The presence of Neandertals in Europe and Western Eurasia before the arrival of anatomically modern humans is well supported by archaeological and paleontological data. In contrast, fossil evidence for Denisovans, a sister group of Neandertals recently identified on the basis of DNA sequences, is limited to three specimens, all of which originate from Denisova Cave in the Altai Mountains (Siberia, Russia). We report the retrieval of DNA from a deciduous lower second molar (Denisova 2), discovered in a deep stratigraphic layer in Denisova Cave, and show that this tooth comes from a female Denisovan individual. On the basis of the number of “missing substitutions” in the mitochondrial DNA determined from the specimen, we find that Denisova 2 is substantially older than two of the other Denisovans, reinforcing the view that Denisovans were likely to have been present in the vicinity of Denisova Cave over an extended time period. We show that the level of nuclear DNA sequence diversity found among Denisovans is within the lower range of that of present-day human populations.

In addition to Denisova 3, two permanent molars (Denisova 4 and Denisova 8) have been identified as originating from Denisovans on the basis of DNA sequence data ( 2 , 17 ); and mtDNA fragments of the Denisovan type were identified in sediments deposited at Denisova Cave ( 26 ). Here, we present analyses of DNA sequences retrieved from a tooth (Denisova 2) that, on the basis of the stratigraphy of the site, is one of the oldest hominin remains discovered at Denisova Cave ( 27 , 28 ).

While Neandertals inhabited Europe and West Asia, Denisovans, who have been identified only from Denisova Cave to date ( 2 , 3 , 16 , 17 ), inhabited Asia ( 2 ) where they overlapped geographically with Neandertals in the Altai region and possibly elsewhere. The two groups must have interacted, as analyses of their genomes have shown that Denisovans interbred with Neandertals and with an unknown archaic hominin group that diverged earlier from the human lineage ( 7 ). Denisovans, or a group related to them, have also contributed genetically to present-day populations in Southeast Asian islands and Oceania and at lower levels to populations across mainland Asia and the Americas ( 2 , 3 , 7 , 18 – 22 ). Denisovan admixture has contributed to several traits in present-day humans ( 20 , 23 , 24 ), including, for example, the adaption of Tibetan populations to life at high altitude ( 25 ).

Genetic analyses of the remains of archaic hominins have yielded insights into their population history and admixture with each other and with modern humans [for example, ( 1 – 12 )]. DNA retrieved from fossils also allows their attribution to a hominin group in the absence of clear archaeological context or informative morphology [for example, ( 13 – 15 )]. One example is a hominin phalanx (Denisova 3) excavated in Denisova Cave (Altai, Russia) in 2008. Although its mitochondrial DNA (mtDNA) was found to fall outside the range of variation of both present-day humans and Neandertals ( 16 ), nuclear sequences retrieved from the specimen showed that it came from a member of a previously unknown sister group of Neandertals, thenceforth named “Denisovans” ( 2 ). The population split time between Neandertals and Denisovans has been estimated to be at least 190 thousand years ago (ka) and perhaps as much as 470 ka ( 7 ).

RESULTS

The Denisova 2 specimen A worn deciduous molar (figs. S1 and S2) was discovered in 1984 in layer 22.1 of the Main Gallery of Denisova Cave and was initially described as a right lower first deciduous molar (dm 1 ) (29). However, Shpakova and Derevianko (30) believed that the tooth was more likely a lower second deciduous molar (dm 2 ), and we concur with their opinion on the basis of the lack of a tuberculum molare and the large size. The crown of the tooth is almost completely worn away, and only a thin rim of enamel is preserved buccally, mesially, and lingually. The only feature of crown morphology preserved is a small remnant of the buccal groove. The roots are mostly resorbed, with only short stumps remaining mesiobuccally and mesiolingually. The exposed pulp cavity shows five diverticles entering the crown. The resorption of the roots and the fact that the specimen exfoliated naturally indicate an age equivalent to about 10 to 12 years in modern humans (for details, see section S1). The strong wear makes most morphological comparisons impossible. However, the cervical mesiodistal and buccolingual diameters are very large, falling outside of the range of variation seen in modern humans and in the range of Neandertals (table S1 and fig. S3).

DNA extraction and sequencing We extracted DNA (31) from ~10 mg of powder removed from the Denisova 2 specimen (fig. S2). One aliquot of the extract was used to produce a single-stranded DNA library, as previously described (32, 33). Another aliquot was converted into a single-stranded DNA library using a modified version of a protocol that enriches the library for DNA molecules carrying uracil residues (34), which result from deamination of cytosine bases in ancient DNA (35–37). This protocol (“mini-U-selection”) enriches for uracils only at the 3′ ends of fragments, but it requires fewer reaction steps and allows for a simpler library preparation than the original method (34). Out of a total of 701 million and 604 million DNA fragments sequenced from the two libraries, 0.06 and 0.46% of all sequences could be mapped to the human genome and exhibited a cytosine (C)–to–thymine (T) substitution at the first or last alignment position (table S2). These substitutions, especially when they occur close to the ends of sequences, are highly indicative of the presence of uracils, which are read as thymines by DNA polymerases (36). The percentage of fragments carrying C-to-T substitutions at the unselected 5′ ends was 9.4% in the former library and 7.3% in the library enriched for uracils. These percentages were 11.1 and 53.8% for the 3′ ends of fragments, respectively (table S3 and figs. S4 and S5), suggesting that authentic ancient DNA is present in both libraries (section S2).

Mitochondrial DNA We used oligonucleotide probes matching a modern human mtDNA sequence to enrich for mtDNA fragments from the library that was not selected for uracil residues (4, 38). Initial inspection of the sequences suggested that the library contained a mixture of contaminating present-day human mtDNA and endogenous sequences that are more similar to a Denisovan mtDNA than to modern human or Neandertal mtDNAs (section S3 and table S4). We therefore aligned the sequences from the mitochondrial capture as well as DNA fragments sequenced without enrichment from all libraries to the Denisova 3 mtDNA genome (16) and identified 86,788 unique mtDNA fragments (table S2). To mitigate the influence of contamination by present-day human mtDNA (section S2), we filtered the 21,537 fragments (table S2) that carried C-to-T differences relative to the Denisova 3 mtDNA genome near the start or end position of sequence alignments (first three or last three positions for sequences from the standard library and first two or last two positions for sequences from the mini-U-selection protocol) (39). Using these fragments, we reconstructed the Denisova 2 mtDNA genome with an average mtDNA coverage of 51-fold (fig. S6). When a given position was required to be covered by at least three fragments and when at least two-thirds of fragments overlapping a position were required to carry an identical base (39), all but 14 positions in the mtDNA genome were resolved (section S3). A maximum likelihood phylogenetic tree shows that the mtDNA of Denisova 2 clusters with the three previously determined Denisovan mtDNAs, to the exclusion of Neandertal and modern human mtDNAs (Fig. 1). It carries 29 nucleotide differences from Denisova 8, 70 nucleotide differences to Denisova 4, and 72 nucleotide differences to Denisova 3 (table S5). Fig. 1 Maximum likelihood tree relating the Denisova 2 mtDNA to other ancient and present-day mtDNAs. The Denisova 2 mtDNA (in red) clusters with the three previously determined Denisovan mtDNAs, to the exclusion of Neandertals and modern humans. Present-day human mtDNA sequences are noted in italics. The tree was rooted using a chimpanzee mtDNA sequence (not shown). Support for each branch is based on 500 bootstrap replications. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Accession codes for the comparative data and the geographical origins of ancient individuals are presented in table S5.

Relative mtDNA dating The tree in Fig. 2 shows the relationships of the four currently known Denisovan mtDNAs using the Middle Pleistocene hominin mtDNA from Sima de los Huesos (39) as an outgroup. A total of 22.5, 33.5, 49.5, and 51.5 substitutions are inferred by parsimony (fractions reflect ambiguous character reconstructions) to have accumulated between the common ancestor and the mtDNAs of Denisova 2, 8, 4, and 3, respectively. In agreement with previous observations (17), Denisova 8 appears to be substantially older than Denisova 3 and 4. The mtDNA of Denisova 2 is more closely related to that of Denisova 8 than to the other two mtDNAs. Notably, 20 substitutions are inferred to have accumulated on the terminal edge leading to Denisova 8, whereas only 9 substitutions are estimated to have done so on the terminal edge leading to Denisova 2, indicating that Denisova 2 is older than Denisova 8. Fig. 2 Phylogenetic tree relating the Denisova 2 mtDNA to other Denisovan mtDNA sequences. The number of substitutions on each branch was inferred by maximum parsimony, and the Middle Pleistocene mtDNA from Sima de los Huesos was used as an outgroup. The schematic representations of the specimens are drawn to scale, shown in the lower right corner. Using a mutation rate of 2.53 × 10−8 substitutions per site per year (95% highest posterior density, 1.76 × 10−8 to 3.23 × 10−8) (6), we estimate that the Denisova 2 individual is between 54.2 and 99.4 thousand years (ky) older than the Denisova 3 individual and between 20.6 and 37.7 ky older than the Denisova 8 individual, whereas the Denisova 3 and 4 individuals were roughly contemporaneous (between 3.7- and 6.9-ky difference). Although the absolute time estimates are dependent on whether the mutation rate of Denisovan mtDNA differs from that of modern human mtDNA, the difference in the number of substitutions between these individuals indicates that Denisova 2 is likely to be older than Denisova 8 and substantially older than Denisova 3 and 4.

Nuclear DNA and sexing For nuclear DNA analyses, DNA sequences from the Denisova 2 specimen were aligned to the human reference genome. We determined the sex of the Denisova 2 individual by counting the number of putatively deaminated DNA fragments that map to the X chromosome and the autosomes. The ratio of sequence coverage per base between the X chromosome and the autosomes is 1.06, indicating that Denisova 2 was a female (fig. S7). To minimize the effect of present-day human contamination (section S2 and table S3), we retained only sequences carrying a C-to-T substitution to the human reference genome at their first or last position (39), leaving 1.08 million DNA sequences (table S2) spanning 47 Mb of the human genome for further analysis.

Attribution to a hominin group Genetic, archaeological, and anthropological evidence indicate that Denisovans, Neandertals, and anatomically modern humans were present in Denisova Cave (2, 3, 7, 16, 17, 28, 40, 41). We computed the proportion of DNA fragments from the Denisova 2 specimen that share derived alleles specific to each branch in a phylogenetic tree relating the high-coverage genomes of a Denisovan (3), a Neandertal (7), and a present-day human from Africa (7). A total of 69,315 fragments overlapped phylogenetically informative positions at which a randomly drawn allele from at least one of these high-coverage genomes is derived (12). Of fragments that overlap positions where both the Denisovan and the Neandertal genomes are derived, 84% (1888 of 2246) share the derived allele. Of fragments that overlap positions where only the Denisovan genome is derived, 49% (2051 of 4160) carry the Denisovan-like allele, whereas the corresponding values for the sharing of Neandertal- and modern human–specific alleles are 6% (252 of 4231) and 5% (307 of 5924), respectively (Fig. 3A). We thus conclude that the Denisova 2 specimen originated from a Denisovan individual. Fig. 3 Attribution of Denisova 2 to a hominin group. (A) For each branch of a phylogenetic tree relating the high-coverage genomes of a Denisovan, a Neandertal, and a present-day human from Africa, the 95% binomial CIs of the proportion of DNA fragments from the Denisova 2 specimen that share a derived allele with that branch are given. (B) The fraction of substitutions inferred to have occurred after the split from the Denisova 2 genome along the branch from the human-chimpanzee (Ch) ancestral sequences to the high-coverage genomes of a Denisovan, a Neandertal, and 12 present-day humans (“X” in the schematic phylogenetic tree shown in the inset) is given. Error bars denote 95% CIs. The corresponding values for two previously sequenced Denisovan teeth, Denisova 4 and Denisova 8 (17), are 92 and 93% of sequences sharing derived alleles with the Neandertal-Denisovan branch, 72 and 60% with the Denisovan branch, 5% with the Neandertal branch, and 2% with the modern human branch, respectively (fig. S8). Thus, Denisova 2 shares fewer derived alleles with the high-coverage Denisova 3 genome than the other two Denisovan genomes (χ2 = 7.6257, P = 0.003 and χ2 = 43.015, P = 2.717 × 10−11 for Denisova 4 and 8, respectively), showing that Denisova 2 is more distantly related to Denisova 3 than Denisova 4 and Denisova 8.