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Archaeogenetic studies have described the formation of Eurasian 'steppe ancestry' as a mixture of Eastern and Caucasus hunter-gatherers. However, it remains unclear when and where this ancestry arose and whether it was related to a horizon of cultural innovations in the 4th millennium BCE that subsequently facilitated the advance of pastoral societies likely linked to the dispersal of Indo-European languages. To address this, we generated genome-wide SNP data from 45 prehistoric individuals along a 3000-year temporal transect in the North Caucasus. We observe a genetic separation between the groups of the Caucasus and those of the adjacent steppe. The Caucasus groups are genetically similar to contemporaneous populations south of it, suggesting that - unlike today - the Caucasus acted as a bridge rather than an insurmountable barrier to human movement. The steppe groups from Yamnaya and subsequent pastoralist cultures show evidence for previously undetected Anatolian farmer-related ancestry from different contact zones, while Steppe Maykop individuals harbour additional Upper Palaeolithic Siberian and Native American related ancestry.

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