Next-generation sequencing (NGS) technologies are revolutionizing the field of ancient DNA (aDNA), and have enabled the sequencing of complete ancient genomes5,6, such as that of Ötzi, a Neolithic human body found in the Alps1. However, very little is known of the genetic composition of earlier hunter-gatherer populations from the Mesolithic period (circa 10,000–5,000 years before present, bp; immediately preceding the Neolithic period).

The Iberian site called La Braña-Arintero was discovered in 2006 when two male skeletons (named La Braña 1 and 2) were found in a deep cave system, 1,500 m above sea level in the Cantabrian mountain range (León, Northwestern Spain) (Fig. 1a). The skeletons were dated to approximately 7,000 years bp (7,940–7,690 calibrated bp)7. Because of the cold environment and stable thermal conditions in the cave, the preservation of these specimens proved to be exceptional (Fig. 1b). We identified a tooth from La Braña 1 with high human DNA content (48.4%) and sequenced this specimen to a final effective genomic depth-of-coverage of 3.40× (Extended Data Fig. 1).

Figure 1: Geographic location and genetic affinities of the La Braña 1 individual. a, Location of the La Braña-Arintero site (Spain). b, The La Braña 1 skeleton as discovered in 2006. c, PCA based on the average of the Procrustes transformations of individual PCAs with La Braña 1 and each of the five Neolithic samples1,3. The reference populations are the Finnish HapMap, FINHM and POPRES. Population labels with labelling of ref. 12 with the addition of FI (Finns) or LFI (late-settlement Finns). Ajv70, Ajv52, Ire8 and Gok4 are Scandinavian Neolithic hunter-gatherers and a farmer, respectively3. Ötzi is the Tyrolean Ice Man1. Full size image Download PowerPoint slide

We used several tests to assess the authenticity of the genome sequence and to determine the amount of potential modern human contamination. First, we observed that sequence reads from both the mitochondrial DNA (mtDNA) and the nuclear DNA of La Braña 1 showed the typical ancient DNA misincorporation patterns that arise from degradation of DNA over time8 (Extended Data Fig. 2a, b). Second, we showed that the observed number of human DNA fragments was negatively correlated with the fragment length (R2 > 0.92), as expected for ancient degraded DNA, and that the estimated rate of DNA decay was low and in agreement with predicted values9 (Extended Data Fig. 2c, d). We then estimated the contamination rate in the mtDNA genome, assembled to a high depth-of-coverage (91×), by checking for positions differing from the mtDNA genome (haplogroup U5b2c1) that was previously retrieved with a capture method2. We obtained an upper contamination limit of 1.69% (0.75–2.6%, 95% confidence interval, CI) (Supplementary Information). Finally, to generate a direct estimate of nuclear DNA contamination, we screened for heterozygous positions (among reads with >4× coverage) in known polymorphic sites (Single Nucleotide Polymorphism Database (dbSNP) build 137) at uniquely mapped sections on the X chromosome6 (Supplementary Information). We found that the proportion of false heterozygous sites was 0.31%. Together these results suggest low levels of contamination in the La Braña 1 sequence data.

To investigate the relationship to extant European samples, we conducted a principal component analysis (PCA)10 and found that the approximately 7,000-year-old Mesolithic sample was divergent from extant European populations (Extended Data Fig. 3a, b), but was placed in proximity to northern Europeans (for example, samples from Sweden and Finland)11,12,13,14. Additional PCAs and allele-sharing analyses with ancient Scandinavian specimens3 supported the genetic similarity of the La Braña 1 genome to Neolithic hunter-gatherers (Ajv70, Ajv52, Ire8) relative to Neolithic farmers (Gok4, Ötzi) (Fig. 1c, Extended Data Figs 3c and 4). Thus, this Mesolithic individual from southwestern Europe represents a formerly widespread gene pool that seems to be partially preserved in some modern-day northern European populations, as suggested previously with limited genetic data2,3. We subsequently explored the La Braña affinities to an ancient Upper Palaeolithic genome from the Mal'ta site near Lake Baikal in Siberia15. Outgroup f 3 and D statistics16,17, using different modern reference populations, support that Mal'ta is significantly closer to La Braña 1 than to Asians or modern Europeans (Extended Data Fig. 5 and Supplementary Information). These results suggest that despite the vast geographical distance and temporal span, La Braña 1 and Mal'ta share common genetic ancestry, indicating a genetic continuity in ancient western and central Eurasia. This observation matches findings of similar cultural artefacts across time and space in Upper Paleolithic western Eurasia and Siberia, particularly the presence of anthropomorphic ‘Venus’ figurines that have been recovered from several sites in Europe and Russia, including the Mal'ta site15. We also compared the genome-wide heterozygosity of the La Braña 1 genome to a data set of modern humans with similar coverage (3–4×). The overall genomic heterozygosity was 0.042%, lower than the values observed in present day Asians (0.046–0.047%), Europeans (0.051–0.054%) and Africans (0.066–0.069%) (Extended Data Fig. 6a). The effective population size, estimated from heterozygosity patterns, suggests a global reduction in population size of approximately 20% relative to extant Europeans (Supplementary Information). Moreover, no evidence of tracts of autozygosity suggestive of inbreeding was observed (Extended Data Fig. 6b).

To investigate systematically the timing of selection events in the recent history of modern Europeans, we compared the La Braña genome to modern populations at loci that have been categorized as of interest for their role in recent adaptive evolution. With respect to two recent well-studied adaptations to changes in diet, we found the ancient genome to carry the ancestral allele for lactose intolerance4 and approximately five copies of the salivary amylase (AMY1) gene (Extended Data Fig. 7 and Supplementary Information), a copy number compatible with a low-starch diet18. These results suggest the La Braña hunter-gatherer was poor at digesting milk and starch, supporting the hypotheses that these abilities were selected for during the later transition to agriculture.

To expand the survey, we analysed a catalogue of candidate signals for recent positive selection based on whole-genome sequence variation from the 1000 Genomes Project13, which included 35 candidate non-synonymous variants, ten of which were detected uniquely in the CEU (Utah residents with northern and western European ancestry) sample 19. For each variant we assessed whether the Mesolithic genome carried the ancestral or derived (putatively adaptive) allele.

Of the ten variants, the Mesolithic genome carried the ancestral and non-selected allele as a homozygote in three regions: C12orf29 (a gene with unknown function), SLC45A2 (rs16891982) and SLC24A5 (rs1426654) (Table 1). The latter two variants are the two strongest known loci affecting light skin pigmentation in Europeans20,21,22 and their ancestral alleles and associated haplotypes are either absent or segregate at very low frequencies in extant Europeans (3% and 0% for SLC45A2 and SLC24A5, respectively) (Fig. 2). We subsequently examined all genes known to be associated with pigmentation in Europeans22, and found ancestral alleles in MC1R, TYR and KITLG, and derived alleles in TYRP1, ASIP and IRF4 (Supplementary Information). Although the precise phenotypic effects cannot currently be ascertained in a European genetic background, results from functional experiments20 indicate that the allelic combination in this Mesolithic individual is likely to have resulted in dark skin pigmentation and dark or brown hair. Further examination revealed that this individual carried the HERC2 rs12913832*C single nucleotide polymorphism (SNP) and the associated homozygous haplotype spanning the HERC2–OCA2 locus that is strongly associated with blue eye colour23. Moreover, a prediction of eye colour based on genotypes at additional loci using HIrisPlex24 produced a 0.823 maximal and 0.672 minimal probability for being non-brown-eyed (Supplementary Information). The genotypic combination leading to a predicted phenotype of dark skin and non-brown eyes is unique and no longer present in contemporary European populations. Our results indicate that the adaptive spread of light skin pigmentation alleles was not complete in some European populations by the Mesolithic, and that the spread of alleles associated with light/blue eye colour may have preceded changes in skin pigmentation.

Table 1: Mesolithic genome allelic state at 10 nonsynonymous variants recently selected in Europeans Full size table

Figure 2: Ancestral variants around the SLC45A2 (rs16891982, above) and SLC24A5 (rs1426654, below) pigmentation genes in the Mesolithic genome. The SNPs around the two diagnostic variants (red arrows) in these two genes were analysed. The resulting haplotype comprises neighbouring SNPs that are also absent in modern Europeans (CEU) (n = 112) but present in Yorubans (YRI) (n = 113). This pattern confirms that the La Braña 1 sample is older than the positive-selection event in these regions. Blue, ancestral; red, derived. Full size image Download PowerPoint slide

For the remaining loci, La Braña 1 displayed the derived, putatively adaptive variants in five cases, including three genes, PTX4, UHRF1BP1 and GPATCH1 (ref. 19), involved in the immune system (Table 1 and Extended Data Fig. 8). GPATCH1 is associated with the risk of bacterial infection. We subsequently determined the allelic states in 63 SNPs from 40 immunity genes with previous evidence for positive selection and for carrying polymorphisms shown to influence susceptibility to infections in modern Europeans (Supplementary Information). La Braña 1 carries derived alleles in 24 genes (60%) that have a wide range of functions in the immune system: pattern recognition receptors, intracellular adaptor molecules, intracellular modulators, cytokines and cytokine receptors, chemokines and chemokine receptors and effector molecules. Interestingly, four out of six SNPs from the first category are intracellular receptors of viral nucleic acids (TLR3, TLR8, IFIH1 (also known as MDA5) and LGP2)25.

Finally, to explore the functional regulation of the genome, we also assessed the La Braña 1 genotype at all expression quantitative trait loci (eQTL) regions associated to positive selection in Europeans (Supplementary Information). The most interesting finding is arguably the predicted overexpression of eight immunity genes (36% of those with described eQTLs), including three Toll-like receptor genes (TLR1, TLR2 and TLR4) involved in pathogen recognition26.

These observations suggest that the Neolithic transition did not drive all cases of adaptive innovation on immunity genes found in modern Europeans. Several of the derived haplotypes seen at high frequency today in extant Europeans were already present during the Mesolithic, as neutral standing variation or due to selection predating the Neolithic. De novo mutations that increased in frequency rapidly in response to zoonotic infections during the transition to farming should be identified among those genes where La Braña 1 carries ancestral alleles.

To confirm whether the genomic traits seen at La Braña 1 can be generalized to other Mesolithic populations, analyses of additional ancient genomes from central and northern Europe will be needed. Nevertheless, this genome sequence provides the first insight as to how these hunter-gatherers are related to contemporary Europeans and other ancient peoples in both Europe and Asia, and shows how ancient DNA can shed light on the timing and nature of recent positive selection.