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Denisova Cave in southern Siberia is the type locality of the Denisovans, an archaic hominin group who were related to lớn Neanderthals1,2,3,4. The dozen hominin remains recovered from the deposits also include Neanderthals5,6 and the child of a Neanderthal & a Denisovan7, which suggests that Denisova Cave sầu was a liên hệ zone between these archaic hominins. However, uncertainties persist about the order in which these groups appeared at the site, the timing and environmental context of hominin occupation, và the association of particular hominin groups with archaeological assemblages5,8,9,10,11. Here we report the analysis of DNA from 728 sediment samples that were collected in a grid-like manner from layers dating to the Pleistocene epoch. We retrieved ancient faunal & hominin mitochondrial (mt)DNA from 685 and 175 samples, respectively. The earliest evidence for hominin mtDNA is of Denisovans, & is associated with early Middle Palaeolithic stone tools that were deposited approximately 250,000 to 170,000 years ago; Neanderthal mtDNA first appears towards the kết thúc of this period. We detect a turnover in the mtDNA of Denisovans that coincides with changes in the composition of faunal mtDNA, and evidence that Denisovans và Neanderthals occupied the site repeatedly—possibly until, or after, the onphối of the Initial Upper Palaeolithic at least 45,000 years ago, when modern human mtDNA is first recorded in the sediments.

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Denisova Cave consists of three chambers (designated Main, East & South Chambers) that contain deposits with stratigraphic sequences extending from the Middle Pleistocene to the Holocene epoch. The Pleistocene deposits have chronologies that have been constructed from the radiocarbon dating of bone, tooth & charcoal5 (to around 50 thousvà years ago (ka)) and optical dating of sediments8 (khổng lồ more than 300 ka). Optical ages for Main & East Chambers (Fig. 1a–c) can be aligned on a common time scale (Extended Data Fig. 1) but excavations are ongoing in South Chamber, where layers are only tentatively recognized. Mitochondrial DNA & nuclear DNA have sầu been recovered from eight hominin fossils, enabling four to lớn be assigned to lớn Denisovans (Denisova 2, Denisova 3, Denisova 4 & Denisova 8)1,2,3,4, three to Neanderthals (Denisova 5, Denisova 9 and Denisova 15)5,6,12, & one to the child of a Neanderthal & a Denisovan (Denisova 11)7. However, there are too few fossils lớn enable the detailed reconstruction of the timing và sequence of hominin occupation, & the association of the early Middle Palaeolithic, middle Middle Palaeolithic & Initial Upper Palaeolithic assemblages identified at the site with specific hominin groups. Moreover, two Denisovan fossils (Denisova 3 & Denisova 4)—but no modern human remains—have been recovered from the Initial Upper Palaeolithic layers, so it is debated whether archaic hominins or modern humans created the associated ornaments & bone tools9,10,11.

Fig. 1: Stratigraphic sequences in Denisova Cave sầu, showing locations of sediment samples collected for mtDNA analysis & results obtained for ancient hominins.


a, East Chamber, southeast protệp tin. b, East Chamber, northwest protệp tin. c, Main Chamber, southeast profile. d, Phylogenetic tree of mtDNA genomes used as references lớn distinguish specific hominin lineages, and estimated placement of the Neanderthal mtDNA lineage identified in sample M76 from Main Chamber layer trăng tròn. Filled circles in ac indicate the locations of individual sediment samples, và colours correspond khổng lồ the hominin mtDNA detected: red (Denisovan), xanh (Neanderthal), yellow (ancient modern human), grey (unidentified ancient hominin) và White (no ancient hominins detected). Other symbols denote samples for which mtDNA could be assigned to lớn one of the specific hominin lineages in d (Denisovan, red open triangles; Neanderthal, xanh open diamonds, crosses and star). Composite stratigraphic sections (modified from a previous publication8) to lớn the left of each protệp tin show modelled start & kết thúc ages (in ka) for sedimentary layers (uncertainties at 95.4% probability). Hominin specimen numbers are shown circled in the layer from which each fossil was recovered: Denisovan (red), Neanderthal (blue) và Neanderthal–Denisovan offspring (both colours)5. Dashed lines in profiles in ab indicate areas in which layer assignment is uncertain8. Background shading denotes the associated archaeological assemblage: early Middle Palaeolithic (eMP) (dark green), middle Middle Palaeolithic (mMP) (light green), Initial Upper Palaeolithic (IUP) (dark orange) và Upper Palaeolithic (UP) (light orange).

A pilot study of DNA preservation in sediments from Denisova Cave sầu identified ancient hominin mtDNA in 12 out of 52 samples13, which suggested a path lớn reconstructing the occupational history of the site at higher resolution than is feasible from the scarce hominin fossil record. Here we report the analysis of 728 sediment samples, collected in a 10–15-cm grid-like pattern from the exposed Pleistocene deposits in all three chambers (Extended Data Figs. 2, 3a, b, Supplementary Information sections 1, 2). Using automated laboratory protocols, DNA was extracted from each sample, converted khổng lồ single-stranded libraries and enriched for mammalian and hominin mtDNA13,14, which we identified khổng lồ the biological-family cấp độ using an established analysis pipeline13.

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We identified ancient mammalian mtDNA in 685 samples (94%) from all sampled layers, including those older than 290 ka (Extended Data Figs. 4, 5a, Supplementary Data 1). DNA retrieved from the deposits in all three chambers shows deamination-induced substitutions that are characteristic of ancient DNA15,16. These substitutions significantly increase with age (Extended Data Figs. 3d, 4a), which argues against extensive post-depositional leaching of DNA across layers13,17. We also observed a significant reduction in average DNA fragment length and the number of mtDNA fragments recovered with increasing age (Extended Data Figs. 3e, f, 4b, c), although variability was greater across layers than for deamination, probably as a result of local differences in the geochemical environment. For example, the fewest ancient DNA fragments (none in some samples) were recovered from the youngest sampled unit (designated pdd-9) in South Chamber (Extended Data Fig. 3f), which is extensively phosphatized8 & has slightly acidic pH values (between 6 and 6.5) (Supplementary Information section 3, Supplementary Data 2).

We detected ancient hominin mtDNA in 175 samples (24%), covering nearly all layers in all three chambers (Fig. 1a–c, Extended Data Figs. 3a, b, 5b). Four samples showed evidence for the presence of predominantly one haplotype & yielded sufficient mtDNA fragments to reconstruct mtDNA consensus sequences that are more than 80% complete (Supplementary Information sections 5, 6). Three of the sequences (samples E202 & E213 from East Chamber (layers 11.4 & 11.4/12.1) and sample M65 from Main Chamber (layer 19)) group with Neanderthals in phylogenetic trees built with previously published hominin mtDNAs (Supplementary Information section 7), specifically with Denisova 5, Denisova 15, Mezmaiskaya 1 & Scladina I-4A (Fig. 1d, Extended Data Fig. 6a). The fourth sequence (sample M71 from Main Chamber (layer 20)) is of the Denisovan type & falls basal to Denisova 2 and Denisova 8, albeit with low bootstrap support (Extended Data Fig. 6b). The most complete mtDNA sequence (over 99% of the genome reconstructed) for a Neanderthal from Main Chamber (M65) has a genetic age estimate of 140 ka (95.4% highest posterior density interval of 181–98 ka) (Supplementary Information section 7), consistent with the time of deposition of layer 19 (151 ± 17 to 128 ± 13 ka; here and below, uncertainties on optical ages are given at 95.4% probability)8.

For the remaining 171 samples, we assigned mtDNA fragments khổng lồ specific hominin groups by counting the number of fragments that support lineage-specific states at diagnostic sites that distinguish between modern human, Neanderthal và Denisovan mtDNA genomes. We distinguished three Neanderthal lineages: the Sima de los Huesos lineage (representing Neanderthals who lived approximately 430 ka in Spain and whose mtDNA is most closely related khổng lồ that of Denisovans)18,19; the Hohlenstein–Stadel (HST) lineage, which falls basal to all other Neanderthal mtDNAs20; và the ‘typical’ Neanderthal mtDNA, known from all other Neanderthals. The presence of ancient modern human mtDNA was evaluated by restricting the analysis to lớn deaminated fragments to lớn mitigate the effect of present-day human DNA contamination. We identified Denisovan và typical Neanderthal mtDNA in 79 và 47 samples, respectively (based on 54–9,093 chất lượng hominin mtDNA fragments), and modern human mtDNA in 35 samples (based on 55–2,200 deaminated fragments) (Fig. 1a–c, Extended Data Fig. 3a, b). We detected DNA from two hominin groups in ten samples, either within a single library or across libraries that were prepared from independent subsamples in some cases (Extended Data Fig. 6c, Supplementary Information section 4). In addition, we identified one sample (M76 from Main Chamber (layer 20)) containing hominin mtDNA fragments that support the branch shared by HST and typical Neanderthal mtDNA, but neither of the branches defining those lineages. This signal cannot be created by mixing mtDNA fragments from Neanderthals, Denisovans & ancient or present-day modern humans. On the basis of simulations with ancestralized Neanderthal mtDNA, the mtDNA in this sample is compatible with the presence of a previously unknown Neanderthal mtDNA lineage that diverged from typical Neanderthal mtDNA between 255 và 230 ka, 20 khổng lồ 45 thous& years after the split of the HST và typical Neanderthal mtDNA lineages (Supplementary Information section 9).

The oldest hominin mtDNA recovered—identified as Denisovan—originates from a sample in Main Chamber layer 21, which began khổng lồ accumulate 250 ± 44 ka. This provides the earliest genetic evidence for hominin occupation in Denisova Cave; Denisova 2 was found in layer 22.1, but is probably intrusive from an overlying layer & has an estimated age of 194–123 ka5. Ahy vọng all 223 samples from the early Middle Palaeolithic layers in Main và East Chambers, 50 contained evidence for Denisovan mtDNA and only three (all from layer đôi mươi in Main Chamber) for Neanderthal mtDNA. Two of these (M174 and M235) contain typical Neanderthal mtDNA & are from areas in which small-scale mixing with overlying sediments may have sầu occurred8; the third (M76) is from the middle of the layer, và carries the previously unknown Neanderthal mtDNA lineage. These results point lớn Denisovans as the first và principal makers of the early Middle Palaeolithic assemblages, which are older than 170 ± 19 ka. Consistent with this interpretation, the detection of Neanderthal mtDNA in a sediment sample from early Middle Palaeolithic layer 14 in East Chamber in the pilot study13 was due to lớn an incorrect assignment, which was later corrected khổng lồ middle Middle Palaeolithic layer 11.4 in this chamber8 (Supplementary Information section 2). Our results also suggest that Neanderthals first occupied Denisova Cave sầu towards the kết thúc of the early Middle Palaeolithic & may therefore have contributed lớn the production of these assemblages in their later stages.

Forty out of 173 samples from the middle Middle Palaeolithic layers in Main và East Chambers (deposited approximately 160–60 ka) yielded Neanderthal and/or Denisovan mtDNA, with both present in six samples (Fig. 1a–c). DNA from both groups also occurs in the deformed Middle Palaeolithic layers in South Chamber (Extended Data Fig. 3b). Notably, sediments deposited between 120 ± 11 và 97 ± 11 ka in Main và East Chambers produced no traces of Denisovan mtDNA, whereas 12 samples contained Neanderthal mtDNA. This suggests that only Neanderthals may have occupied the cave sầu during that period, and possibly for most of Marine Isotope Stage (MIS) 5 (Fig. 2).