studied groups herein, using Yoruba, Ust’Ishim, Kostenki, Mal’ta and Han as
outgroups, included three reference ancient sources related to Eastern Hunter-Gatherers (EHG), Natufians and Altaians, i.e., a
three-way admixture. The three-way migration model fit well for the tested populations and had also been supported by [1] previ-
ously; additionally, it accommodated the greatest number of test populations without fail compared to other tested models where
we used different combinations of sources and outgroups (Figure 3; Data S1).
Additionally, we experimented with two separate proximal models on the analyzed target groups, to assess the relative fitting and
ancestry contributions from temporally close ancient neighbors (Data S1). Specifically, we used Yamnaya or Srubnaya as one recent
ancient source, the other sources being Central European Middle Neolithic and Altaians. In case of some targets that did not fit the
model with three components, we added Iranian Neolithic (Iran_N) or EHG as additional sources (Figure 3; Data S1).
2. Inferring the ancestry proportion of ancient individuals
In order to infer the admixture proportions of ancient individuals, we applied the ChromoPainter/NNLS pipeline [58–60]. Since Chro-
moPainter does not allow for the presence of missing data, we iteratively painted, using the ‘unlinked mode’, every target ancient
individual as ‘recipient’ together with one representative individual from a given putative source.
In details, we tested two different temporal approaches. In the distal analysis every target individual was painted with a represen-
tative of EHG (Eastern Hunter-Gatherer, ‘‘I0061’’) and Natufian (‘‘I1072’’). In order to allow for an eastern contribution into the analyzed
populations we added one randomly selected individual from the Altaian population. All the remaining modern individuals were used
as donors.
In addition, we tested two separate proximal models, evaluating the contribution of more recent sources to the analyzed popula-
tions. In details, we used Yamnaya (‘‘Yamnaya’’) or Srubnaya (‘‘I0232’’) together with Central European Middle Neolithic (‘‘I0172’’) and
Altaians (‘‘altai7’’).
The resulting painting profiles, which summarize the fraction of the individual’s DNA coalescing most recently with each donor in-
dividual, were summarized at a population level summing over individuals from the same group (Figure 3; Data S1).
Let Xg, and Yp be a vector summarizing the proportion of DNA that source and target individuals copies from each of the modern
donor groups as inferred by ChromoPainter, we reconstructed Yp = b1X1 + b2X2 + b3X3 + ... b4X4 using a slight modification of the nnls
P
function in R and implemented in GT, under the conditions of bg R 0 and bg = 1.
We also evaluated the fit of the proximal test by exploring the distribution of the squared residuals for each analysis, examining
which model yields the lowest value of residuals [23].
3. Correlation with distance from Altai
For each sample group we compared the genome-wide fraction of Altaian ancestry estimated through qpAdm and ChromoPainter/
NNLS with great circle distance from Altai, using for each group the median locations of each sample inferred from the literature or
available from the current study, and taking the coordinates of Altai as Lat 49 Lon 89. The groups were stratified into three time bins
(pre-Scythian, Scythian and Sarmatian or post-Scythian) according to the available chronological and associated cultural informa-
tion. Taking the Scythian samples as reference, distance from Altai appeared as a good predictor of Altaian ancestry regardless of the
method used to estimate it (qpAdm: r2 = 0.63, dashed line in Figure 4; CP/NNLS: r2 = 0.58, solid line in Figure 4). Notably, the
post-Scythian Chernyakhiv culture and Late Sarmatian samples fit within this broader pattern, except for the CP/NNLS result of
the Chernyakhiv samples, while pre-Scythians have consistently less Altaian ancestry given their geographic location (except Cim-
merians who have been shown previously to already carry eastern genetic ancestry despite predating the Scythians [2]).

Outgroup f3 and D statistics

For calculating autosomal outgroup f3 statistics, the ancient sample-set included 950 individuals from 172 populations and the mod-
ern sample-set 1,423 individuals from 90 populations from Europe, Caucasus, Near East, Siberia, Central Asia and East Asia, and the
Yorubas as an outgroup (Figure S3; Table S3; Data S1). Heterozygous positions were converted to homozygous by randomly
choosing one of the alleles at each position to enable comparison between pseudo-haploid ancient samples and diploid modern
samples. The data was converted to EIGENSTRAT format using the program convertf from the EIGENSOFT 5.0.2 package [55].
Outgroup f3 statistics of the form f3(Yoruba; Ukr_BA/Scy_Ukr/Scy_Kaz/Chern, modern/ancient) were computed using the
ADMIXTOOLS 1.1 [57] program qp3Pop.
D statistics of the form D(Yoruba, Ukr_BA/Scy_Ukr/Scy_Kaz/Chern; Ukrainians, modern/ancient) were calculated on the same
EBC-chipDB as outgroup f3 statistics (Table S3; Data S1). The ADMIXTOOLS 1.1 [57] package program qpDstat was used.

f4 statistics
f4 statistics of the form f4(Yorubas, mixing population; study population, study population) were calculated on the same EBC-chipDB
as outgroup f3 statistics (Data S1). The ADMIXTOOLS 1.1 [57] package program qpDstat and the option f4mode: YES was used.

Inspecting sites highly differentiated between modern Europeans and East Asians
Given the detectable Asian component in most Scythian and Sarmatian samples, we inquired whether phenotype informative
markers which are associated with morphological features often linked to macro-geographic regions were over- or underrepresented
in the sampled individuals. We specifically looked for the presence or absence of the ‘Asian’ allele of the SNP rs3827760 in the

Current Biology 29, 1–12.e1–e10, July 22, 2019 e9

 €rve et al., Shifts in the Genetic Landscape of the Western Eurasian Steppe Associated with the Beginning and End
Please cite this article in press as: Ja
of the Scythian Dominance, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.06.019

ectodysplasin A receptor EDAR gene (the variant causing thick, straight hair) and compared it to all the SNPs reported as highly differ-
entiated (delta derived allele frequency, DDAF, R 0.8) between modern European and East Asian samples of the 1000 Genomes
Project by [186]. We then inspected individual bam files for the presence or absence of the allele reported as predominantly ‘Asian’
by [186] and reported the number of samples where at least one such allele was present (numerator) over the total number of samples
where at least one read was available (denominator) in Table S4.
Of the samples of this study, we found that only one, from the Scy_Ukr group, had the ‘Asian’ allele (one allele out of two reads in
total) for the rs3827760 EDAR SNP (Table S4). There were a few other incidental findings of ‘Asian’ alleles for the non-EDAR markers
tested in all sample groups except Chern (Table S4). The relatively high percentage of ‘Asian’ alleles among Cimmerians was mostly
due to the Thraco-Cimmerian sample MJ-12 that surprisingly displayed 55% of the ‘Asian’ alleles, even though it positioned among
SE. Europeans in PCA (Figures 2 and S1) and had barely any Asian component in ADMIXTURE (Figure S2).

Metagenomic screening for pathogen reads

Raw datasets were trimmed with cutadapt [43]. We removed reads below 30 bp and trimmed bases below a quality score of 20 from
the end of all reads using the integrated NextSeq trimming option, which accounts for erroneous guanine calls resulting from two-
color chemistry sequencing platforms. The data was then deduplicated using ParDre [61]. Deduplicated and trimmed FASTQ files
were subsequently analyzed with the taxonomic classifier Kraken2 [62] using a dusted reference database. Kraken2 uses exact
k-mer matches to assign reads to the lowest common ancestor (LCA) of sequences matching the given k-mer. As a follow up we
also analyzed the data using Metaphlan2 [63], which has a different approach to Kraken2. It relies on unique clade-specific marker
genes to compute metagenomic profiles.
As the result of our investigation of the shotgun data for the presence of the plague pathogen Yersinia pestis we found that none of
the analyzed samples contained significant amounts of reads matching genomes of the bacterium or of its ancestor Yersinia
pseudotuberculosis.

DATA AND CODE AVAILABILITY

The accession number for the ancient DNA sequences reported in this paper is the European Nucleotide Archive: PRJEB32764. The
DNA sequences of this study are also available from the EBC data repository (http://evolbio.ut.ee).

e10 Current Biology 29, 1–12.e1–e10, July 22, 2019

Current Biology, Volume 29

Supplemental Information

Shifts in the Genetic Landscape of the Western

Eurasian Steppe Associated with the Beginning
and End of the Scythian Dominance
Mari Järve, Lehti Saag, Christiana Lyn Scheib, Ajai K. Pathak, Francesco Montinaro, Luca
Pagani, Rodrigo Flores, Meriam Guellil, Lauri Saag, Kristiina Tambets, Alena
Kushniarevich, Anu Solnik, Liivi Varul, Stanislav Zadnikov, Oleg Petrauskas, Maryana
Avramenko, Boris Magomedov, Serghii Didenko, Gennadi Toshev, Igor Bruyako, Denys
Grechko, Vitalii Okatenko, Kyrylo Gorbenko, Oleksandr Smyrnov, Anatolii Heiko, Roman
Reida, Serheii Sapiehin, Sergey Sirotin, Aleksandr Tairov, Arman Beisenov, Maksim
Starodubtsev, Vitali Vasilev, Alexei Nechvaloda, Biyaslan Atabiev, Sergey Litvinov, Natalia
Ekomasova, Murat Dzhaubermezov, Sergey Voroniatov, Olga Utevska, Irina Shramko, Elza
Khusnutdinova, Mait Metspalu, Nikita Savelev, Aivar Kriiska, Toomas
Kivisild, and Richard Villems
 Saa

Kha Modern population medians

Kar CentralSteppe_EMBA
Vep
Lat Sel Nen
Lit Fin Ket Ngn Okunevo_EMBA
Est
Kom
NRu Udm Mar Afanasievo
Pol CRu
Mrd
Chk
Andronovo
Blr Chv
SRu Kor
Poltavka
Ukr
Swe
Evn
Potapovka
Slk Ttr
Orc Sintashta
Dol
Ger Bsh Ykg Srubnaya
Sln Shr
Hun Evk
Khk Yamnaya_Kalmykia
Bos Yak
Fre
Yamnaya_Samara
FrB Srb Tuv
PC2 (0.68%)

Alt Ukraine_Eneolithic
Mac
Rom
Yamnaya_Ukraine
Bul Bur
Gag Kaz Srubnaya-Alakulskaya
Kyr Kal
Mon
NIt Ak_Moustafa_MLBA1
Orq
Uzb Aktogai_MLBA
Tus Nog
Grk Kazakh_Mys_MLBA
Tjk Uyg
Dau
Sar Hzh Krasnoyarsk_MLBA
Lzg Psh
Che
MglXib Maitan_MLBA_Alakul
Kum
Blk
Ady NOs Zevakinskiy_LBA
Trm Tu
Ukr_BA
Tur Jap
Scy_Ukr
Abh Aze Han
Krd Chern
Cyp Irn
Grg
Arm Sar_Cau
Leb
Syr ScySar_SU
Scy_Kaz
Jor
Late Srubnaya
Pal
Cimmerians
PC1 (4.99%)

Figure S1. Principal component plot of the samples of this study together with published Bronze Age samples from the Eurasian
Steppe on a background of modern Eurasians. Related to Figure 2. PCA obtained by projecting the ancient samples onto the
eigenvectors calculated based on 537,802 autosomal SNPs in 1,422 modern Eurasians. To improve readability, the modern
populations have been plotted as population medians (after outlier removal). Ancient individuals (in colour): asterisks – samples
from [S1], squares – samples from [S2], small rectangles – samples from [S3], large rectangles – samples from [S4], crosses –
samples from [S5], diamonds – samples from this study. Modern populations (in grey) by regional groups clockwise from top left:
EUROPE: Bsh=Bashkirs; Blr=Belarusians; Bos=Bosnians; Bul=Bulgarians; Chv=Chuvash; Cyp=Cypriots; Est=Estonians; Fin=Finns;
Fre=French; FrB=French Basques; Gag=Gagauzes; Ger=Germans; Grk=Greeks; Hun=Hungarians; Kar=Karelians; Kom=Komis;
Lat=Latvians; Lit=Lithuanians; Mac=Macedonians; Mar=Maris; Mrd=Mordovians; NIt=North Italians; Orc=Orcadians; Pol=Poles;
Rom=Romanians; NRu=Northern Russians; CRu=Central Russians; SRu=Southern Russians; Saa=Saami; Sar=Sardinians;
Srb=Serbians; Slk=Slovaks; Sln=Slovenians; Swe=Swedes; Ttr=Tatars; Tus=Tuscans; Udm=Udmurts; Ukr=Ukrainians; Vep=Vepsas;
SIBERIA: Alt=Altaians; Bur=Buryats; Chk=Chukchis; Dol=Dolgans; Evk=Evenkis; Evn=Evens; Ket=Kets; Khk=Khakases; Kha=Khanty;
Kor=Koryaks; Mon=Mongolians; Nen=Nenets; Ngn=Nganasans; Sel=Selkups; Shr=Shors; Tuv=Tuvinians; Yak=Yakuts;
Ykg=Yukaghirs; EAST ASIA: Dau=Daur; Han=Han; Hzh=Hezhen; Jap=Japanese; Mgl=Mongola; Orq=Oroqens; Tu=Tu; Uyg=Uygurs;
Xib=Xibo; CENTRAL ASIA: Irn=Iranians; Kaz=Kazakhs; Krd=Kurds; Kyr=Kyrgyzians; Psh=Pashtun; Tjk=Tajiks; Trm=Turkmens;
Uzb=Uzbeks; NEAR EAST: Jor=Jordanians; Leb=Lebanese; Pal=Palestinians; Syr=Syrians; Tur=Turks; CAUCASUS: Abh=Abkhasians;
Ady=Adygei; Arm=Armenians; Aze=Azeris; Blk=Balkars; Che=Chechens; Grg=Georgians; Kal=Kalmyks; Kum=Kumyks; Lzg=Lezgins;
Nog=Nogais; NOs=North Ossetians.
 Sumbar_LBA Chukchis
Tepe_Hissar_C Koryaks
Shahr_I_Sokhta_BA3 Evens
Shahr_I_Sokhta_BA2 Evenkis
Shahr_I_Sokhta_BA1 Yakuts
Sappali_Tepe_BA Yukaghirs
Gonur2_BA Shors
Gonur1_BA Selkups
Parkhai_LBA Nganasans
Parkhai_MBA Kets
Parkhai_EBA Dolgans
Sarazm_EN Tuvinians
Tepe_Anau_EN Buryats
Parkhai_EN Japanese
Geoksiur_EN Mongolians
Ganj_Dareh_N Mongola
Bustan_BA Oroqens
Dzharkutan1_BA Hezhen
Darra_i_kur_MBA Daur
Dzharkutan2_BA Xibo
Hajji_Firuz_C Tu
Hajji_Firuz_BA Han
Kanai_MBA Sakilli
NLithuania Paniya
Lithuania_BA Malayan
Latvia_BA North_Kannadi
Lithuania_CWC Sindhi
Latvia_CWC Makrani
Estonia_CWC Brahui
Latvia_CCC_EHG Balochi
Estonia_CCC Burusho
Latvia_CCC_WHG Pashtun
Lithuania_HG Pathan
Latvia_HG Hazara
Estonia_HG Khakases
Ukraine_Eneolithic Altaians
Hungarian_Med Uygurs
Sweden_LNBA Kazakhs
Northern_LNBA Kyrgyzians
Hungary_BA Uzbeks
HallstattBylany Tajiks
Central_LNBA Turkmens
Bell_Beaker Kalmyks
Trypillia Nogais
Sweden_TRB Kumyks
Remedello Lezgins
LBK_EN Chechens
Iberia_MN North_Ossetians
Iberia_EN Adygei
Iberia_Chl Balkars
Iberia_BA Abkhasians
Hungary_EN Georgians
Globular_Amphora Armenians
Central_MN Tatars
Cardial_EN Bashkirs
WHG Chuvash
Iron_Gates_HG Hantis
Bichon Nenets
SHG Udmurts
Romania_HG Komis
Ukraine_Neolithic Mordovians
Ukraine_Mesolithic Maris
SidelkinoEHG_ML Vepsas
EHG Saami
Nomad_His Swedes
Karakhanid Karelian
Kipchak Finns
Karluk Estonians
Kimak Latvians
Nomad_Med Lithuanians
Nomad_Hun-Sarmatian Russians_North
Nomad_HP Russians_Central
Turk Russians_South
TianShanHun Belarusians
XiongNu_WE Ukrainians
XiongNu Poles
GoldenHordeAsian Slovaks
GoldenHordeEuro Gagauzes
Kazakh_His Romanians
SaltovoMayaki Hungarians
Kangju Bulgarians
Wusun Slovenians
LateSarmatian Serbians
Scy_Kaz_MJ-54 Bosnians
Scy_Kaz_MJ-53 Macedonians
Scy_Kaz_MJ-52 Greeks
Scy_Kaz_MJ-51 Cypriots
ScySar_SU_MJ-56 Tuscans
ScySar_SU_MJ-44 North_Italians
ScySar_SU_MJ-43 Sardinians
ScySar_SU_MJ-42 French_Basques
ScySar_SU_MJ-41 Germans
ScySar_SU_MJ-40 French
ScySar_SU_MJ-39 Orcadians
ScySar_SU_LS-13 Azeris
Sar_Cau_MJ-38 Iranians
Chern_MJ-37 Kurds
Chern_MJ-36 Turks
Chern_MJ-19 Lebanese
Scy_Ukr_MJ-47 Syrians
Scy_Ukr_MJ-46 Jordanians
Scy_Ukr_MJ-35 Palestinians
Scy_Ukr_MJ-34
Scy_Ukr_MJ-33 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Scy_Ukr_MJ-16
Scy_Ukr_MJ-15
Scy_Ukr_MJ-14
Scy_Ukr_MJ-13
Cimmerians_MJ-32
Cimmerians_MJ-31
Cimmerians_MJ-12
Late_Srubnaya_MJ-08
Ukr_BA_MJ-09
Ukr_BA_MJ-06
Scythian_East
CentralSaka
NoDateTianShanSaka
TianShanSaka
Tagar
Nomad_IA
Scythian_IA
Sarmatian_SU
Sarmatian
Scythian_PC
HungarianScythian
Cimmerian
Poprad
Turkmenistan_IA
Zevakinskiy_LBA
Zevakinskiy_MLBA
Zevakinskiy_BA
Srubnaya
Srubnaya-Alakulskaya
Sintashta_MLBA
Sintashta
Petrovka
Preobrazhenka_MLBA
Alpamsa_MLBA_Alakul
Molaly_MLBA
Taldysay_MLBA2
Taldysay_MLBA1
Solyanka_MLBA
Kyzlbulak_MLBA2
Kyzlbulak_MLBA1
Oy_Dzhaylau_MLBA
Satan_MLBA_Alakul
Ak_Moustafa_MLBA1
Maitan_MLBA_Alakul
Lisakovskiy_MLBA_Alakul
Krasnoyarsk_MLBA
Kazakh_Mys_MLBA
Kashkarchi_BA
Karagash_MLBA
Kairan_MLBA
Dashti_Kozy_BA
Dali_MLBA
Andronovo
Aktogai_MLBA
Yamnaya_Ukraine
Yamnaya_Samara
Yamnaya_Kalmykia
Yamnaya_Bulgaria
Yamnaya
Russia_EBA
Potapovka
Poltavka
Okunevo_EMBA
CentralSteppe_EMBA
Glazkovo
Kurma_EBA
Shamanka_EBA
UstIda_LN
Lokomotiv_EN
Shamanka_EN
Afanasievo
Namazga_CA
Dali_EBA
Botai
Samara_Eneolithic
West_Siberia_N
Kostenki
Mal'ta
Ust-Ishim
Levant_BA
Anatolia_ChL
Levant_N
Anatolia_N
Natufian
LchashenMetsamor
Alan
Armenia_MLBA
Armenia_EBA
Iran_ChL
Armenia_ChL
Iran_LN
Iran_N
Iran_HotuIIIb
CHG
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Figure S2. ADMIXTURE analysis results at K=5 with ancient individuals projected onto the modern genetic structure. Related to
STAR Methods section “ADMIXTURE”. Left panel: ancient populations (ancestral component proportions averaged across each
population, except for the samples of this study that are presented individually). Right panel: modern populations (ancestral
component proportions averaged across each population).
 f3 with ancient populations f3 with modern populations

Figure S3. Outgroup f3 results. Related to STAR Methods section “Outgroup f3 and D statistics” and to Data S1. Left panel:
outgroup f3 results in the form of f3(Yorubas; sample group of this study; ancient population) with different sample groups of this
study plotted against each other. Right panel: outgroup f3 results in the form of f3(Yorubas; sample group of this study; modern
population) with different sample groups of this study plotted against each other. HG – hunter-gatherers; EF – early farmers; LNBA
– Late Neolithic and Bronze Age; IMA – Iron Age and Middle Ages; NCA – Neolithic and Copper Age; EMBA – Early–Middle Bronze
Age; MLBA – Middle–Late Bronze Age.
 Group / Endogenous Average Average Average mtDNA Average Y Average X mtDNA Male X cont. estimate
Genetic Y chr haplo-
Sample label in this DNA genome read mtDNA haplo- chr chr C>T cont. Method Method
sex group Average
study content coverage length coverage group coverage coverage estimate 1 2
MJ-06 Ukr_BA 21.84% 0.184 60 XX 21.2 H2a1 0.003 0.185 21.55% 0.57%
MJ-09 Ukr_BA 8.90% 0.228 57 XX 32.8 H13a2c 0.003 0.219 24.96% 0.49%
Late
MJ-08 5.64% 0.138 63 XX 62.9 T2a1b1a 0.001 0.136 20.86% 0.66%
Srubnaya
MJ-12 Cimmerians 82.73% 0.299 56 XX 14.0 H35 0.004 0.290 22.83% 0.71%
MJ-31 Cimmerians 42.07% 0.217 62 XY 18.0 U5a1b1 0.065 R1a-Z645 0.113 23.45% 1.30% 1.17% 3.37% 2.27%
MJ-32 Cimmerians 42.86% 0.296 62 XY 60.5 U2e2 0.087 R1a2c-B111 0.150 8.98% 0.57% 0.37% 0.01% 0.19%
MJ-13 Scy_Ukr 76.27% 0.265 62 XX 25.7 H11b1 0.003 0.251 15.02% 0.53%
MJ-14 Scy_Ukr 24.52% 0.120 62 XY 35.7 H6a1b 0.035 R1a-Z645 0.060 6.40% 0.49% N/A N/A
MJ-15 Scy_Ukr 9.67% 0.166 60 XY 56.9 U2e2 0.047 R1a-M417 0.082 10.70% 0.57% N/A N/A
MJ-16 Scy_Ukr 69.67% 0.282 58 XY 37.8 T2b 0.082 J2a8-B437 0.143 11.74% 0.41% 1.07% 0.01% 0.54%
U5a2a2
MJ-33 Scy_Ukr 12.93% 0.078 62 XY 33.2 0.023 R1a-M417 0.039 10.46% 0.76% N/A N/A
a
MJ-34 Scy_Ukr 87.29% 0.500 60 XY 44.9 W3a1 0.146 R1a2-Z93 0.253 11.04% 1.25% 0.16% 0.26% 0.21%
MJ-35 Scy_Ukr 28.28% 0.149 65 XY 30.7 X4 0.045 Q1c-L332 0.077 10.93% 0.95% N/A N/A
MJ-46 Scy_Ukr 80.35% 0.173 52 XX 13.8 J1d6 0.002 0.170 19.95% 1.18%
MJ-47 Scy_Ukr 42.14% 0.073 55 XX 8.4 T2 0.001 0.067 10.73% (4.55%)
MJ-19 Chern 6.23% 0.210 62 XX 42.3 H1n6 0.003 0.202 13.32% 1.60%
MJ-36 Chern 4.15% 0.020 61 XX 10.1 H1c 0.000 0.019 13.65% 1.58%
MJ-37 Chern 19.56% 0.140 66 XX 30.5 T2g1 0.001 0.135 9.85% 1.03%
MJ-38 Sar_Cau 15.72% 0.092 54 XY 17.0 W 0.026 Q1c-L332 0.045 15.46% 0.96% N/A N/A
R1a1e-
LS-13 ScySar_SU 80.82% 0.460 60 XY 44.1 W3a 0.136 0.237 12.48% 1.42% 0.44% 0.01% 0.22%
CTS1123
MJ-39 ScySar_SU 12.50% 0.104 64 XY 22.9 T1a1 0.031 R1a-Z645 0.054 12.44% 1.42% N/A N/A
U5a2+1 E2b1-
MJ-40 ScySar_SU 6.46% 0.036 65 XY 17.1 0.011 0.018 16.09% 1.23% N/A N/A
6294 PF6746
U5b2a1
MJ-41 ScySar_SU 65.84% 0.529 65 XX 72.1 0.005 0.514 5.72% 0.47%
a2
MJ-42 ScySar_SU 84.33% 0.525 57 XY 32.6 T1a1d 0.150 R1a-Z645 0.261 11.05% 1.42% 1.26% 0.37% 0.81%
MJ-43 ScySar_SU 78.60% 0.456 66 XX 59.4 C1e 0.004 0.442 5.53% 1.03%
U5b2a1
MJ-44 ScySar_SU 96.83% 0.717 67 XX 46.6 0.007 0.689 10.41% 1.80%
a1
MJ-56 ScySar_SU 71.71% 0.301 61 XX 36.1 U5b2c 0.003 0.284 8.09% 1.32%
MJ-51 Scy_Kaz 24.39% 0.085 56 XY 19.2 U5a1f1 0.025 R1a-M198 0.042 11.08% 0.00% N/A N/A
J1b-
MJ-52 Scy_Kaz 26.64% 0.092 55 XY 29.7 A23 0.026 0.046 10.23% 1.10% N/A N/A
P58(3/10)
MJ-53 Scy_Kaz 5.17% 0.022 57 XY? 3.9 U5b2b 0.006 R1-? 0.011 6.19% (0.53%) N/A N/A
MJ-54 Scy_Kaz 9.04% 0.027 50 XX 11.0 F2 0.000 0.026 5.94% 0.00%
AVERAGE: 40.10% 0.225 60 32.0 0.032 0.170 12.81% 0.92% 0.71%

Table S2. Sequencing results, DNA damage and contamination estimates and mitochondrial and Y chromosome haplogroups of the samples of this study. Related to STAR Methods sections
“Mapping”, “aDNA authentication”, “Calculating general statistics and determining genetic sex”, “Determining mtDNA haplogroups” and “Y chromosome variant calling and haplotyping”.
 Chr Position rsID Gene Anc EUR ASN DDAF Ukr_BA Late Srubnaya Cimmerians Scy_Ukr Chern Sar_Cau ScySar_SU Scy_Kaz
1 1243896 rs61766198 ACAP3 T T C 0,803130247 0/1 0/0 1/1 0/0 0/0 0/0 0/2 0/0
1 234342067 rs12036383 SLC35F3 A G A 0,830557964 1/1 0/0 0/0 0/1 0/0 0/0 1/3 0/0
2 26113913 rs78404020 na A A G 0,844114988 0/1 0/1 0/0 0/0 0/0 0/1 0/0 0/1
2 109513601 rs3827760 EDAR A A G 0,86 0/1 0/0 0/2 1/2 0/0 0/0 0/2 0/0
2 109543883 rs922452 EDAR C C T 0,902001033 1/2 0/0 0/2 0/1 0/0 0/1 0/1 0/1
2 216321788 rs1250221 na C C G 0,800191893 0/1 0/0 0/0 0/1 0/0 0/0 0/1 1/1
3 108192751 rs4365635 MYH15 T T C 0,846107718 1/1 0/0 1/1 0/2 0/0 0/0 0/2 0/0
5 33951693 rs16891982 SLC45A2 C G C 0,962663985 0/0 1/1 2/3 0/0 0/1 0/0 0/4 1/1
6 2745352 rs6927195 MYLK4 G C G 0,926185951 0/0 1/1 0/2 0/0 0/0 1/1 0/2 0/2
7 71689925 rs71551254 CALN1 A C A 0,806820488 0/0 0/1 1/1 0/0 0/0 0/0 0/0 2/2
8 12720349 rs11778591 na A A C 0,808033655 0/1 0/1 0/0 0/1 0/0 1/1 0/3 0/0
9 7385490 rs2148360 na T T C 0,839511412 1/1 0/1 1/1 0/0 0/1 0/0 1/1 0/0
10 28398905 rs10826399 MPP7 G T G 0,799762902 0/0 0/0 0/1 0/0 0/0 0/0 0/2 0/0
14 97324289 rs12434466 VRK1 A A G 0,810566083 0/1 0/1 1/2 0/0 0/0 0/1 0/1 0/0
15 48426484 rs1426654 SLC24A5 G A G 0,982056202 0/0 0/0 0/2 0/1 0/1 0/1 0/5 0/1
16 86082532 rs61691136 na C T C 0,817932727 0/0 0/1 1/2 0/1 0/0 0/0 0/2 0/0
17 4400392 rs11657785 na C T C 0,841720944 0/0 0/0 0/1 0/0 0/0 0/1 0/1 1/1
20 568696 rs6053171 na G G T 0,890169198 0/0 0/0 0/0 0/0 0/0 0/0 0/5 0/1
Total: 4/11 2/8 8/21 1/10 0/3 2/7 2/37 5/11

Table S4. Presence or absence of alleles highly differentiated between modern Europeans and modern East Asians in the sample groups of this study. Related to STAR Methods section
“Inspecting sites highly differentiated between modern Europeans and East Asians”. The numerator of each cell shows the number of samples displaying at least one read carrying the Asian
allele while the denominator reports the total number of individuals with at least one sequencing read at a given site. The bottom row shows the total of each column. Red cells denote sites
for which at least one Asian allele was observed in a given sample group. Chr=Chromosome; Anc=Ancestral allele; EUR=European modal allele; ASN=East Asian modal allele; DDAF=Delta of
derived allele frequencies as reported in [S23].
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