Genetics of Ice Age Europe

To these two clear-cut conflict sites we can now add another with unequivocal osteoarchaeological evidence of indiscriminate lethal violence, torture and mutilation, and disposal of the corpses in a commingled and chaotic mass grave (Fig. 1). Although isolated cases of interpersonal violence are known from much older periods (27, 28), as are some pictorial representations of possible violent behavior (29), direct evidence for targeted collective violence is very rare in the preagricultural record of Central Europe. One of the most frequently discussed sites in this regard is the Ofnet Cave in Germany, which shows the deposition of selected human remains with clear evidence of perimortem injuries and cut marks in Mesolithic times (30, 31). At that site, however, only detached and ochre-covered heads with articulated vertebrae were deposited carefully along with animal teeth ornaments in a special location, indicating much more complex behavior than a massacre (32).



Assessing the overall antemortem disease load, we find lesions typical for the LBK: probable signs of tuberculosis in some ribs (50), traces of vitamin C deficiency (48), healed rib and long-bone fractures, a likely well-healed cranial surgery following trauma (Fig. 2A) (59), and osteomyelitis. Joint and dental diseases remain rare. The general health status therefore is as expected for an LBK group from Central Europe, as previously determined from larger population samples.



The lesions that provide the most telling evidence for the interpretation of this mass grave feature are the very frequent perimortem cranial and postcranial fractures (49). These features characterize the assemblage in a way comparable to the other known LBK massacre sites of Talheim and Asparn/Schletz (24⇓–26). Unequivocal perimortem blunt-force injuries affect most cranial bones and can be securely attributed in part to the known adze weapon-tools of the LBK (Fig. 2 B–D). Because most crania were only partly preserved, incomplete, and fragmentary, exact impact sites could not always be determined with confidence. Instead, the overall patterning of perimortem fracture zones was analyzed per cranial element for subadults and adults separately. The highest percentage of traumatized bone was found in the left parietal for both groups, a classic location for blows delivered in face-to-face confrontations during interpersonal violence (60, 61). Injuries to the other larger cranial elements are prevalent as well (Table 1). In the subadult sample, the left halves of the occipital and the frontal are the second and third most frequent cranial injuries (Table 2).



In addition to the extensive cranial trauma, a very high number of perimortem-fractured long bones were recorded (Fig. 3), but traces of carnivore activity could not be identified. Again, because of the high overall fragmentation, an element count based upon anatomically identified and isolated bone units was used for analysis (Table 3). The differences in fracture frequency between the major long bones are very apparent. On average, 19% of the fragments of the upper extremity bones show perimortem fracture [including at least one case of intended amputation of a humerus (Fig. 3D)], but the fracture frequency is much higher for the distal elements of the lower extremity. Some 31–42% of fibula units and a staggering 53–63% of tibia units show perimortem fractures, double to triple the percentage found in the upper limb bones. The lowest overall frequency was found in the femur, where a maximum of only 7% of identified units show perimortem fractures. This pattern clearly reveals a highly significant bias toward perimortem fragmentation of the distal segment of the lower limb and, especially, the tibia (χ2 = 56.011; P < 0.001). Suggestive but not definite lesions that could be attributed to arrow injuries were found in two vertebrae.

Early Neolithic Mass Graves as Evidence for Lethal Collective Violence
Building on both the evidence previously available for the LBK and the evidence presented here, we suggest that the repeated occurrence of almost indiscriminate massacres, the possible abduction of selected members, and the patterns of torture, mutilation, and careless disposal all fit into the concept of prehistoric warfare as currently understood within anthropology (54, 64, 75). Particular LBK groups were singled out for as yet unknown reasons, attacked with brute force, and annihilated by others, probably close neighbors and very likely other LBK groups of the wider region (25, 76). As has been shown, even within the overall quite homogenous-appearing LBK, recognizable boundaries did exist in many places (77⇓⇓–80). These borders most probably were a result of the spread of different groups without close social or biological kinship ties to one another who came in to close contact as a consequence of the LBK colonization pattern (4, 80). In fact, because the LBK was the first complete Neolithic culture in Central Europe (3), today all farmers of this time and region are classified as members of the LBK by default, regardless of how these people defined themselves and how they differentiated themselves from their contemporaries. Alternative cultural attributions, based almost exclusively on pottery styles, arise only with the decline of the LBK in its final phases (4). The suggested regional differentiation, the possible collapse of previous exchange systems, and increasing defensive architecture are all compatible with increasing levels of widespread social tensions and the looming threat of utmost violence (3). The massacres now known from three widely separated localities but dating to a rather short period give direct evidence that outbreaks of lethal collective violence unquestionably occurred repeatedly within the later LBK (19, 24⇓–26). Although the particular triggers for each massacre might have been different, the overarching patterns of extreme violence and the atypical treatment of the dead are recognizably similar (35). In this context, it is especially telling that all three of the unequivocal massacre sites currently known date to the later phases of the LBK (17, 25), but there is no evidence for comparable levels of violence in the earlier periods.

The Kilianstädten massacre, which occurred within an archaeologically suggested border zone of different LBK subgroups (43, 80), with its high potential for intergroup conflict (54, 76), provides an illuminating example of characteristics of nonstate warfare identified earlier from the ethnographic record; the abduction of younger women and the torture, mutilation, and killing of enemies can be seen clearly in the osteological record of Early Neolithic Central Europe (54). Although some earlier works supporting the notion of widespread warfare during the later LBK were based, at least in part, on a premature interpretation of several LBK sites (15, 16, 41) that now are interpreted differently (20⇓⇓–23), the evidence has become more conclusive again with the Kilianstädten site. Importantly, more skeletal remains, the only direct evidence for collective lethal violence, are now available (16, 41, 49). Although the underlying supraregional causes for the recognized increase in mass violence in the late LBK undoubtedly were complex and multifactorial, a significant increase in population followed by adverse climatic conditions (drought), possibly coupled with the inability of long-settled farmers to practice the avoidance behavior by which hunter-gatherers typically evade conflict (75), seems to have been an important component of the overall picture (4). As previous research has shown, climatic changes, especially those leading to increasing unpredictability of or even significant decreases in agricultural production, have played major roles in the change and collapse of societies throughout human history (4, 81, 82). Ecological imbalance and perceived or actual resource stress were suggested previously as some of the main reasons for massacres and warfare in general (55, 64, 83), and at the end of the LBK aggression might have been aggravated further by patrilineally determined social inequality, especially with regard to access to coveted, high-quality farmland, food, and possibly prestige goods (9, 13, 14, 76, 84).

Published online before print August 17, 2015, doi: 10.1073/pnas.1504365112

Natural selection has reduced Neanderthal ancestry over the last 45,000 years
We used two previously published statistics3,7,21 to ask if the proportion of Neanderthal ancestry in Eurasians changed over the last 45,000 years. Whereas on the order of 2% of present-day Eurasian DNA is of Neanderthal origin (Extended Data Table 2), the ancient modern human genomes carry significantly more Neanderthal DNA (Figure 2) (P≪10−12). Using one statistic, we estimate a decline from 4.3–5.7% from a time shortly after introgression to 1.1–2.2% in Eurasians today (Figure 2). Using the other statistic, we estimate a decline from 3.2–4.2% to 1.8–2.3% (Extended Data Figure 1, Extended Data Table 3). Because all the European samples we analyzed dating to between 37,000 and 14,000 years ago are consistent with descent from a single founding population, admixture with populations with lower Neanderthal ancestry cannot explain the steady decrease in Neanderthal-derived DNA that we detect during this period, showing that natural selection against Neanderthal DNA must have driven this phenomenon (Figure 2). We also obtain an independent line of evidence for selection from our observation that the decrease in Neanderthal-derived alleles is more marked near genes than in less constrained regions of the genome (P=0.010) (Supplementary Information section 3; Extended Data Table 3)22–25.


Figure 2
Decrease of Neanderthal ancestry over time

Y chromosomes, mitochondrial DNA and phenotypically important mutations
We used the proportion of sequences mapping to the Y chromosome to infer sex (Extended Data Table 4; Supplementary Information section 4), and determined Y chromosome haplogroups for the males. We were surprised to find haplogroup R1b in the ~14,000-year-old Villabruna individual from Italy. While the predominance of R1b in western Europe today is owes its origin to Bronze Age migrations from the eastern European steppe9, its presence in Villabruna and in a ~7,000-year-old farmer from Iberia9 document a deeper history of this haplotype in more western parts of Europe. Additional evidence of an early link between west and east comes from the HERC2 locus, where a derived allele that is the primary driver of light eye color in Europeans appears nearly simultaneously in specimens from Italy and the Caucasus ~14,000-13,000 years ago. Extended Data Table 5 presents results for additional alleles of known phenotypic importance. When analyzing the mitochondrial genomes we note the presence of haplogroup M in a ~27,000-year-old individual from southern Italy (Ostuni1) in agreement with the observation that this haplogroup, which today occurs in Asia and is absent in Europe, was present in pre-Last Glacial Maximum Europe and became lost during the Ice Age26. We also find that the ~33,000 year old Muierii2 from Romania carries a basal version of haplogroup U6, in agreement with the hypothesis that the presence of derived versions of this haplogroup in North Africans today is due to back-migration from western Eurasia27.


Figure 3

Genetic clustering of the ancient specimens
This dataset provides an unprecedented opportunity to study the population history of Upper Paleolithic Europe over more than 30,000 years. In order to not prejudice any association between genetic and archaeological groupings among the individuals studied, we first allowed the genetic data alone to drive the groupings of the specimens and only afterward examined their associations with archaeological cultural complexes. We began by computing f3-statistics14 of the form f3(X, Y; Mbuti), which measure shared genetic drift between a pair of ancient individuals after divergence from an outgroup (here Mbuti from sub-Saharan Africa), which allowed us to observe clear clusters of samples (Figure 3A; Extended Data Figure 2). Through Multi-Dimensional Scaling (MDS) analysis of this matrix (Figure 3B), as well as through D-statistic analyses28 (Supplementary Information section 5), we identified five clusters of individuals with substantial shared genetic drift, which we name after the oldest individual with >1.0-fold coverage in each cluster (Supplementary Information section 5; Table 1; Extended Data Table 1). In contrast, we were not able to identify clear structure among these samples based on model-based clustering29,30, which may reflect the fact that many of the samples are so ancient that present-day patterns of human variation are not very relevant to understanding their patterns of genetic differentiation4,13. The “Vestonice Cluster” is composed of 14 pre-Ice Age individuals from 34,000-26,000 years ago, who are all associated with the archaeologically defined Gravettian culture. The “Mal’ta Cluster” is composed of three individuals from the Glacial Maximum 24,000-17,000 years ago from the Lake Baikal region of Siberia. The “El Mirón Cluster” is composed of 6 Late Glacial individuals from 19,000-14,000 years ago, who are all associated with the Magdalenian culture. The “Villabruna Cluster” is composed of 13 post-Ice Age individuals from 14,000-7,000 years ago, associated with the Azilian, Epipaleolithic and Mesolithic cultures. The “Satsurblia Cluster” is composed of two individuals from 13,000-10,000 years ago from the northern Caucasus2. There were ten samples that we did not assign to any cluster, either because of evidence of representing distinct early lineages, (Ust’-Ishim, Oase1, Kostenki14, GoyetQ116-1, Muierii2, Cioclovina1, Kostenki12), or because they were admixed between major clusters (Karelia or Motala12), or of very different ancestry (Stuttgart). To classify the ancestry of additional low coverage samples, we built an admixture graph that fits the allele frequency correlation patterns among high coverage samples28 (Supplementary Information section 6; Figure 4a). We fit each low coverage sample into the graph in turn, including all fragments from every individual rather than just ones with evidence of cytosine deamination, accounting for contamination bias by modeling (Supplementary Information section 7).