Gene-flow from steppe individuals into Cucuteni-Trypillia associated populations indicates long-standing contacts and gradual admixture

www.nature.com/scientificreports OPEN Gene-flow from steppe individuals into Cucuteni-Trypillia associated populations indicates longstanding contacts and gradual admixture Alexander Immel1, Stanislav Țerna2, Angela Simalcsik 3, Julian Susat1, Oleg Šarov4, Ghenadie Sîrbu 5, Robert Hofmann6, Johannes Müller6*, Almut Nebel1 & Ben Krause-Kyora 1* The Cucuteni-Trypillia complex (CTC) flourished in eastern Europe for over two millennia (5100–2800 BCE) from the end of the Neolithic to the Early Bronze Age. Its vast distribution area encompassed modern-day eastern Romania, Moldova and western/central Ukraine. Due to a lack of existing burials throughout most of this time, only little is known about the people associated with this complex and their genetic composition. Here, we present genome-wide data generated from the skeletal remains of four females that were excavated from two Late CTC sites in Moldova (3500–3100 BCE). All individuals carried a large Neolithic-derived ancestry component and were genetically more closely related to Linear Pottery than to Anatolian farmers. Three of the specimens also showed considerable amounts of steppe-related ancestry, suggesting influx into the CTC gene-pool from people affiliated with, for instance, the Ukraine Mesolithic. The latter scenario is supported by archaeological evidence. Taken together, our results confirm that the steppe component arrived in eastern Europe farming communities maybe as early as 3500 BCE. In addition, they are in agreement with the hypothesis of ongoing contacts and gradual admixture between incoming steppe and local western populations. In the archaeological record of eastern Europe, the first evidence for an agrarian lifestyle appeared in the 6th millennium BCE, when the Neolithic societies of the Danube basin (e.g. Linear Pottery [Linearbandkeramik, LBK] and Starčevo) began to spread to the Carpathian region1–3. Following these early foundations, a new society, the Cucuteni-Trypillia complex (CTC) emerged in a vast area that encompassed modern-day eastern Romania, Moldova and western/central Ukraine (Trypillia; Fig. 1). CTC flourished in eastern Europe for about over two millennia (5100–2800 BCE) from the end of the Neolithic to the Early Bronze Age, and is commonly divided into an Early, Middle and Late period4,5 (Fig. 1). Due to its geographic location, CTC was at the nexus of several contemporaneous societies, such as the Lengyel, Funnel Beaker (FBC, also Trichterbecher TRB) and the Globular Amphorae (GAC) cultures (Fig. 1)6. CTC is characterized by a wealth of material finds, attesting to a strong farming economy, a high level of social organization and advanced metallurgy as well as by large proto-urban mega-sites that may have housed hundreds or thousands of inhabitants during the Middle period (4100–3600 BCE). However, subsequently these settlements were mostly abandoned4, and there is archaeological evidence that individuals of the Late CTC interacted with populations that lived in the vast grasslands, or steppes, of Eurasia, such as the Early Bronze Age Yamnaya pastoralists7. Despite the important role of CTC in prehistoric eastern Europe, little is known about the genetic composition of the people associated with this complex, the extent of biological contacts with their neighbors or the level 1 Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany. 2“High Anthropological School” University, Chişinău, Republic of Moldova. 3“Olga Necrasov” Centre for Anthropological Research, Iași, Romania. 4Institute for the History of Material Culture, Russian Academy of Sciences, Saint Petersburg, Russian Federation. 5Institute of Cultural Heritage, Academy of Sciences of the Republic of Moldova, Chișinău, Republic of Moldova. 6Institute of Prehistoric and Protohistoric Archaeology, Kiel University, Kiel, Germany. *email: -kiel. de; Scientific Reports | (2020) 10:4253 | https://doi.org/10.1038/s41598-020-61190-0 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. (a) Map with the Moldovan sites Pocrovca V and Gordinești I from where the individuals presented in this study were recovered. Also shown is Verteba Cave in Ukraine where the CTC individuals presented in Mathieson et al.9 were discovered. (b–d) Temporal and geographic distribution of archaeological cultures mentioned in this study shown for the (b) Early (5100–4600 BCE), (c) Middle (4600–3600 BCE) and (d) Late (3600–2800 BCE) CTC. (Figure is based on a map made with Natural Earth (naturalearthdata.com) and was modified using Adobe Illustrator, Illustrator CS 6 (adobe.com)). of continuity with successive cultural groups. This gap in the genetic landscape can be attributed to a remarkable lack of CTC burials. Human remains have mainly been recovered from Late CTC contexts6, and ancient DNA (aDNA) studies have so far only been performed on specimens from a Trypillian site called Verteba Cave in Ukraine (Fig. 1). A mitochondrial DNA (mtDNA) study on eight Verteba individuals (3700–2900 cal. BCE) revealed in six cases maternal lineages typical of Anatolian and central European farmers. Two specimens had haplogroup U8b1 that may have been derived from European hunter-gatherers6,8. A subsequent genome-wide analysis in four males from Verteba (3900–3600 cal. BCE) confirmed the large Neolithic (~80%) and smaller hunter-gatherer (~20%) ancestry components9. Here, we present genome-wide data generated from the human remains of four females that were excavated from two Late CTC burials in the present-day Republic of Moldova (Fig. 1). The specimens dated to 3500– 3100 cal. BCE, i.e. several hundred years later than the previously investigated males from Verteba Cave9. The incorporation of these additional data sets obtained from new specimens, sites and time points allows us to draw a more nuanced picture of the population movements and dynamics during this important period in the prehistory of eastern Europe. Results We generated genome-wide shotgun sequencing data from three adults that were recovered from a multiple burial at the site of Pocrovca V (individuals: Pocrovca 1, Pocrovca 2, Pocrovca 3) and from a child interred in the Gordinești I flat necropolis (individual: Gordinești) in northern Moldova (Fig. 1, Supplementary Table 1). The specimens date to the Late CTC period (3500–3100 cal. BCE). Bioinformatic data analyses showed that all four individuals were females and carried the mitochondrial haplogroups U4, K1, T1 and T2, respectively (Table 1). Kinship analyses revealed no relatedness among them. When we screened the sequence data for known human blood-borne pathogens such as Yersinia pestis, Mycobacterium tuberculosis and Mycobacterium leprae, no signs of an infection were detected. A principal component analysis of the four Moldova females together with previously published data sets of ancient Eurasians9–13 showed that Gordinești, Pocrovca 1 and Pocrovca 3 grouped with later dating Bell B (...truncated)