Between 45,000 and 35,000 years ago, anatomically modern humans spread across Europe, while the Neanderthals, present since before 300,000 years ago, disappeared. How this process occurred has long been debated1,3,4,5. Comparisons between the Neanderthal genome and the genomes of present-day humans have shown that Neanderthals contributed approximately 1–3% of the genomes of all people living today outside sub-Saharan Africa6,7 suggesting that human populations ancestral to all non-Africans mixed with Neanderthals. The size of segments of Neanderthal ancestry in present-day humans suggests that this occurred between 37,000 and 86,000 years ago8. However, where and how often this occurred is not understood. For example, Neanderthals share more alleles with East Asians and Native Americans than with Europeans, which may reflect additional interbreeding in the ancestors of eastern non-Africans9,10,11,12. Surprisingly, analyses of present-day genomes have not yielded any evidence that Neanderthals mixed with modern humans in Europe, despite the fact that Neanderthals were numerous there and cultural interactions between the two groups have been proposed13,14.

More direct insight into the interactions between modern and archaic humans can be obtained by studying genomes from modern humans who lived at a time when they could have met Neanderthals. Recent analyses of genomes from a ∼43,000–47,000-year-old modern human from western Siberia15 and a ∼36,000–39,000-year-old modern human from eastern Europe16 showed that Neanderthal gene flow into modern humans occurred before these individuals lived. The Siberian individual’s genome contained some segments of Neanderthal ancestry as large as 6 million base pairs (bp), suggesting that some Neanderthal gene flow could have occurred a few thousand years before his death15.

We report genome-wide data from a modern human mandible, Oase 1, found in 2002 in the Peştera cu Oase, Romania. The age of this specimen has been estimated to be ∼37,000–42,000 years by direct radiocarbon dating2,17,18. Oase 1 is therefore one of the earliest modern humans in Europe. Its morphology is generally modern but some aspects are consistent with Neanderthal ancestry19,20,21. Subsequent excavations uncovered a cranium from another, probably contemporaneous individual, Oase 2, which also carries morphological traits that could reflect admixture with Neanderthals17,19.

We prepared two DNA extracts from 25 mg and 10 mg of bone powder removed from the inferior right ramus of Oase 1. We treated an aliquot of each of these extracts with Escherichia coli uracil-DNA glycosylase (UDG), an enzyme that removes uracils from the interior parts of DNA molecules, but leaves a proportion of uracils at the ends of the molecules unaffected. Uracil residues occur in DNA molecules as a result of deamination of cytosine residues, and are particularly prevalent at the ends of ancient DNA molecules9,22. Among the DNA fragments sequenced from these two extracts, 0.18% and 0.06%, respectively, could be mapped to the human reference genome. We prepared three additional DNA libraries from the extract containing 0.18% human-like molecules, but omitted the UDG treatment to increase the number of molecules in which terminal C-to-T substitutions could be seen and used to identify putatively ancient fragments. Because the fraction of endogenous DNA is so small, we used hybridization to DNA probes to isolate human DNA fragments from the libraries23. Applying this strategy to the mitochondrial genome allowed the mitochondrial (mt)DNA from the five libraries to be sequenced to an average coverage of 803-fold (Supplementary Note 1). At the 3′ ends of the DNA fragments, cytosine residues appeared as thymine residues relative to the human mtDNA reference in 21% of fragments, reflecting appreciable levels of cytosine deamination. This suggests that at least some of the human mtDNA is of ancient origin. We determined mtDNA consensus sequences in two ways: using all mtDNA fragments, and using only deaminated fragments that carry C-to-T substitutions at either end relative to the consensus mtDNA sequence based on these fragments, an approach known to enrich for endogenous DNA9,24,25,26. The mtDNA sequence based on all fragments clusters with present-day Europeans (Extended Data Fig. 1) (Supplementary Note 1). In contrast, the mtDNA sequence based on deaminated fragments is related to a large group of present-day Eurasian mtDNAs (haplogroup N) but diverges from these before they diverged from each other. This Oase 1 mtDNA carries a few private mutations on the basis of which its age can be estimated to be 36,330 years before present (14,520–56,450; 95% confidence interval). Using six positions at which the mtDNA sequence differs from at least 99% of 311 present-day humans, we estimate the contamination among all mtDNA fragments to be 67% (95% confidence interval 65–69%). When we restrict to mtDNA fragments that carry terminal C-to-T substitutions, the contamination estimate is 4% (95% confidence interval of 2–9%) (Supplementary Note 1).

To isolate nuclear DNA from Oase 1, we used three sets of oligonucleotide probes that cover about two million sites that are single nucleotide polymorphisms (SNPs) in present-day humans and captured DNA molecules from the five libraries. Of the SNPs targeted, 51% (n = 1,038,619) were covered by at least one DNA fragment, and 13% (n = 271,326) were covered by at least one fragment with a terminal C-to-T substitution. To estimate nuclear DNA contamination, we tested whether Oase 1 DNA fragments with or without evidence of deamination share more alleles with present-day Europeans or with East Asians. We found that Europeans share significantly fewer alleles with Oase 1 fragments that are deaminated than with Oase 1 fragments that are not, consistent with European contamination of 17–30% (Supplementary Note 1). On the basis of these findings and those from mtDNA, we restricted all subsequent analyses to DNA fragments that carry terminal C-to-T substitutions. After doing this, we found that we captured targeted SNPs from the X and Y chromosomes at a similar rate, indicating that Oase 1 carried both an X and a Y chromosome and thus that he was male. The Y chromosome alleles belong to the F haplogroup, which is carried by most males in Eurasia today (Supplementary Note 2).

To determine the relationship of the Oase 1 individual to present-day populations, we first tested whether he shared more alleles with particular present-day individuals from different populations using D-statistics, which provides a robust estimate of admixture almost regardless of how SNPs for analysis are chosen27. We find that Oase 1 shared more alleles with present-day East Asians and Native Americans than with present-day Europeans, counter to what might naively be expected for an ancient individual from Europe (Fig. 1) (5.2 ≤ |Z| ≤ 6.4; Extended Data Table 1). However, it has been suggested that Europeans after the introduction of agriculture derive a part of their ancestry from a ‘basal Eurasian’ population that separated from the initial settlers of Europe and Asia before they split from each other28. Therefore, we replaced present-day Europeans with Palaeolithic and Mesolithic European individuals in these analyses. We then find that the Oase 1 individual shares equally many alleles with these early Europeans as with present-day East Asians and Native Americans (Fig. 1) (|Z| ≤ 1.5 in Extended Data Table 1). Restricting this analysis to transversion polymorphisms, which are not susceptible to errors induced by cytosine deamination, does not influence this result (Extended Data Table 2 and Supplementary Note 3). This suggests that the Oase 1 individual belonged to a population that did not contribute much, or not at all, to later Europeans. This contrasts, for example, with the ∼36,000–39,000-year-old Kostenki 14 individual from western Russia, who was more closely related to later Europeans than to East Asians (1.9 ≤ |Z| ≤ 13.7; Extended Data Table 1)16.

Figure 1: Allele sharing between the Oase 1 individual and other genomes. Each point indicates the extent to which the Oase 1 genome shares alleles with one or other of a pair of genomes from different populations indicated above and below (see Extended Data Table 1 for numbers). Z-scores with an absolute value greater than 2 indicate an excess of allele sharing (grey). Full size image Download PowerPoint slide

To assess whether the ancestors of the Oase 1 individual mixed with Neanderthals, we tested whether the Altai Neanderthal genome shares more alleles with the Oase 1 genome than with sub-Saharan Africans. We find this to be the case (|Z| = 7.7; Supplementary Note 4). We then asked whether the amount of Neanderthal ancestry in the Oase 1 genome is similar to that in present-day non-Africans. Surprisingly, the Neanderthal genome shares more alleles with the Oase 1 individual than it does with any present-day people in Eurasia that we tested, indicating that he carries more Neanderthal-like DNA than present-day people (5.0 ≤ |Z| ≤ 8.2; Extended Data Table 3). We also observe more Neanderthal-like alleles in the Oase 1 individual when we compare him to four early modern humans: an 8,000-year-old individual from Luxembourg, and three individuals from Russia who vary in age between 24,000 and 45,000 years (3.6 ≤ |Z| ≤ 6.8; Extended Data Table 3). Thus, the Oase 1 individual appears to have carried more Neanderthal-like DNA than any other modern human analysed to date. This observation cannot be explained by residual present-day human contamination among the DNA fragments that carry terminal C-to-T substitutions, because all modern humans studied to date carry less Neanderthal ancestry than the Oase 1 genome, and thus contamination would lower, rather than increase, the apparent Neanderthal ancestry.

We estimated the proportion of Neanderthal DNA in the Oase 1 genome using three different statistics7,29 (Supplementary Note 4). Although the results differ, they all yield point estimates between 6.0% and 9.4% (Table 1). For one of the statistics, none of the 90% confidence intervals for Neanderthal ancestry in the other modern human samples overlap with the confidence interval in Oase 1. When we restrict analysis to transversion SNPs, the point estimates of Neanderthal ancestry are even higher (range of 8.4% to 11.3%) (Extended Data Table 4).

Table 1: Estimated fraction of the Oase 1 genome that derives from Neanderthals Full size table

To study the spatial distribution of Neanderthal DNA across the Oase 1 genome, we designed capture probes for around 1.7 million nucleotide positions at which nearly all individuals in a sub-Saharan African population carry one allele whereas Neanderthal genomes carry a different allele. We used these probes to isolate DNA fragments from the Oase 1 individual. A total of 78,055 sites were covered by deaminated DNA fragments from the Oase 1 individual and were also covered by DNA fragments sequenced from the ∼36,000–39,000-year-old Kostenki 14 individual from western Russia16, the ∼43,000–47,000-year-old individual from Ust’-Ishim in Siberia15, and three present-day human genomes from China, France and Sudan (Supplementary Note 5). Because the Dinka from Sudan are thought to have little or no Neanderthal ancestry7, we subtracted the number of alleles that match the Neanderthals in the Dinka individual (485) from the number in the other genomes to estimate the number of alleles attributable to Neanderthal ancestry. The resulting numbers of putative Neanderthal alleles are 3,746 in the Oase 1 individual, 1,586 and 1,121 in the Ust’-Ishim and Kostenki 14 individuals, respectively, and 1,322 and 1,033 in the Chinese and the European individuals (Extended Data Table 5). Thus, the Neanderthal contribution to the Oase 1 genome appears to be between 2.3- and 3.6-fold larger than to the other genomes analysed. Assuming that the Neanderthal contribution to the European individual is 2% (ref. 7), this suggests that 7.3% of the Oase 1 genome is of Neanderthal origin. When the numbers of alleles matching the Neanderthal genome are compared per chromosome (Extended Data Table 5), the highest numbers are always observed for the Oase 1 genome, except in the case of chromosome 21, in which the Ust’-Ishim individual carries a large segment of likely Neanderthal ancestry.

We plotted the positions of Neanderthal-like alleles across the Oase 1 genome (Fig. 2). We detect three segments that are over 50 centimorgans (cM) in size, suggesting that the Neanderthal contribution to the Oase 1 individual occurred so recently in his family tree that chromosomal segments of Neanderthal origin had little time to break up due to recombination. To estimate the date of the most recent Neanderthal contribution to the Oase 1 genome, we studied the size spans of seven segments of the genome that appeared to be recently derived from Neanderthals. Their genetic lengths suggest that the Oase 1 individual had a Neanderthal ancestor as a fourth-, fifth- or sixth-degree relative (Supplementary Note 5). This would predict that an average of 1.6% to 6.3% of the Oase 1 genome derived from this recent Neanderthal ancestor. Visual inspection of the Oase 1 genome suggests that in addition to these seven segments, other smaller segments also carry Neanderthal-like alleles (Fig. 2). When we remove the seven longest segments, the estimate of Neanderthal ancestry in Oase 1 drops from 7.3% to 4.8%, which is still around twice the 2.0–2.9% estimated for the French, Han, Kostenki and Ust’-Ishim individuals in this remaining part of the genome. This additional Neanderthal ancestry could reflect an older Neanderthal admixture into the ancestors of Oase 1, or that we failed to find all segments of recent Neanderthal ancestry.

Figure 2: Spatial distribution of alleles matching Neanderthals in modern humans. Coloured vertical lines indicate alleles shared with Neanderthals and no colour indicates alleles shared with the great majority of West Africans. D, Dinka; F, French; H, Han; K, Kostenki 14; O, Oase 1; U, Ust’-Ishim. The seven grey bars indicate segments of putative recent Neanderthal ancestry. This analysis is based on 78,055 sites. Numbers refer to chromosomes. Full size image Download PowerPoint slide

The Oase 1 genome shows that mixture between modern humans and Neanderthals was not limited to the first ancestors of present-day people to leave Africa, or to people in the Near East; it occurred later as well and probably in Europe. The fact that the Oase 1 individual had a Neanderthal ancestor removed by only four to six generations allows this Neanderthal admixture to be dated to less than 200 years before the time he lived. However, the absence of a clear relationship of the Oase 1 individual to later modern humans in Europe suggests that he may have been a member of an initial early modern human population that interbred with Neanderthals but did not contribute much to later European populations. To better understand the interactions between early modern and Neanderthal populations, it will be important to study other specimens that, like Oase 1, have been suggested to carry morphological traits suggestive of admixture with Neanderthals30.