We sequenced the genomes of 15 skeletons from a 5,000-y-old mass grave in Poland associated with the Globular Amphora culture. All individuals had been brutally killed by blows to the head, but buried with great care. Genome-wide analyses demonstrate that this was a large extended family and that the people who buried them knew them well: mothers are buried with their children, and siblings next to each other. From a population genetic viewpoint, the individuals are clearly distinct from neighboring Corded Ware groups because of their lack of steppe-related ancestry. Although the reason for the massacre is unknown, it is possible that it was connected with the expansion of Corded Ware groups, which may have resulted in violent conflict.

The third millennium BCE was a period of major cultural and demographic changes in Europe that signaled the beginning of the Bronze Age. People from the Pontic steppe expanded westward, leading to the formation of the Corded Ware complex and transforming the genetic landscape of Europe. At the time, the Globular Amphora culture (3300–2700 BCE) existed over large parts of Central and Eastern Europe, but little is known about their interaction with neighboring Corded Ware groups and steppe societies. Here we present a detailed study of a Late Neolithic mass grave from southern Poland belonging to the Globular Amphora culture and containing the remains of 15 men, women, and children, all killed by blows to the head. We sequenced their genomes to between 1.1- and 3.9-fold coverage and performed kinship analyses that demonstrate that the individuals belonged to a large extended family. The bodies had been carefully laid out according to kin relationships by someone who evidently knew the deceased. From a population genetic viewpoint, the people from Koszyce are clearly distinct from neighboring Corded Ware groups because of their lack of steppe-related ancestry. Although the reason for the massacre is unknown, it is possible that it was connected with the expansion of Corded Ware groups, which may have resulted in competition for resources and violent conflict. Together with the archaeological evidence, these analyses provide an unprecedented level of insight into the kinship structure and social behavior of a Late Neolithic community.

In 2011, archaeological excavations near the village of Koszyce in southern Poland uncovered a ca. 5,000-y-old mass grave (Fig. 1) associated with the Globular Amphora culture and containing the remains of 15 men, women, and children who had been killed, but carefully buried with rich grave goods (1). Closer study of the skeletons (2) revealed that the individuals had all been killed by blows to the head, possibly during a raid on their settlement. To shed light on this Late Neolithic community and the events that unfolded at Koszyce 5,000 y ago, we sequenced their genomes to between 1.1- and 3.9-fold coverage (Table 1) and performed genome-wide analyses to explore their genetic ancestry and kinship relations. In addition, we obtained 16 radiocarbon dates (SI Appendix, section 4 and Dataset S1) to narrow down the date of the massacre to 2880–2776 BCE (SI Appendix, Fig. S5). We also provide a detailed description of the injuries (SI Appendix, section 3), and strontium isotope measurements of dental enamel provide information on mobility and residence patterns (SI Appendix, section 5). Together, the analyses enable us to draw up a remarkably detailed picture of this Late Neolithic community, including their genetic ancestry, physical appearance, kinship structure, and social organization.

Results and Discussion

DNA was isolated from teeth and petrous bones, using established protocols, and the libraries were sequenced on Illumina HiSeq 2500 platforms. The sequence data showed all the hallmarks of damaged ancient DNA, and modern contamination was estimated to be very low (Table 1). The human endogenous DNA contents ranged between 13% and 75% (Dataset S3). According to the sequencing data, eight of the individuals in the grave were males and seven were females (Table 1). This is consistent with previously published results based on skeletal traits, with the exception of one male (individual 4) who had previously been identified as a probable female and five juveniles who could not be sexed previously (2). Data on phenotypic traits based on imputed genotypes (Dataset S5) revealed that the individuals had mostly brown eyes, dark or dark-blonde hair, and intermediate to dark skin.

Genetic Affinities. To investigate their genetic ancestry, we merged the 15 Koszyce genomes with the Human Origins data set (3), as well as 168 previously published ancient genomes (Dataset S6). In addition, we included genome-wide data for nine individuals from four contemporary, neighboring sites in southern Poland belonging to the Globular Amphora culture and its Złota group variant that we sequenced to between 0.2- and 1-fold coverage (Dataset S3). We then performed a principal component analysis and found that all 24 Globular Amphora/Złota group individuals clustered with other previously sequenced Globular Amphora individuals (4, 5) and other Neolithic groups (Fig. 2A). This confirms earlier suggestions that the Globular Amphora people belonged to the Neolithic gene pool of Europe, as typified by early Anatolian farmers (4, 5). Fig. 2. Genetic affinities of the Koszyce individuals and other GAC groups (here including Złota) analyzed in this study. (A) Principal component analysis of previously published and newly sequenced ancient individuals. Ancient genomes were projected onto modern reference populations, shown in gray. (B) Ancestry proportions based on supervised ADMIXTURE analysis (K = 3), specifying Western hunter-gatherers, Anatolian Neolithic farmers, and early Bronze Age steppe populations as ancestral source populations. LP, Late Paleolithic; M, Mesolithic; EN, Early Neolithic; MN, Middle Neolithic; LN, Late Neolithic; EBA, Early Bronze Age; PWC, Pitted Ware culture; TRB, Trichterbecherkultur/Funnelbeaker culture; LBK, Linearbandkeramik/Linear Pottery culture; GAC, Globular Amphora culture; Złota, Złota culture. To further investigate the ancestry of the Globular Amphora individuals, we performed a supervised ADMIXTURE (6) analysis, specifying typical western European hunter-gatherers (Loschbour), early Neolithic Anatolian farmers (Barcın), and early Bronze Age steppe populations (Yamnaya) as ancestral source populations (Fig. 2B). The results indicate that the Globular Amphora/Złota group individuals harbor ca. 30% western hunter-gatherer and 70% Neolithic farmer ancestry, but lack steppe ancestry. To formally test different admixture models and estimate mixture proportions, we then used qpAdm (7) and find that the Polish Globular Amphora/Złota group individuals can be modeled as a mix of western European hunter-gatherer (17%) and Anatolian Neolithic farmer (83%) ancestry (SI Appendix, Table S2), mirroring the results of previous studies (4, 5).

Kinship and Consanguinity. Analyses of ancient genomes can provide detailed information on the kinship structures and social organization of past communities (8⇓–10). At Koszyce, mitochondrial DNA (mtDNA) analysis revealed the presence of six different maternal lineages, whereas analysis of the nonrecombining region of the Y chromosome showed that all males carried the same Y chromosome haplotype: I2a-L801 (Table 1). We then estimated genomic runs of homozygosity (ROH) and found that the Koszyce individuals were not particularly inbred. Although a slightly larger section of the Koszyce genomes is contained within ROH compared with typical modern European populations (SI Appendix, Fig. S7), this signal is mainly driven by an increased fraction of short ROH (<2 Mb), which is indicative of ancestral restrictions in population size rather than recent inbreeding. On the basis of genome-wide patterns of allelic identity-by-state (IBS), we computed kinship coefficients between all pairs of individuals and applied established cutoff values for possible kinship categories (Materials and Methods). We find that the Koszyce burial represents a large extended family connected via several first- and second-degree relationships (Fig. 3 and SI Appendix, Fig. S9). Fig. 3. Kinship. (A) Artistic reconstruction of the Koszyce mass burial based partly on phenotypic traits inferred from the ancient genomes (reconstruction by Michał Podsiadło); (B) Schematic representation of the burial and pedigree plots showing kinship relations between the Koszyce individuals inferred from genetic data. (C) kinship network based on kinship coefficients inferred from IBS scores for pairs of Koszyce individuals showing first- and second-degree relationships. Kinship coefficients and R scores are reported in Dataset S7 and plotted in SI Appendix, Fig. S9. Overall, we identified four nuclear families in the grave, which are for the most part represented by mothers and their children (Fig. 3). Closely related kin were buried next to each other: a mother was buried cradling her child, and siblings were placed side by side. Evidently, these individuals were buried by people who knew them well and who carefully placed them in the grave according to familial relationships. For example, individual 14, the oldest individual in the grave, was buried close to her two sons (individuals 5 and 15), whereas individual 8, a 30–35-y-old woman, was buried with her teenage daughter (individual 9) and 5-y-old son (individual 13). Using genome-wide patterns of IBS, we were also able to reconstruct more complex relationships: individuals 5, 10, 11, and 15 all appear to be brothers, and yet they do not have the same mother (individual 14 is the mother of individuals 5 and 15, but not 10 and 11), suggesting that they might be half-brothers. However, all four of them share the same mitochondrial DNA haplotype, suggesting that their mothers might also have been related. Interestingly, the older males/fathers are mostly missing from the grave, suggesting that it might have been them who buried their kin. The only father present in the grave is individual 10, whose partner and son are placed together opposite him in the grave. In addition, there is a young boy (individual 7), aged 2–2.5 y, whose parents are not in the grave, but he is placed next to other individuals to whom he is closely related through various second-degree relationships. Finally, there is individual 3, an adult female, who does not seem to be genetically related to anyone in the group. However, her position in the grave close to individual 4, a young man, suggests that she may have been as close to him in life as she was in death. These biological data and burial arrangements show that the social relationships held to be most significant in these societies were identical with genetic and reproductive relationships. However, they also demonstrate that nuclear families were nested in larger, extended family groups, either permanently or for parts of the year.

Social Organization, Residence Patterns, and Subsistence Strategies. The presence of unrelated females and related males in the grave is interesting because it suggests that the community at Koszyce was organized along patrilineal lines of descent, adding to the mounting evidence that this was the dominant form of social organization among Late Neolithic communities in Central Europe (11, 12). Usually, patrilineal forms of social organization go hand in hand with female exogamy (i.e., the practice of women marrying outside their social group). Indeed, several studies (11, 12) have shown that patrilocal residence patterns and female exogamy prevailed in several parts of Central Europe during the Late Neolithic. At Koszyce, there is no clear difference in enamel 87Sr/86Sr ratios between males and females (SI Appendix, Fig. S6) that would suggest that the females are nonlocal. However, the high diversity of mtDNA lineages, combined with the presence of only a single Y chromosome lineage, is certainly consistent with a patrilocal residence system. Social organization is most often aligned with settlement and subsistence patterns, and several studies (13⇓–15) suggest that Globular Amphora communities and other related groups specialized in animal husbandry, often with a main focus on cattle, and that they moved around the landscape to seek new pastures for their animals at different times of the year (see SI Appendix, section 1 for a more detailed discussion). This form of mobility is likely to have included fission-fusion dynamics in which a larger social unit, similar to the extended family, would split up into smaller groups, perhaps nuclear families, for certain purposes and parts of the year (16). This dynamic could explain the relatively high variation we observe in the 87Sr/86Sr isotope signatures at Koszyce. Similar to strongly patrilineal modes of social organization, such pastoral economic strategies have often been linked to Corded Ware groups that introduced steppe genetic ancestry into Europe (7, 17), and the two (social organization and economic strategy) are probably linked: Pastoral ways of life involve a high level of mobility within vaguely defined territories and with the groups’ main economic capital, their animal herds, exposed across the landscape, and thus harbor a significant potential for conflict with neighboring groups. One ethnographically known cultural response to this situation is to adopt an aggressive strategy toward competing groups in which male dominance, including patrilineal kin alliance, and warrior-like values prevail (18). Although we cannot be certain that the people at Koszyce shared these values, we show that they were organized around patrilineal descent groups, demonstrating that this form of social organization was already present in communities before the expansion of the Corded Ware complex in Central and Eastern Europe (13, 14).