The Yamnaya Steppe Herders from Russia

Population genetic analysis
The studied Conqueror group very likely represent real populations, as 24 of the 29 samples came from 4 nearby cemeteries (Karos 1,2,3 and Kenézlő) with identical archaeological and anthropological groupings and the studied Magyarhomorog individuals were also categorized as belonging to the same early Conqueror elite. The Avar group was assembled from several different cemeteries of a wider timespan, thus they cannot represent the Avar period population of the Carpathian Basin, however their relatively homogenous Hg distribution indicate that the Avar elite embodied the same east Eurasian sub-population throughout their reign, so it appeared to be meaningful to include them in the analysis.

In order to find the most similar populations to our studied ones, we compared the Hg distribution of the Avar and Conqueror elite to that of 78 modern Eurasian populations (Supplementary Table S4) and represented their relations on MDS plot displayed on Fig. 5. Only one individual was included in the analysis from first degree relatives. In order to increase resolution, we separately calculated the frequencies of each sub-Hg-s which are present in our samples, while frequencies of all other subclades were combined as listed in Supplementary Table S4. Similar Hg distributions are mapped into neighboring positions on the MDS plot, which clearly separates populations according to their geographic locations. Along the x axis east Eurasian populations map to the right while Europeans are compressed at the left. Along the y axis Siberian populations are sequestered at the top, while East Asian ones at the bottom of the graph. The Avars are obviously mapped to the Siberian side with smallest weighted Euclidean distances from Koryaks (KRY), Teleuts (TEL), Khantys (KHT), Komis, (KOM) and Dolgans (DLG). The Conquerors are positioned between eastern Europeans, Central Asians and Siberians but their exact relations are hard to make out because of the crowding at the European side.


Figure 5

In order to better discern the position of the Conquerors we redraw the MDS plot without the eastern Asian and Siberian populations (Fig. 6). Though having removed subset of the data MDS imaging in two-dimensions inevitably rearranged the remaining components, but the general organization of the population clusters remained unchanged: Western-Northern Europeans map to the left, Central-eastern European and Balkan people around the middle, Central Asians to the top right, and Caucasus-Middle East populations to bottom right. Conquerors remained between Central Asians and eastern Europeans, with smallest weighted Euclidean distances from Bashkirs (BSH), Hungarians (HUN), Tajiks (TJK), Estonians (EST), Kazakhs (KAZ), Uzbeks (UZB) and Slovaks (SVK).


Figure 6

The considerable frequency of Hg N1a in Conquerors and especially in Avars facilitates another analysis in which the frequency distribution of their N1a subbranches can be compared to that of all Eurasian populations carrying this Hg, which is described in22. The N1a database of22 contains several relevant Eurasian populations missing from Supplementary Table S3, moreover the Y dataset of Supplementary Table S4 has a low resolution of N1a subclades, thus this analysis is expected to provide additional information. We combined the N1a dataset published in22 with our data as presented in Supplementary Table S5 and performed Principal Component Analysis (PCA) as shown on Fig. 7.


Figure 7

PCA also separates populations according to their geographical positions, as identical colors appear in the same segments of the graph. The Avars map to the top right corner, close to eastern Siberian Chukchis (CHK), Yukaghirs (YUK), Eskimos (ESK) and Transbaikalian Buryats (BUR). The Conquerors (CONQ) map to the middle of the graph, very close to eastern European Belarusians (BLR), Ukrainians (UKR), Russians (RUS) and Polish (POL), not far from Volga-Uralic region Bashkirs (BSH), Volga Tatars (TAT), Komi Permians (KOMP) and Mordvins (MRD), also surrounded by Central Asian groups; Karanogays (KNO), Karakalpaks (KARA), Uzbeks (UZB) as well as Crimean Tatars (TATC), Mongols (MON), Selkups (SEL) and Dolgans from Taymyr (DLGT)

Abstract
For millennia, the Pontic-Caspian steppe was a connector between the Eurasian steppe and Europe. In this scene, multidirectional and sequential movements of different populations may have occurred, including those of the Eurasian steppe nomads. We sequenced 35 genomes (low to medium coverage) of Bronze Age individuals (Srubnaya-Alakulskaya) and Iron Age nomads (Cimmerians, Scythians, and Sarmatians) that represent four distinct cultural entities corresponding to the chronological sequence of cultural complexes in the region. Our results suggest that, despite genetic links among these peoples, no group can be considered a direct ancestor of the subsequent group. The nomadic populations were heterogeneous and carried genetic affinities with populations from several other regions including the Far East and the southern Urals. We found evidence of a stable shared genetic signature, making the eastern Pontic-Caspian steppe a likely source of western nomadic groups.

INTRODUCTION
The Pontic-Caspian steppe (PCS), stretching from the southern Urals to the western North Pontic lands, was the stage of various demographic changes in the past, and several of those remain unknown. During the Bronze and Iron Age, the area was inhabited by a succession of nomadic populations that had significant impact on the cultural development of both Asia and Europe (1, 2). Possibly the best known of these groups is the Yamnaya. Recent genomic studies have revealed cross-continental Early Bronze Age migrations (~3000 BCE) of the nomadic people associated with the Yamnaya horizon (3, 4). The migration introduced the Caucasus genetic component to the genetic landscape of Europe. In Central Europe, Yamnaya ancestry first appeared among people from the Corded Ware complex and has since been found in many subsequent ancient and present-day populations. However, the Pontic-Caspian steppe was critical not only for Early Bronze Age Yamnaya migrations but also because of succeeding movements and population transformations that took place in the developed classical stage of the Late Bronze and Iron Ages between 1800 BCE and 400 CE. This period covered the development of the Srubnaya and Alakulskaya Cultures (~1800–1200 BCE), associated with small settlement sites distributed from the Urals to the Dnieper valley (1). From around 1000 BCE, pre-Scythian nomadic populations started to appear in the western Pontic-Caspian steppe including the Cimmerians known from historical sources (5). Despite regional variation and local peculiarities, the Cimmerians were not associated with any uniform type of archaeological material culture (6). In the seventh century BCE, they were succeeded by the Scythians, who plausibly pushed the Cimmerians into Asia Minor (7). Between 700 and 300 BCE, the Scythians, representing mobile pastoral nomads of a new militaristic type (1), dominated the Pontic-Kazakh steppe, occupying an area from the Altai to the Carpathian Mountains. Their decline began around 300 BCE and was caused by intensifying hostile relations with the Macedonians in the West and the invasion of the Sarmatians from the East. The Sarmatians and the Scythians are thought to have coexisted for a few centuries, but eventually, the former group prevailed (2), resulting in the Scythian downfall. The Sarmatians are believed to comprise a number of groups of similar nomadic background (8), and they became the politically most influential force within the eastern fringes of the Roman Empire at the time. Their decline (~400 CE) was associated with the attack of the Goths and the subsequent invasion of the Huns (8).

The genomic structure of the Bronze and Iron Age (1800 BCE–400 CE) populations in the Pontic-Caspian steppe has not been fully resolved. While earlier genomic studies have suggested close links between the Srubnaya and the central European Late Neolithic and Bronze Age populations (9), our knowledge of the genetic origins of the Cimmerians is limited. Genetic analyses of maternal lineages of Scythians suggest a mixed origin and an east-west admixture gradient across the Eurasian steppe (10–12). The genomics of two early Scythian Aldy-Bel individuals (13) showed genetic affinities to eastern populations of Central Asia (12). However, population interactions and the origin of Scythians of the Pontic-Caspian steppe remain poorly understood. Similarly, little is known about the origins and genetic affinities of the Sarmatians. Genomic studies suggest that the latter group may have been genetically similar to the eastern Yamnaya and Poltavka Bronze Age groups (12). To investigate the demographic dynamics in the Pontic-Caspian steppe, we generated and analyzed genomes of the Late Bronze and Iron Age individuals from the region (Fig. 1, A and B).



Fig. 1 Radiocarbon ages and geographical locations of the ancient samples used in this study.
Figure panels presented counterclockwise: (A) Bar plot visualizing approximate timeline of presented and previously published individuals. (B) Map showing the locations of ancient individuals sequenced in this study and the locations of previously published ancient individuals used in comparative analyses. (C) Principal component analysis (PCA) plot visualizing 35 Bronze Age and Iron Age individuals presented in this study and in published ancient individuals (table S5) in relation to modern reference panel from the Human Origins data set (41).

RESULTS
We produced genome-wide sequence data with genome coverage between 0.01× and 2.9× per individual for 35 Bronze Age and Iron Age individuals from the Pontic-Caspian steppe from four chronologically sequential cultural groups, which comprise Srubnaya-Alakulskaya individuals (n = 13), Cimmerians (n = 3), Scythians (n = 14), and Sarmatians (n = 5), with radiocarbon dates between ca. 1900 BCE and 400 CE (Fig. 1, A and B; tables S1 to S3; and fig. S1, A and B). All DNA libraries displayed damage patterns typical of ancient DNA (fig. S2) (14). To ensure data integrity, we calculated mitochondrial DNA (mtDNA)–based contamination levels using distribution of private polymorphisms in mtDNA (15) and a Bayesian likelihood method (16). The former yielded point estimates of contamination between 0 and 10% [95% confidence intervals (CIs) between 0 and 17%], and the latter method revealed that all individuals carried sequences with >89% probability of being authentic (table S4). Thus, we included all sequenced individuals in the downstream analyses.

Late Bronze Age (LBA) Srubnaya-Alakulskaya individuals carried mtDNA haplogroups associated with Europeans or West Eurasians (17) including H, J1, K1, T2, U2, U4, and U5 (table S3). In contrast, the Iron Age nomads (Cimmerians, Scythians, and Sarmatians) additionally carried mtDNA haplogroups associated with Central Asia and the Far East (A, C, D, and M) (table S3) (11, 18). The absence of East Asian mitochondrial lineages in the more eastern and older Srubnaya-Alakulskaya population suggests that the appearance of East Asian haplogroups in the steppe populations might be associated with the Iron Age nomads, starting with the Cimmerians. The Y chromosome haplogroup variation in 17 of 18 males was limited to two major haplogroup lineages within the macrohaplogroup “R” (table S3). The Srubnaya-Alakulskaya individuals carried the Y haplogroup R1a, which showed a major expansion during the Bronze Age (19). It has previously been found in Bronze Age individuals from the Krasnoyarsk Kurgan in Siberia (20). The Iron Age nomads mostly carried the R1b Y haplogroup, which is characteristic of the Yamnaya of the Russian steppe (4). An exception was a Cimmerian individual (cim358) who carried the Q1* lineage associated with the east (table S3).

Genetic relationships between Eurasian steppe nomads and present-day populations
PCA on the autosomal genomic data (Fig. 1C and table S5) revealed the following: (i) Srubnaya-Alakulskaya individuals exhibited genetic affinity to northern and northeastern present-day Europeans (fig. S3), and these results were also consistent with outgroup f3 statistics (table S6 and fig. S4A). (ii) The Cimmerian individuals, representing the time period of transition from Bronze to Iron Age, were not homogeneous regarding their genetic similarities to present-day populations according to the PCA. F3 statistics confirmed the heterogeneity of these individuals in comparison with present-day populations (table S6 and figs. S3 and S4C). (iii) The Scythians reported in this study, from the core Scythian territory in the North Pontic steppe (12), showed high intragroup diversity. In the PCA, they are positioned as four visually distinct groups compared to the gradient of present-day populations (Fig. 1C): (i) A group of three individuals (scy009, scy010, and scy303) showed genetic affinity to north European populations, hereafter referred to as a north European (NE) cluster. (ii) A group of four individuals (scy192, scy197, scy300, and scy305) showed genetic similarities to southern European populations, hereafter referred to as a south European (SE) cluster. (iii) A group of three individuals (scy006, scy011, and scy193) located between the genetic variation of Mordovians and populations of the North Caucasus, hereafter referred to as a steppe cluster (SC). In addition, one Srubnaya-Alakulskaya individual (kzb004), the most recent Cimmerian (cim357), and all Sarmatians fell within this cluster. In contrast to the Scythians, and despite being from opposite ends of the Pontic-Caspian steppe, the five Sarmatians grouped close together in this cluster. (iv) A group of three Scythians (scy301, scy304, and scy311) formed a discrete group between the SC and SE and had genetic affinities to present-day Bulgarian, Greek, Croatian, and Turkish populations, hereafter referred to as a central cluster (CC). All PCA results were consistent with outgroup f3 statistics (table S6 and figs. S3 and S4, B and D). Finally, one individual from a Scythian cultural context (scy332) is positioned outside of the modern West Eurasian genetic variation (Fig. 1C) but shared genetic drift with East Asian populations (table S6 and fig. S4B).