Supplementary Materials for

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1 advances.sciencemag.org/cgi/content/full/4/10/eaat4457/dc1 Supplementary Materials for Ancient genomes suggest the eastern Pontic-Caspian steppe as the source of western Iron Age nomads Maja Krzewińska*, Gülşah Merve Kılınç*, Anna Juras, Dilek Koptekin, Maciej Chyleński, Alexey G. Nikitin, Nikolai Shcherbakov, Iia Shuteleva, Tatiana Leonova, Liudmila Kraeva, Flarit A. Sungatov, Alfija N. Sultanova, Inna Potekhina, Sylwia Łukasik, Marta Krenz-Niedbała, Love Dalén, Vitaly Sinika, Mattias Jakobsson, Jan Storå, Anders Götherström* *Corresponding author. (M.K.); (G.M.K.); (A.G.) The PDF file includes: Published 3 October 2018, Sci. Adv. 4, eaat4457 (2018) DOI: /sciadv.aat4457 Archaeological context information Fig. S1. Radiocarbon dating and diet. Fig. S2. Nucleotide misincorporation patterns at last 30-bp sequences. Fig. S3. PCA with modern populations. Fig. S4. Outgroup f3 statistics. Fig. S5. PCA with Bronze Age individuals. Fig. S6. PCA with Paleolithic and Mesolithic individuals. Fig. S7. PCA with Neolithic individuals. Fig. S8. PCA with Chalcolithic individuals. Fig. S9. PCA with Iron Age individuals. Fig. S10. ADMIXTURE analysis. References (56 60) Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/4/10/eaat4457/dc1) Table S1 (Microsoft Excel format). Archaeological information for individuals used in this study. Table S2 (Microsoft Excel format). Stable isotope and radiocarbon dating results information for individuals used in this study. Table S3 (Microsoft Excel format). Sequencing statistics and mitochondrial variants for individuals sequenced in this study.

2 Table S4 (Microsoft Excel format). Mitochondrial contamination estimates for individuals sequenced in this study. Table S5 (Microsoft Excel format). Ancient sample data set details including the number of SNPs overlapping with modern reference. Table S6 (Microsoft Excel format). Outgroup f3 statistics for individuals sequenced in this study. Table S7 (Microsoft Excel format). Summary f4 statistics between Srubnaya and Andronovo, Afanasievo, and Sintashta. Table S8 (Microsoft Excel format). Summary f4 statistics between Srubnaya-Alakulskaya tested as a single population or individuals and Karasuk and other Bronze Age populations. Table S9 (Microsoft Excel format). Summary f4 statistics between Srubnaya-Alakulskaya tested as a single population or individuals and Yamnaya and other Bronze Age populations. Table S10 (Microsoft Excel format). Diversity estimates in Bronze Age populations. Table S11 (Microsoft Excel format). Summary f4 statistics between Cimmerians, Scythians, and other ancient populations. Table S12 (Microsoft Excel format). Summary f4 statistics between Cimmerians, Sarmatians, and other ancient populations. Table S13 (Microsoft Excel format). F3 support for different components changing with time in Cimmerians as observed in ADMIXTURE. Table S14 (Microsoft Excel format). Summary f4 statistics between Srubnaya/Srubnaya- Alakulskaya and Andronovo, Afanasievo, and Sintashta. Table S15 (Microsoft Excel format). Summary f4 statistics between Scythians tested as population, Yamnaya, and other Bronze Age populations. Table S16 (Microsoft Excel format). Summary f4 statistics between Cimmerians tested as a population, Srubnaya/Srubnaya-Alakulskaya, and other Bronze Age populations. Table S17 (Microsoft Excel format). Summary f4 statistics of multiway comparisons of individuals form the study together with Srubnaya. Table S18 (Microsoft Excel format). Summary f4 statistics between Sarmatians and other Bronze Age populations.

3 Archaeological context information Nomadic world of the Eurasian steppe zone The Eurasian steppe zone is a vast region united by shared ecology. The extensive grasslands of the steppe are able to support large herds of grazing animals such as cattle, horses, sheep and goats, which in the past facilitated the development of nomadic herding. The horse became an essential element of the nomadic herders life allowing people to follow their herds moving between seasonal pastures. Horse domestication, invention of horse-back riding and using steppe resources for the maintenance of large herds of hoses was of key importance in the emergence of the Western Nomads providing an unprecedented mobility and military advantage over the settled populations in the area (1). Economically, they were nomadic pastoralists that often depended on sedentary populations for acquisition of pottery and iron tools. Metal trading and iron production at the time occurred in settlements of various types. The nomadic groups relied on trade with the sedentary groups as there was no place for specialized workshops within the mobile societies (2). Prior to emergence of the Nomadic peoples the steppe zone was inhabited by various Bronze Age populations (7). They had different genetic origin but shared similar subsistence strategies where keeping domestic animals and small scale primitive farming was practiced alongside hunting and gathering. The Bronze Age populations of the Eurasian steppe are divided into two main social and cultural complexes; the Srubnaya (Timber Grave) and the Andronovo (Alakulskaya) cultures. The Srubnaya complex was dispersed the Western steppe region while the Andronovo (Alakulskaya) complex was associated with the Eastern Eurasian steppe, beyond the Ural Mountains. The Volga-Kama region, south of the Urals was a shared contact zone between the two groups. In the developed classical stage of Late Bronze Age (LBA) people had begun using horses for transportation. The skill was crucial for increased mobility of the steppe zone

4 inhabitants in the 10 th century BCE which was further induced by a changing climate and a search for more hospitable conditions. This increased mobility gradually resulted in the adaptation of nomadic cattle breeding as the main form of subsistence among some of the LBA groups. Between the 10 th and the 8 th century BCE the mobile herders dominated the steppe and started using iron. Also, a development of a more stratified social structure is seen. The Iron Age Nomads first appear in the eastern fringes of Europe in the early first millennium BC. The steppe nomads consisted of various ethnically and culturally distinct groups united by certain common cultural elements, including differently shaped and sized barrow or kurgan burials, similar weaponry (including bows, arrows and swords), presence of bronze cauldrons and mirrors, shared animalistic art motifs, similar horse bridles and olenniye kamni (tree-legged carved stone stelae) (2). The Iron Age nomadic groups were part of what has been referred to as the Scytho-Siberian world suggesting a certain degree of unity, but despite a number of commonly shared characteristics there were distinct local variation between those groups. The contact zones between the nomadic world and both the eastern and the western states are sources of available historical information about the nomadic peoples, including Persian, Chinese or Byzantine sources. However, little historical information is available from the heart of the nomadic world such as southern Urals. Srubnaya (Timber Grave culture) The Srubnaya culture is known from the eastern part of the Pontic-Caspian Steppe region. It was an LBA complex dated between BCE, characterized by a burial tradition in which the dead were interred in individual, often timber-framed burials and were accompanied by animal remains. Despite its name the timber construction is not as common in Srubnaya burials as previously thought (2). The Srubnaya complex succeeds the earlier Catacomb, Poltavaka, Sintashta and Potapovka cultures. The latter two may have been direct

5 predecessors of both Srubnaya and Andronovo (Alakulskaya), which became the dominating cultural complexes over both the eastern and the western parts of the steppe region in LBA. The two LBA cultures coexisted for centuries and in the southern Urals there is evidence of extensive contacts with many sites with an archeological record exhibiting traits and characteristics of both traditions (1, 56, 57). According to many scholars representatives of the Srubanya cultural complex were the predecessors of the Cimmerians (6). Cimmerians The Cimmerians managed to create the first extensive union of the nomadic tribes in Southern Russia (7). According to the written sources the Cimmerians were occupants of the North Black Sea region and directly preceded the Scythians. Little is known about their origins and the definition of Cimmerian is to some extent controversial due to little archaeological uniformity allowing for a confident identification of Cimmerian sites. For that reason, few human remains have been identified as Cimmerian thus far. The Cimmerians appear in the North Pontic Region (NPR) in the developed classical stage of late Bronze Age and beginning of the Iron Age (2). They probably mastered the art of iron coasting sometime in the 10 th century BCE. According to Herodotus the Cimmerians were driven from NPR sometime between the 8 th and 7 th centuries BCE by incoming Scythians that followed the retreating Cimmerians to Asia Minor (7). Scythians The Scythians are probably the most famous of the nomadic peoples, nowadays defined by characteristic animal art forms and the kurgan burial tradition. For the Black Sea Scythians most evidence for an origin point to Central Asia based on the absence of animalistic art forms in the local Black Sea Late Bronze Age populations (known from East Asian Bronze

6 Age populations), the records of Herodotus reports, and the finds of horse harnesses that are earlier in the Asiatic burials, etc. However, Grakov suggested that the Scythians rather were genetically linked to the Black Sea Late Bronze Age populations and that the culture was autochthonous to North Pontic-Caspian Steppe region. Finally, a third hypothesis, supporting a polycentric origin, suggests an independent local development with stable contacts between neighboring groups. Based on similarities in material culture there seems the have been well developed connections in Early Iron Age between eastern and west steppe populations. However, to the contemporaries, Scythians could have been any of the nomadic tribes from the East (1). The area of Scythian influence stretched from Altai to the Carpathian Mountains, and from Caucasus in the South to the North European Plain to the North. Scythians were horseback warriors, settlers, craftsmen and tradesmen who formed Great Scythia in the 6 th century BC. Known for their military skills Scythians sometimes also served as mercenaries: for instance in ancient Greece. The 3 rd century BCE was characterized by intensifying hostile relations with Macedonians in the west and the invasion of the Sarmatians from the east, which eventually led to the Scythian downfall. The Sarmatians and the Scythians coexisted for a few centuries but eventually the Sarmatians succeeded the Scythians in the region (1). Sauromatians and Sarmatians The Sarmatians, became the politically most influential nomadic group in the eastern fringes of the Roman Empire. Herodotus reported that Sarmatians were related to the Scythians, though they had a different belief system. Those new tribes probably arose in response to Alexander the Great s expansive campaigns. The Sauromatians (Sarmatians) first appear in 6 th century BCE and the term is usually used to refer to numerous nomadic tribes that inhabited the territories to the east of Scythia. The cultural and social relationships between the Sauromatians and Sarmatians are unclear. Chronologically, the entire period of the Sauro-

7 Sarmatian culture is roughly divided into four stages: the Sauromatian period (6 th -4 th century BCE), the Early Sarmatian period (4 th -2 nd century BCE), the Middle Sarmatian (2 nd century BCE to 2 nd century CE) period, and the Late Sarmatian period (2 nd -4 th century CE). According to K. F. Smirnov the origin of Sauro-Sarmatian tribes should be sought in the Andronovo and Srubnaya (Timber Grave) cultures. It has been suggested that the Early Sarmatians emerged from nomadic peoples of southern Urals under the influence from trans- Uralian and Kazakh steppe peoples (7). Description of the sites and individuals Kazburun 1/Muradym 8 ( BCE) The Muradym 8 settlement is situated between the Urshak River and Bely Kluch in Bashkorstan territory (57). The steppe plane between Dejma and Urshak Rivers is marked by extensive settlement remains of the developed classical stage of Late Bronze Age, and formed the contact zone between the Srubnaya and the Alakulskaya cultural complexes. The Kazburun 1 site is a barrow burial ground associated with a Muradymovo settlement site (ca 1,500 BCE), located close to the Usmanovo village. The burial ground consists of 33 barrows (six barrows were destroyed during the construction of the Usmanovo-Turumbet highway). The barrows are located in four clusters; in the south-west (nine mounds), in the north-west (nine barrows), in the central part (tree barrows) and in the south-east (six mounds) (56). Both the settlement and the burial ground are associated with the same population, however, the area is, as mentioned above, a mixing zone between Srubnaya and Andronovo (Alakulskaya). The observed mixed burial customs and relatively short site occupation (see section: 2. Radiocarbon dating) suggested the population may have consisted of people with various

8 backgrounds. We analyzed four individuals from the Muradym 8 settlement and nine individuals from three barrows format the Kazburun 1 burial ground. Glinoe ( BCE) The Glinoe site is located in the Slobodzeya district, in the southeastern part of Moldova ( N, E). It consists of 114 Scythian barrows dated from the end of the 4th century BCE to the 2nd century BCE. The assessment of the chronology of the cemetery and cultural affiliation of the buried individuals are based on burial inventory found during the excavations (mainly amphorae and epigraphic data, as well as ceramics and lamps). The majority of the Scythian barrows contained single graves, rarely double graves, while multiple graves occurred even less frequently. Human skeletal remains were deposited at the depth of 1-6 m below the ground level. Ten of the excavated individuals were included in present study; K103B1, sample: scy192; K75B1, sample: scy193; K50B1S1, sample: scy197; K89B3S2, sample: scy300; K89B1, sample: scy301; K75B2, sample: 303; K81B1S1, sample: scy304; K87B1, sample: scy305; K43B1, sample: scy311; K65B1, sample: scy332. Glinoe Sad The Glinoe SAD archaeological site is located in the south-eastern part of present-day Moldova, about 2 km from the Glinoe site ( N E). The cemetery consists of 10 barrows dated from Early Bronze Age to Early Iron Age (from the 2nd half of the 4th millennium BCE to the 4th century BCE). In 2015 the barrow no 4 containing a total of 18 burials was excavated. Three of them were Cimmerian secondary burials: K4B13, K4B16, K4B17. The assessment of the cultural affiliation of the recovered individuals was made on the basis of grave goods. In the burial K4B16, a hand-made round-shaped pot with a narrow neck was found. It is a typical kitchen vessel of the burials of the Chernogorovka culture

9 (which is the historical name of the Cimmerians) of the second half of the 8th century BCE to the first half of the 7th century BCE (58). In the present study 2 out of 3 Cimmerian individuals were included: K4B16 (sample: cim357) and K4B17 (sample: cim358). As the skeletal remains of the third individual were poorly preserved, adna analyses could not be performed. The skeleton of the first individual belonged to a male aged years, whereas the skeleton of the second one represented a male aged years. The burial mound of this barrow was totally destroyed by plowing. Mokra The Mokra archaeological site is located on the left bank of the Lower Dniester in the Rybnitsa district, in the north-eastern part of Moldova ( N E). This site is dated from the Early Bronze Age to the Early Iron Age (from the end of 4th millennium BCE to 8th century BCE). In 1994 excavations of barrow no 1 were conducted. This barrow embraced in total 18 burials. One of them was a secondary Cimmerian (K1B16, sample: cim359) burial. The individual was a female aged years. The archaeological affiliation of this individual was assessed according to two vessels (large pots) found during the excavations. One of them is polished and has ledges in the lower part of the body. At the second vessel s shoulder there is a geometric ornament, stamped with a spiked stamp. The vessels can be dated within a wide range from the 9th century BCE to the 8th century BCE (58).

10 Starosillya ( BCE) and Nesterivka ( BCE), Ukraine The Scythian individuals SCY006 and SCY009-SCY011 from mainland Ukraine included kurgan groups from Starosillya, Cherkasy Region (SCY006, SCY009, SCY010, 7 th century BCE) and Nesterivka, Cherkasy Region (dated to 4 th century BCE). Starosillya kurgan group The Kurgan burial ground of the Scythian period near the village of Starosillya (Gorodishche District, Cherkasy Region) was excavated by the Forest-Steppe Right-Bank Expedition of the Institute of Archaeology of the National Academy of Sciences of Ukraine from 1984 onwards. Two complexes with barrow groups were identified, in total containing about 400 mounds. The individuals selected for DNA analysis come from mounds 1 and 3 from the first burial group, investigated in 1997 by the expedition of the Cherkasy Regional Archaeological Inspection (59). The burial goods are few, and are partially or completely destroyed by plowing. The burials were carried out in pits of rectangular shape, oriented along the line NW-SE, covered with wooden partly burnt logs, with a gently sloping entrance from the south-eastern side. Most often the burial mounds contained one burial, but sometimes the skeletal remains were found in concentration, which suggests the dismemberment of bodies before the burial. Anthropological research has not yet been published. In mound 1, a deep scoop, a miniature pot, a round spindle and a bronze nail pin were found. The burial mound of burial no 3 consisted of a ladle, a cup, two bronze pins, glass beads, paste, ceramics and lead, a ceramic spindle and a bronze pendant in the form of a ring. In addition, remains of dyes of bright red color were found in this burial. Based on the inventory and burial rites, the Scythian burials at Starosillya can be dated to the 7 th century BC.

11 Nesterivka Burial During the construction of the Kyiv-Odessa Highway in , on the outskirts of the village of Nesterivka, in the Mankivsky District of Cherkasy Region, a late-scythian burial containing a golden treasure was discovered during rescue excavations by the Cherkasy Regional Archaeological Inspection. The burial was of a woman belonging to the Scythian military nomadic nobility, topologically dated to the IV century BC. The burial was of the socalled catacomb type. At first, a mine was excavated to a depth of 7.4 m, from which the burial chamber was extended. A total of 181 gold objects were discovered within the burial, including two pendants to the headgear, as well as embroidery bands for clothing. Six of the bands had images of human face, one contained a plant-geometric ornament. Together with the woman, a horse was also buried. Additional material culture finds included 16 silver jewelry harnesses, as well as a bronze lantern, 10 arrows, ceramic loom weight and spindle. The burial appeared to have been looted in ancient times. Cherniy Yar The burial site Cherniy Yar is located in the south of Orenburg region in the Southern Urals (N51 04,4090' E55 04,0210'). It consists of about 70 barrows dated from the Bronze Age to the Middle Ages. In 2007 a unique burial belonging to the Late Sarmatian culture, was discovered in barrow no 42 (60). The barrow was 1 m high and 30 m in diameter. The burial represents a deep square grave surrounded by a rampart and ditch and covered by a wooden funerary building. It is a double burial of a man and a woman. The woman's age is estimated between 55 and 60, the man s years. The skulls were artificially deformed during their lifetime. In ancient times the barrow had been robbed, the bones were found scattered all over the grave at a depth of m below the ground level. The items found in the burial are: an imported glass vessel, a Chinese mirror of the Han period in a wooden case, a sharpening

12 stone, a lot of gold and bronze sewn plaques, a pendant, a bronze cauldron, ceramic vessels imported from the Caucasus and Central Asia, ceramic spindle whorls, a sword-belt silver plaque with a bronze buckle. Temyasovo The analyzed individual were recovered from barrow no 9 of the Temyasovo-1 burial ground, located in the Baymak District, east of the Temyasovo village, in the Republic of Bashkortostan, Russia. The burial ground contained 13 burial mounds. The remains of three graves lying parallel to each other were found under one mound excavated from the burial chamber. Anthropological research has shown that they belong to an adolescent aged years, a woman and a man of mature age. The skulls have apparent traces of artificial deformation of circular type. Anthropological analyses of the skeletal remains and dental traits indicate a mixture of morphological traits. The distribution of various genetic traits suggested possibility of a kinship burial. Analysis of activity related osteological markers, suggests that the man and woman were horsemen. The discovered inventory comprised of a variety of products: a bronze fibula with articulatedcurved back with a curl at the end, a biconical whorl, a brooch, a navicular earring, a long knife with a bone handle, and a composite bronze cauldron with loop-handles. Analyses of available analogs and prevalence of chronologically distinct artefacts allows to date the complex to the III century and attribute it to the Hun-Sarmatian time. Archaeological context details and radiocarbon dates for investigated individuals are given in table S1.

13 Supplementary Figures Fig. S1. Radiocarbon dating and diet. (A) Calibrated radiocarbon dates. (B) Diet.

14 Fig. S2. Nucleotide misincorporation patterns at last 30-bp sequences. The reduced pattern as seen in kzb002 is a result of UGD treatment.

15 Fig. S3. PCA with modern populations. Ancient individuals sequenced in this study are projected on present day genetic variation. Iron Age individuals including Cimmerians, Scythians and Sarmatians are plotted as triangles. Bronze Age individuals, Srubnaya- Alakulskaya, are plotted as pentagons.

16 Fig. S4. Outgroup f3 statistics. (A) Outgroup f3-statistics calculated in the form of f3(yoruba, SrubnayaAlakulskaya_individual, Modern_population) projected on a geographical map. Warmer colors show more genetic drift shared between Srubnaya- Alakulskaya individual and modern population. (B) Outgroup f3-statistics calculated in the form of f3(yoruba, Scythian_individual, Modern_population) projected on a geographical map. Warmer colors show more genetic drift shared between Scythian individual and modern

17 population. (C) Outgroup f3-statistics calculated in the form of f3(yoruba, Cimmerian_individual, Modern_population) projected on a geographical map. Warmer colors show more genetic drift shared between Cimmerian individual and modern population. (D) Outgroup f3-statistics calculated in the form of f3(yoruba, Scythian_individual, Modern_population) projected on a geographical map. Warmer colors show more genetic drift shared between Scythian individual and modern population. Fig. S5. PCA with Bronze Age individuals. Ancient individuals sequenced in this study and published Bronze Age individuals are projected on present day genetic variation. Iron age individuals including Cimmerians, Scythians and Sarmatians are plotted as triangles. Bronze Age individuals including Srubnaya-Alakulskaya and published samples are plotted as pentagons. Present-day individuals are plotted as gray points.

18 Fig. S6. PCA with Paleolithic and Mesolithic individuals. Ancient individuals sequenced in this study and published Paleolithic and Mesolithic individuals are projected on present day genetic variation. Iron Age individuals including Cimmerians, Scythians are plotted as triangles. Bronze Age individuals, Srubnaya-Alakulskaya, are plotted as pentagons. Paleolithic and Mesolithic individuals are plotted as circles. Present-day individuals are plotted as gray points.

19 Fig. S7. PCA with Neolithic individuals. Ancient individuals sequenced in this study and published Neolithic individuals are projected on present day genetic variation. Iron Age individuals including Cimmerians, Scythians are plotted as triangles. Bronze Age individuals, Srubnaya-Alakulskaya, are plotted as pentagons. Neolithic individuals are plotted as squares. Present-day individuals are plotted as gray points.

20 Fig. S8. PCA with Chalcolithic individuals. Ancient individuals sequenced in this study and published Chalcolithic individuals are projected on present day genetic variation. Iron Age individuals including Cimmerians, Scythians are plotted as triangles. Bronze Age individuals, Srubnaya-Alakulskaya, are plotted as pentagons. Chalcolithic individuals are plotted as circles. Present-day individuals are plotted as gray points.

21 Fig. S9. PCA with Iron Age individuals. Ancient individuals sequenced in this study and published Iron Age individuals are projected on present day genetic variation. Iron Age individuals including Cimmerians, Scythians and Sarmatians and published Iron Age samples are plotted as triangles. Bronze Age individuals, Srubnaya-Alakulskaya, are plotted as pentagons. Present-day individuals are plotted as gray points.

22 Fig. S10. ADMIXTURE analysis. (A) ADMIXTURE analysis for K=2 to K=15. (B) ADMIXTURE results for modern populations projected on a world map for K=15.