Since the initial identification of the Denisovans a decade ago, only a handful of their physical remains have been discovered. Here we analysed ~3,800 non-diagnostic bone fragments using collagen peptide mass fingerprinting to locate new hominin remains from Denisova Cave (Siberia, Russia). We identified five new hominin bones, four of which contained sufficient DNA for mitochondrial analysis. Three carry mitochondrial DNA of the Denisovan type and one was found to carry mtDNA of the Neanderthal type. The former come from the same archaeological layer near the base of the cave’s sequence and are the oldest securely dated evidence of Denisovans at 200 ka (thousand years ago) (205–192 ka at 68.2% or 217–187 ka at 95% probability). The stratigraphic context in which they were located contains a wealth of archaeological material in the form of lithics and faunal remains, allowing us to determine the material culture associated with these early hominins and explore their behavioural and environmental adaptations. The combination of bone collagen fingerprinting and genetic analyses has so far more-than-doubled the number of hominin bones at Denisova Cave and has expanded our understanding of Denisovan and Neanderthal interactions, as well as their archaeological signatures.
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The mtDNA consensus sequences generated for the current study are available in NCBI GenBank under accession numbers MT576650–MT576653.
Raw MALDI-TOF files from ZooMS analysis of the hominin bones DC4969 (Denisova 17), DC7277 (Denisova 18), DC8846 (Denisova 19), DC7795 (Denisova 20) and DC8591 (Denisova 21) converted to open source format. Files have been uploaded to: https://doi.org/10.17617/3.44.
MicroCT scan files of the hominin bones DC4969 (Denisova 17), DC7277 (Denisova 18), DC8846 (Denisova 19) and DC7795 (Denisova 20). Files have been uploaded to: https://doi.org/10.17617/3.45.
Reich, D. et al. Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature 468, 1053–1060 (2010).
Krause, J. et al. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature 464, 894–897 (2010).
Prüfer, K. et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017).
Browning, S. R., Browning, B. L., Zhou, Y., Tucci, S. & Akey, J. M. Analysis of human sequence data reveals two pulses of archaic Denisovan admixture. Cell 173, 53–61.e9 (2018).
Jacobs, G. S. et al. Multiple deeply divergent Denisovan ancestries in Papuans. Cell 177, 1010–1021.e32 (2019).
Slon, V. et al. A fourth Denisovan individual. Sci. Adv. 3, e1700186 (2017).
Slon, V. et al. The genome of the offspring of a Neanderthal mother and a Denisovan father. Nature 561, 113–116 (2018).
Sawyer, S. et al. Nuclear and mitochondrial DNA sequences from two Denisovan individuals. Proc. Natl Acad. Sci. USA 112, 15696–15700 (2015).
Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222–226 (2012).
Chen, F. et al. A late middle Pleistocene Denisovan mandible from the Tibetan Plateau. Nature 569, 409–412 (2019).
Zhang, D. et al. Denisovan DNA in late Pleistocene sediments from Baishiya Karst Cave on the Tibetan Plateau. Science 370, 584–587 (2020).
Buckley, M., Collins, M., Thomas-Oates, J. & Wilson, J. C. Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 3843–3854 (2009).
Brown, S. et al. Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis. Sci. Rep. 6, 23559 (2016).
Buckley, M. et al. Species identification of archaeological marine mammals using collagen fingerprinting. J. Archaeol. Sci. 41, 631–641 (2014).
Welker, F. et al. Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne. Proc. Natl Acad. Sci. USA 113, 11162–11167 (2016).
Charlton, S. J. L., Alexander, M., Collins, M. J., Milner, N. & Craig, O. E. Finding Britain’s last hunter-gatherers: a new biomolecular approach to ‘unidentifiable’ bone fragments utilising bone collagen. J. Archaeol. Sci. 73, 55–61 (2016).
Douka, K. et al. Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave. Nature 565, 640–644 (2019).
Devièse, T. et al. Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition. Proc. Natl Acad. Sci. USA 114, 10606–10611 (2017).
Jacobs, Z. et al. Timing of archaic hominin occupation of Denisova Cave in southern Siberia. Nature 565, 594–599 (2019).
Agadjanian, A. K. The dynamics of bioresources and activity of the paleolithic man, using the example of northwestern Altai Mountains. Paleontol. J. 40, S482–S493 (2006).
Bolikhovskaya, N. S. & Shunkov, M. V. Pleistocene environments of Northwestern Altai: vegetation and climate1. Archaeol. Ethnol. Anthropol. Eurasia 42, 2–17 (2014).
Shunkov, M. V., Kozlikin, M. B. & Derevianko, A. P. Dynamics of the Altai Paleolithic industries in the archaeological record of Denisova Cave. Quat. Int. https://doi.org/10.1016/j.quaint.2020.02.017 (2020).
Derevianko, A. P., Shunkov, M. V. & Kozlikin, M. B. Who were the Denisovans? Archaeol. Ethnol. Anthropol. Eurasia 48, 3–32 (2020).
Zavala, E. I. et al. Pleistocene sediment DNA reveals hominin and faunal turnovers at Denisova Cave. Nature 595, 399–403 (2021).
Slon, V. et al. Neandertal and Denisovan DNA from Pleistocene sediments. Science 356, 605–608 (2017).
Vasiliev, S. K., Shunkov, M. V. & Kozlikin, M. B. Preliminary results for the balance of megafauna from Pleistocene layers of the east gallery, Denisova Cave. Probl. Archaeol. Ethnogr. Anthropol. Sib. Adjac. T. 19, 32–38 (2013).
van Doorn, N. L., Hollund, H. & Collins, M. J. A novel and non-destructive approach for ZooMS analysis: ammonium bicarbonate buffer extraction. Archaeol. Anthropol. Sci. 3, 281 (2011).
Brown, S. et al. Zooarchaeology by mass spectrometry (ZooMS) for bone material – AmBiC protocol v1 https://www.protocols.io/view/zooarchaeology-by-mass-spectrometry-zooms-for-bone-bffdjji6 (2020).
Brown, S. et al. Zooarchaeology through the lens of collagen fingerprinting at Denisova Cave. Sci. Rep. 11, 15457 (2021).
Buckley, M. & Kansa, S. W. Collagen fingerprinting of archaeological bone and teeth remains from Domuztepe, South Eastern Turkey. Archaeol. Anthropol. Sci. 3, 271–280 (2011).
Immel, A. et al. Effect of X-ray irradiation on ancient DNA in sub-fossil bones – Guidelines for safe X-ray imaging. Sci. Rep. 6, 32969 (2016).
Krause, J. et al. Neanderthals in central Asia and Siberia. Nature 449, 902–904 (2007).
Vernot, B. et al. Excavating Neandertal and Denisovan DNA from the genomes of Melanesian individuals. Science 352, 235–239 (2016).
Peter, B. M. 100,000 years of gene flow between Neanderthals and Denisovans in the Altai mountains. Preprint at bioRxiv https://doi.org/10.1101/2020.03.13.990523v1 (2020).
Reich, D. et al. Denisova admixture and the first modern human dispersals into Southeast Asia and Oceania. Am. J. Hum. Genet. 89, 516–528 (2011).
Mafessoni, F. et al. A high-coverage Neandertal genome from Chagyrskaya Cave. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2004944117 (2020).
Bordes, L. et al. Raman spectroscopy of lipid micro-residues on Middle Palaeolithic stone tools from Denisova Cave, Siberia. J. Archaeol. Sci. 95, 52–63 (2018).
Zaidner, Y. & Weinstein-Evron, M. The end of the Lower Paleolithic in the Levant: the Acheulo-Yabrudian lithic technology at Misliya Cave, Israel. Quat. Int. 409, 9–22 (2016).
Barkai, R. & Gopher, A. Cultural and biological transformations in the Middle Pleistocene Levant: a view from Qesem Cave, Israel. in Dynamics of Learning in Neanderthals and Modern Humans Vol 1: Cultural Perspectives (eds Akazawa, T. et al.) 115–137 (Springer, 2013).
Strohalm, M., Hassman, M., Kosata, B. & Kodícek, M. mMass data miner: an open source alternative for mass spectrometric data analysis. Rapid Commun. Mass Spectrom. 22, 905–908 (2008).
Rohland, N., Glocke, I., Aximu-Petri, A. & Meyer, M. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nat. Protoc. 13, 2447–2461 (2018).
Korlević, P. et al. Reducing microbial and human contamination in DNA extractions from ancient bones and teeth. Biotechniques 59, 87–93 (2015).
Gansauge, M.-T., Aximu-Petri, A., Nagel, S. & Meyer, M. Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nat. Protoc. https://doi.org/10.1038/s41596-020-0338-0 (2020).
Kircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res. 40, e3 (2012).
Fu, Q. et al. DNA analysis of an early modern human from Tianyuan Cave, China. Proc. Natl Acad. Sci. USA 110, 2223–2227 (2013).
Maricic, T., Whitten, M. & Pääbo, S. Multiplexed DNA sequence capture of mitochondrial genomes using PCR products. PLoS ONE 5, e14004 (2010).
Renaud, G., Stenzel, U. & Kelso, J. leeHom: adaptor trimming and merging for Illumina sequencing reads. Nucleic Acids Res. 42, e141 (2014).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Green, R. E. et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134, 416–426 (2008).
Prüfer, K. et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505, 43–49 (2014).
Rougier, H. et al. Neandertal cannibalism and Neandertal bones used as tools in Northern Europe. Sci. Rep. 6, 29005 (2016).
Briggs, A. W. et al. Targeted retrieval and analysis of five Neandertal mtDNA genomes. Science 325, 318–321 (2009).
Gansauge, M.-T. & Meyer, M. Selective enrichment of damaged DNA molecules for ancient genome sequencing. Genome Res. 24, 1543–1549 (2014).
Skoglund, P. et al. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proc. Natl Acad. Sci. USA 111, 2229–2234 (2014).
Posth, C. et al. Deeply divergent archaic mitochondrial genome provides lower time boundary for African gene flow into Neanderthals. Nat. Commun. 8, 16046 (2017).
Hajdinjak, M. et al. Reconstructing the genetic history of late Neanderthals. Nature 555, 652–656 (2018).
Peyrégne, S. et al. Nuclear DNA from two early Neandertals reveals 80,000 years of genetic continuity in Europe. Sci. Adv. 5, eaaw5873 (2019).
Wood et al. A new date for the Neanderthals from El Sidrón cave (Asturias, Northern Spain). Archaeometry 55, 148–158 (2013).
Meyer, M. et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505, 403–406 (2014).
Fu, Q. et al. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514, 445–449 (2014).
Fu, Q. et al. An early modern human from Romania with a recent Neanderthal ancestor. Nature 524, 216–219 (2015).
Fu, Q. et al. The genetic history of Ice Age Europe. Nature 534, 200–205 (2016).
Devièse, T. et al. Compound-specific radiocarbon dating and mitochondrial DNA analysis of the Pleistocene hominin from Salkhit Mongolia. Nat. Commun. 10, 274 (2019).
Sikora, M. et al. Ancient genomes show social and reproductive behavior of early Upper Paleolithic foragers. Science 358, 659–662 (2017).
Green, R. E. et al. A draft sequence of the Neandertal genome. Science 328, 710–722 (2010).
Horai, S. et al. Man’s place in Hominoidea revealed by mitochondrial DNA genealogy. J. Mol. Evol. 37, 89 (1993).
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
Stecher, G., Tamura, K. & Kumar, S. Molecular evolutionary genetics analysis (MEGA) for macOS. Mol. Biol. Evol. 37, 1237–1239 (2020).
Swofford, D. L. PAUP: phylogenetic analysis using parsimony, version 4.0 b10. (Sinauer, 2002).
Suchard, M. A. et al. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 4, vey016 (2018).
Darriba, D. et al. ModelTest-NG: a new and scalable tool for the selection of DNA and protein evolutionary models. Mol. Biol. Evol. 37, 291–294 (2020).
Baele, G. et al. Improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty. Mol. Biol. Evol. 29, 2157–2167 (2012).
Baele, G., Li, W. L. S., Drummond, A. J., Suchard, M. A. & Lemey, P. Accurate model selection of relaxed molecular clocks in Bayesian phylogenetics. Mol. Biol. Evol. 30, 239–243 (2013).
Fu, Q. et al. A revised timescale for human evolution based on ancient mitochondrial genomes. Curr. Biol. 23, 553–559 (2013).
Kass, R. E. & Raftery, A. E. Bayes factors. J. Am. Stat. Assoc. 90, 773–795 (1995).
Lisiecki, L. E. & Raymo, M. E. A Pliocene–Pleistocene stack of 57 globally distributed benthic δ18O records. Paleoceanography 20, PA1003 (2005).
We thank the European Research Council, the Max Planck Society, the Oxford Radiocarbon Accelerator Unit (ORAU) and the Institute of Archeology and Ethnography, Russian Academy of Sciences Siberian Branch for their ongoing support. M. O’Reilly from the Max Planck Institute for the Science of Human History and I. Cartwright from the University of Oxford photographed the hominin fossils. We also thank in particular the volunteers who helped us sample the material (M. Jenkins, E. Gillespie, L. Bell, M. Caldarola, R. Heikkila, L. Doody, S. Amirova, G. Church, L. Koster, R. Holmes, L. Ghent, P. Ewles-Bergeron, N. Siemens, M. Sandilands and J. Zavodski); V. Slon, S. Peyrégne, E. Essel, S. Nagel and J. Richter from the Max Planck Institute for Evolutionary Anthropology for discussions and laboratory work. This work received funding from the ERC under the European Union’s Horizon 2020 Research and Innovation Programme grant agreement no. 715069 (FINDER) to K.D. and under the European Union’s Seventh Framework Programme (FP7/2007–2013) grant agreement no. 324139 (PalaeoChron) to T.H. and grant agreement no. 694707 (100 Archaic Genomes) to S.P. The archaeological field studies were funded by the Russian Foundation for Basic Research (no. 20-29-01011).
The authors declare no competing interests.
Peer review information Nature Ecology and Evolution thanks Virginia Harvey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Brown, S., Massilani, D., Kozlikin, M.B. et al. The earliest Denisovans and their cultural adaptation. Nat Ecol Evol 6, 28–35 (2022). https://doi.org/10.1038/s41559-021-01581-2
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