Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis


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Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis

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Title:
Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis
Series Title:
Scientific Reports
Creator:
Brown, Samantha
Higham, Thomas
Slon, Viviane
Pääbo, Svante
Meyer, Matthias
Katerina, Douka
Brock, Fiona
Comeskey, Daniel
Procopio, Noemi
Shunkov, Michael
Derevianko, Anatoly
Buckley, Michael
Publication Date:
Language:
English

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Subjects / Keywords:
Hominin ( local )
Hominin Bone ( local )
Desinova Cave, Siberia ( local )
Collagen Fingerprinting ( local )
Mitochondrial DNA Analysis ( local )
Archaic Humans ( local )
Middle Paleolithic ( local )
Upper Paleolithic ( local )
Genre:
serial ( sobekcm )

Notes

Abstract:
DNA sequencing has revolutionised our understanding of archaic humans during the Middle and Upper Palaeolithic. Unfortunately, while many Palaeolithic sites contain large numbers of bones, the majority of these lack the diagnostic features necessary for traditional morphological identification. As a result the recovery of Pleistocene-age human remains is extremely rare. To circumvent this problem we have applied a method of collagen fingerprinting to more than 2000 fragmented bones from the site of Denisova Cave, Russia, in order to facilitate the discovery of human remains. As a result of our analysis a single hominin bone (Denisova 11) was identified, supported through in-depth peptide sequencing analysis and found to carry mitochondrial DNA of the Neandertal type. Subsequent radiocarbon dating revealed the bone to be >50,000 years old. Here we demonstrate the huge potential collagen fingerprinting has for identifying hominin remains in highly fragmentary archaeological assemblages, improving the resources available for wider studies into human evolution.
Original Version:
Scientific Reports, Vol. 6, no. 23559 (2016-03-29).

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This item is licensed with the Creative Commons Attribution License. This license lets others distribute, remix, tweak, and build upon this work, even commercially, as long as they credit the author for the original creation.
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K26-00022 ( USFLDC: LOCAL DOI )
k26.22 ( USFLDC: LOCAL Handle )

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2 are either teeth or small in size, generally phalanges, which are less likely to suer fragmentation leading to the loss of diagnostic features. Such fragmentation, which is due to both environmental taphonomy and carnivore or human activity, results in a high percentage of the bones excavated from this and many other archaeological sites which cannot be identied on the basis of their morphology3,8. Within the East Gallery of Denisova Cave alone, excavations between 2005 and 2013 yielded approximately 135,600 bones; however 128,591 could not be identied8.Here we apply a method of species identification by collagen peptide mass fingerprinting, known as Zooarchaeology by Mass Spectrometry (ZooMS), to 2,315 archived unidentiable bone fragments from Denisova Cave. ese non-diagnostic bones were selected from amongst material excavated from the cave’s East Gallery in 2014. e remains varied in size, generally ranging between 3–5þ t cm, with bones which were large enough to be useful for additional analyses (i.e. radiocarbon and DNA analysis) preferentially selected. In the recent past, ZooMS analysis has been successful in discriminating between a diverse range of mammalian groups, including domesticated taxa9,10, wild terrestrial taxa11,12, and marine fauna13, as well as some non-mammalian taxa13,14. To achieve this ZooMS uses peptide mass ngerprinting to analyse the dominant protein in bone, Type 1 collagen (COL1) which is known for its longevity, particularly in cooler climates. e method has yielded collagen nger-prints in specimens dating back to ~3.5 million years15. COL1 is comprised of three polypeptide chains, known as alpha chains, which are constructed of a repeating pattern of amino acids. ese chains carry variation in their amino acid sequence, particularly in the alpha 2 (I) chain (COL1þ n 2), and are visible in the analysis and measure-ment of collagen peptides9. Measurement is conducted using Matrix-assisted Laser Desorption/Ionisation Mass Spectrometry (MALDI-MS), in which peptides are converted into their respective mass-to-charge (m/z) values. Comparison of these values with homologous values of known fauna allows for identication. A previous study9 proposed that seven specic collagen peptides were appropriate for zooarchaeological analysis, but with addi-tional taxonomic groups this number has been expanded upon13. Although occasionally at the family/sub-family level11, these have been shown to most frequently identify a specimen to its genus and in some cases to species level15. e identication of hominin remains in this study is therefore based on the matching of previously pub-lished markers for ‘human’9,12 peptides, the majority of which could also be present in other primates due to lack of wider inclusion in these earlier studies. In the case of Denisova cave there is a very low probability of other primates being the source of hominin markers of course, due to their geographical distribution.‡•—Ž–•‘‘•…”‡‡‹‰ˆ‘”Š‘‹‹”‡ƒ‹•äþ e 2,315 bones included in this study were excavated from layers 11, 12, 14 and 17 in Denisova Cave’s East Gallery. From each specimen, a bone chip ranging from 20–50þ t mg was removed and the bone demineralized in order to release acid-soluble proteins, which were then ultraltered and enzymatically digested into peptides16. In a single case (sample DC1227) the ngerprint identied contained all 6 of the peptide markers previously identied as human markers: m/z 1235.6, 1477.7, 1580.8, 2115.1, 2832.4, 2869.5 and 2957.4 (Fig.1). However, since the original publications9,12 identifying these six markers, several have been found to appear in other taxa including afrosoricids and xenarthrans17. erefore in order to conrm this peptide mass ngerprint (PMF)-based identication and the identity of the peptides present throughout the n-gerprint, the sample was then analysed for peptide sequencing by LC-Orbitrap-Elite tandem mass spectrometry18. Figure 1.þ MALDI-ToF mass spectrum of digested collagen from DC1227. Previously published human markers are labelled A–G. All numbered peaks represent conrmed sequencing-matched peptides observed in human collagen (except E which is only known through similarity to homologous markers in other species9).

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3 ese analyses conrmed the dominance of human collagen in the sample (all annotated peaks in Fig.1 were matched as Homo COL1A1 and COL1A2 peptides) to the exclusion of all other known mammal sequences (including other primates), identifying two new sequence PMF markers unique to Hominoidea at m/z 2121.0 and m/z 2568.2 (sequence spectra shown in Supplementary Figures S1 and S2 respectively). e LC-MS/MS data (which also yielded at least 13 non-collagenous human proteins; see Supplementary Data), specically the com-bination of particular collagen peptides (e.g., the tandem spectra shown in Supplementary Figures S1–S12) was able to resolve this identication to the genus level of Homo, even when compared with collagen sequences from closely related primates including the other great ape genera19.e specimen identied as originating from a hominin, DC1227, was excavated from square A-2, layer 12 of the East Gallery. Prior to sampling, the bone weighed 1.68 g, with a maximum length of 24.7þ t mm and width of 8.39þ t mm. Around 36þ t mg was taken for ZooMS analysis (Fig.2). e bone appears quite unremarkable, without any morphological features or evidence for purposeful modication, it was therefore easily overlooked in osteo-logical analysis.‹…”‘…ƒ‘ˆwxx}äþ Prior to further destructive sampling for radiocarbon and mitochondrial DNA analysis, micro-computed tomography (micro-CT) was performed. Given the rarity of such a discov-ery, a micro-CT was deemed appropriate in order to identify areas that had suered from visible degradation and should therefore be avoided in future analysis. e results revealed the sample to be highly dense, with no signs of bone degradation despite a series of diagenetic micro-cracks running through its length. ree of these sub-micron cracks run in close proximity to one another through the bone, however they do not form a ssure and do not appear to have compromised the structure of the bone. e scan also highlighted the extent of acid etching and pitting on the bones surface, which may be the result of being passed through the digestive system of a carnivore (Fig.3). ere are a number of carnivores identied at the site; given the prevalence of hyaenas at Denisova Cave, it seems likely that the bone was subjected to acid etching via the stomach acids of a hyaena8,20.ƒ†‹‘…ƒ”„‘ƒƒŽ›•‹•‘ˆwxx}äþ Prior to our work, all hominin bones discovered at the site were too small for direct radiocarbon dating, meaning that age determinations for these individuals have rested solely on their position relative to other dated materials, fauna and sediments4. is method assumes that the hom-inin remains have not been subject to post-depositional movement due to bioturbation or other taphonomic inuences. is is particularly problematic when considering the limited chronological data that has so far been published for the site. Age determinations for the East Gallery are conned to a single layer (layer 11) with a series of radiocarbon dates on bones that exhibit cut marks or other human modications, and potentially reveal some degree of mixing in that layer4. Radiocarbon analysis of DC1227 was therefore undertaken in order to determine that the specimen had not been reworked or intruded from higher up the sequence. While no scientic dating has so far been published for the older layer 12 of the East Gallery, studies of mammalian fauna suggest the layer is associated with species of steppe inhabitants, perhaps representing oxygen isotope stage (OIS) 4, which ended ~57,000 years ago21,22. If DC1227 was found in situ, it would be expected to return an age beyond the upper limit of radiocarbon dating (þ n 50,000 years).Radiocarbon dating was carried out at the Oxford Radiocarbon Accelerator Unit (ORAU) following standard procedures and protocols23 yielding an age estimate of þ n 49,900 years BP (OxA-32241), indicating that the bone is older than the maximum measureable limit for the radiocarbon dating of bone collagen. e result is entirely consistent with its inferred geoarchaeological age with respect to the stratigraphic sequence at the site. Figure 2.þ Photograph of DC1227, detailing each visible surface of the bone.

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4 Isotopic measurements of carbon and nitrogen yielded a þ n 13C value of þ n 17.3‰ and a þ n 15N value of 16.4‰. Hominins in the region typically return nitrogen isotopic values between 13–15‰, shown for example amongst the Neandertals of Okladnikov Cave24. e elevated values of DC1227 could indicate a variety of dietary anom-alies, including a diet rich in protein derived from higher trophic level organisms such as freshwater sh25,26. Further research is required in order to place the elevated isotopic values in a proper context and this will be reported in future. Such investigations into the isotopic composition of the hominin and associated fauna from Denisova have the potential to reveal important information about the diets of Palaeolithic hominins living in the Altai and such research is currently underway at the University of Oxford.‹–‘…Š‘†”‹ƒŽ–f•‡“—‡…‡•ˆ”‘•’‡…‹‡wxx}äþ We extracted DNA from 30.9þ t mg of bone powder from DC122727. An aliquot of the extract was converted into a single-stranded DNA library28, which was enriched for hominin mitochondrial DNA fragments using human mitochondrial probes29. e isolated DNA fragments were sequenced and mapped to the revised Cambridge human mitochondrial ref-erence sequence (rCRS). In total, we identied 282,502 unique mtDNA fragments longer than 35 base pairs (Supplementary Table S1).To assess whether some of the mtDNA fragments were of ancient origin, we made use of the fact that cyto-sine (C) bases at the ends of DNA molecules over time tend to undergo deamination30 and as a result are read as thymines (T) by DNA polymerases. Ancient DNA fragments aligned to a reference sequence thus tend to carry high frequencies of apparent C to T substitutions at their 5þ n and 3þ n ends31–33. Of the fragments starting or ending at positions where the rCRS base is a C, 32.2% and 31.3% carried Ts at their 5þ n and 3þ n ends, respectively (Supplementary Figure S13), indicating that ancient hominin DNA molecules are present in DC1227.To determine whether the endogenous mtDNA of DC1227 is most closely related to modern human, Neandertal or Denisovan mitochondrial genomes, we restricted the analysis to sequences carrying a C to T substitution relative to the rCRS at one of their ends34 to diminish the inuence of putative contamination by present-day human DNA (Supplementary Information). Using 36,665 such sequences (Supplementary Table S1), the mitochondrial genome of specimen DC1227 was reconstructed with an average coverage of 130-fold, leaving 63 positions covered by two or fewer sequences and four where fewer than two thirds of sequences carried the same base. us, 67 positions could not be condently called.When comparing the DC1227 mtDNA to complete Neandertal mtDNA determined to date, it carries ve base dierences to the Neandertal mtDNA of Okladnikov 233 found approximately 60 km from Denisova Cave, 12 to 17 dierences to Neandertals from western and southern Europe, and 31 dierences to Mezmaiskaya 1 from the Caucasus35 and to a Neandertal found in Denisova Cave5. In comparison, the mtDNA of DC1227 diers by between 174 and 354 bases to the mtDNAs of other hominin groups (Supplementary Table S2). In a phylogenetic analysis (Fig.4), the DC1227 mitochondrial genome thus falls within the variation of the ten Neandertals, to the exclusion of 311 present-day humans, ten ancient modern humans, two Denisovans, and a Middle Pleistocene hominin from Spain. We conclude that the DC1227 individual carried a mitochondrial genome of the Neandertal type and refer to it henceforth as Denisova 11.‘…Ž—•‹‘•e crucial importance of genetic analysis for palaeoanthropology is apparent in the insights that have been made in the recent years. e dearth of human skeletal material from Pleistocene-age deposits constitutes a major drawback for future applications of these methods. By utilising assemblages of bone already excavated from archaeological sites it may be possible to identify hominin remains useful for a variety of scientic investigations. Figure 3.þ Micro-CT Scan of DC1227, (a,b) surface of DC1227, (c) projection through the length of DC1227, (d,e) projections through the width of DC1227.

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5 Collagen ngerprinting using ZooMS is highly eective at identifying such material for further scientic inves-tigation and it is this kind of multi-disciplinary approach that holds the key to broadening our understanding of Pleistocene human populations. At Denisova Cave we have shown the feasibility of successfully carrying out such research. Here, and in the wider Altai region, there is the additional possibility of attaining high genetic coverage due to the favourable climatic and post-depositional conditions. Ongoing work is now focusing on screening additional material from this important site in an attempt to identify more hominin remains. e rapid nature of ZooMS, and its amenability to high-throughput methodology, has the potential to identify undiagnostic and fragmented hominin remains at key sites. is is the rst time a novel combination of ZooMS, radiocarbon dat-ing and ancient DNA analysis have been performed in order to identify and analyse human fragments from an archaeological site. Increased utility of this combined methodology could ultimately provide the source material necessary to further our understanding of human evolution.‡–Š‘†•‘‘äþ Between 20–50þ t mg of bone were taken from each of the bones within the assemblage and deminer-alised in 0.6þ t M hydrochloric acid (HCl) for 18þ t hours. e resulting residue was removed into 30þ t kDa molecular weight cut-o (MWCO) ultralters and centrifuged at 3700þ t rpm for 1þ t hour. e ltrate was then twice washed through with 500þ t L of 50þ t mM ammonium bicarbonate (AmBic) and further centrifuged at 3700þ t rpm for half an hour aer each treatment. e nal residue was resuspended with additional AmBic (200þ t L), half of which was removed to create a backup sample set before digestion. e remaining 100þ t L was then treated with 0.2þ t g trypsin (sequencing grade; Promega UK) and incubated at 37þ t °C for 18þ t hours. e resulting solution was then mixed with a matrix solution of 1þ t l of þ n -cyano-4-hydroxycinnamic acid solution (10þ t mg/mL in 50% acetonitrile (ACN)/0.1% triuoroacetic acid (TFA)), allowed to co-crystalise and analysed using a Bruker Ultraex II (Bruker Daltonics, Bremen) MALDI Tof/Tof mass spectrometer. e resulting mass spectra were screened for the human markers9,12 within FlexAnalysis soware.For peptide sequencing, the pellet following demineralisation was further extracted for 18þ t h at 4þ t °C in a buer containing 100þ t mM Tris and 6þ t M GuHCl at pHþ t 7.4 and then ultraltered into 50þ t mM AmBic as described above. Figure 4.þ Neighbor-joining tree relating the DC1227 mtDNA to other ancient and present-day mtDNAs. A chimpanzee mtDNA was used as an outgroup (not shown). Support for each branch is based on 500 bootstrap replications. See Table S2 for the geographic origin of the ancient specimens.

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6 Following reduction and alkylation, the proteins were digested with trypsin as above and then acidied to 0.1% TFA and desalted, puried and concentrated with 100þ t L C18 reversed-phase Zip-Tips (Millpore)18, eluting with 50% ACN/0.1% TFA. Following purication the sample was lyophilised by evaporation and resuspended in 20þ t L 0.1% TFA. e sample peptides were then analysed by LC/MS/MS using an UltiMate®þ n 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA, USA) coupled to an Orbitrap Elite (ermo Fisher Scientic, Waltham, MA, USA) mass spectrometer (120þ t k resolution, full scan, positive mode, normal mass range 350–1500). Peptides were separated using an Ethylene Bridged Hybrid (BEH) C18 analytical column (75þ t mmþ t þ n t 250þ t m i.d., 1.7þ t M; Waters) with a gradient from 92% A (0.1% formic acid in water) and 8% B (0.1% formic acid in acetonitrile) to 33% B in 44þ t min at a ow rate of 300þ t nLþ t min–1 and automatically selected for fragmentation by data-dependent analysis; six MS/MS scans (Velos ion trap, product ion scans, rapid scan rate, Centroid data; scan event: 500 count minimum signal threshold, top 6) were acquired per cycle, dynamic exclusion was employed, and 1 repeat scan (i.e. two MS/MS scans total) was acquired in a 30þ t s repeat duration with that precursor being excluded for the subsequent 30þ t s (activation: collision-induced dissociation (CID), 2þ n default charge state, 2þ t m/z isolation width, 35þ t eV normalized collision energy, 0.25 Activation Q, 10.0þ t ms activation time). e data consisting of 11,651 peptide ions were searched against the SwissProt database (www.ebi.ac.uk/swissprot/) for matches to primary protein sequences using the Mascot search engine (version 2.5.1; Matrix Science, London, UK) including the xed carbamidomethyl modication of cysteine and the variable modications for deamidation, and oxidation of lysine, proline and methionine residues to account for common PTMs and diagenetic alterations. Enzyme specicity was limited to trypsin/P with up to 2 missed cleavages allowed, mass toler ances were set at 5þ t ppm for the precursor ions and 0.5 Daltons for the fragment ions, all spectra were considered as having either 2þ n or 3 þ n precursors and the peptide ion score cut o was set at 42 as identied by the initial Mascot output.…ƒ‹‰äþ e CT scan was undertaken using a Nikon XT H 225 micro-scanner with a transmission tar get. Attempts to keep the dosage as low as possible were made in order to avoid any damage to the sample, so the scan was run at 70kv and 80þ t A. In total 1,448 projects were taken at two frames per projection, with an exposure set at 100ms and magnication at þ n 7.2. Data was reconstructed using CT Pro 3D soware, and processed with VG Studio Max 2.1 soware.‹–‘…Š‘†”‹ƒŽƒŽ›•‹•äþ DNA extraction and library preparation.þ 30.9þ t mg of bone powder was removed from DC1227 using a dentistry drill. DNA was extracted using a silica-based protocol designed to retrieve short DNA molecules27 ,36. 10þ t l of the DNA extract (E3128) were converted into a single-stranded DNA library (A9301), as described28,36. e number of DNA molecules in the library was assessed by digital droplet PCR (BioRad QX 200), using 1þ t l as input for an EvaGreen (BioRad) assay with primers IS7 and IS837. e library was barcoded with two unique indexes36,38 and amplied using AccuPrime Pfx DNA polymerase (Life Technologies)39. Amplication products were puried using the MinElute PCR purication kit (Qiagen); and quantied on a NanoDrop ND-1000 (NanoDrop Technologies) photospectrometer. Mitochondrial capture and sequencing.þ e amplied library (A9317) was enriched by a bead-based hybridization capture protocol29 using 52-mer probes40 designed in single base pair tiling on the rCRS (National Center for Biotechnology Information [NCBI] reference NC_012920) in two rounds of capture, using 1þ t g and 0.5þ t g of input DNA, respectively. e captured library (L5502) was sequenced on a MiSeq (Illumina) platform, using paired-end runs (2þ t þ n t 76 cycles) with double-index conguration38. One DNA extraction blank and one library preparation blank were carried along the entire procedure as negative controls. Sequence processing and mapping.þ Base calling was performed using Bustard (Illumina). Adapter sequences were trimmed, and forward and reverse reads were merged into single sequences41. Sequences lacking perfect matches to the expected barcodes were discarded. Mapping to the reference genome was carried out using BWA42, with parameters “-n 0.01 -o 2 -l 16500”þ t 7. PCR duplicates were removed by merging sequences with identical alignment start and end coordinates, using bam-rmdup (https://github.com/udo-stenzel/biohazard). Sequences longer than 35 bases with a mapping quality greater than 30 were retained for further analyses. Phylogenetic analysis.þ Sequences carrying a terminal C to T substitution were used to reconstruct the DC1227 mtDNA. Terminal Ts at the rst or last positions of each sequence were converted to Ns to reduce the impact of damage-derived sequence errors in consensus calling. A consensus base was determined if a position was covered by at least three sequences, and if at least 67% of sequences, i.e. more than two thirds overlapping it carried an identical base34. e mtDNA was aligned to the mtDNAs of 311 present-day humans43, 10 ancient modern humans31 ,44–47 ten Neandertals5 ,33,35,48,49, two Denisovans3 ,4 a Middle Pleistocene hominin34 and a chimpanzee (Pan troglodytes, NC_001643)50 by MAFFT51. e number of base dierences between sequences and a Neighbor-Joining tree with 500 bootstrap replications52 based on these dierences were produced using MEGA653.‡ˆ‡”‡…‡•1.þt Bolihovsaya, N. S. & Shunov, M. V. Pleistocene environments of northwestern Altai: Vegetation and climate. Arch. Ethno. Anthr. Eurasia 42, 2–17 (2014). 2.þt Agadjanian, A. . e dynamics of bioresources and activity of the Palaeolithic man, using the example of northwestern altai mountains. Palaeontolog. J. 40, S482–S493 (2005). 3.þt rause, J. et al. e complete mitochondrial DNA genome of an unnown hominin from southern Siberia. Nature 464, 894–897 (2010).

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7 4.þt eich, D. et al. Genetic history of an archaic hominin group from Denisova cave in Siberia. Nature 468, 1053–1060 (2010). 5.þt Prüfer, . et al. e complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505(7481), 43–49 (2014). 6.þt Dereviano, A. P., Shunov, M. V. & Volov, P. V. A Paleolithic Bracelet from Denisova Cave, Archaeology. Arch. Ethno. Anthr. Eurasia 34, 13–25 (2008). 7.þt Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338(6104), 222–6 (2012). 8.þt Vasiliev, S. ., Shunov, M. V. & ozliin, M. B. Preliminary esults for the Balance of Megafauna from Pleistocene Layers of the East Gallery, Denisova Cave. Problems of Archaeology, Ethno. and Anth. Siberia and Adjacent Territories 19, 32–38 (2013). 9.þt Bucley, M., Collins, M., omas-Oaies, J. & Wilson, J. C. Species identication by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-ight mass spectrometry. apid Commun. Mass Sp. 23, 3843–3854 (2009). 10.þt Bucley, M. et al. Distinguishing between archaeological sheep and goat bones using a single collagen peptide. J. Archaeol. Sci. 37(1), 13–20 (2010). 11.þt Bucley, M., Larin, N. & Collins, M. Mammoth and Mastodon collagen sequences; survival and utility. Geochim. Cosmochim. Ac. 75(1), 2007–2016 (2011). 12.þt Bucley, M. & ansa, S. W. Collagen ngerprinting of archaeological bone and teeth remains from Domuztepe, South Eastern Turey. Archaeol. Anthropol. Sci. 3, 271–280 (2011). 13.þt Bucley, M. et al. Species identication of archaeological marine mammals using collagen ngerprinting. J. Archaeol. Sci. 41, 631–641 (2014). 14.þt ichter, . . et al. Fish ‘n chips: ZooMS peptide mass ngerprinting in a 96 well plate format to identify sh bone fragments. J. Archaeol. Sci. 38(7), 1502–1510 (2011). 15.þt ybczynsi, N. et al. Mid-Pliocene warm-period deposits in the High Arctic yield insight into camel evolution. Nature Comms. 4, 1550 (2013). 16.þt van der Sluis, L. G. et al. Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius. Palaeogeogr. Palaeoclimatol. Palaeoecol . 416, 80–91 (2014). 17.þt Bucley, M. & Melton, N. D., Montgomery, J. Proteomics analysis of ancient food vessel stitching reveals þ n 4000-year-old mil protein. apid Commun. Mass Sp. 27, 531–538 (2013). 18.þt Wadsworth, C. & Bucley, M. Proteome degradation in fossils: Investigating the longevity of protein survival in ancient bone. apid Commun. Mass Sp. 28, 605–615 (2014). 19.þt Bucley, M. Ancient collagen reveals evolutionary history of the endemic south american ‘ungulates’. Proc. . Soc. B. 282, 1806 (2015). 20.þt Agadjanian, A. . & Serdyu, N. V. e history of mammalian communities and paleogeography of the Altai mountains in the Paleolithic. Paleontol. J. 39, S645–S821 (2005). 21.þt Chlachula, J. Pleistocene climate change, natural environments and Palaeolithic occupation of the Altai area, west-central Siberia. Quatern. Int. 80–81, 131–167 (2001). 22.þt Bolihovsaya, N. S. & Shunov, M. V. Pleistocene environments of northwestern Altai: Vegetation and climate. Archaeol. Ethn. Anth. Eurasia 42, 2–17 (2014). 23.þt Broc, F., Higham, T., Ditcheld, P. & amsey, C. B. Current pretreatment methods for ams radiocarbon dating at the Oxford adiocarbon Accelerator Unit (OAU). adiocarbon 52, 103–112 (2010). 24.þt Dobrovolsaya, M. V. & Tiunov, A. V. Stable isotope (13C/12C and 15N/14N) evidence for late Pleistocene homininies’ palaeodiets in Gorny Altai in Proceedings of the International Symposium: Characteristic Features of the Middle to Upper Palaeolithic Transition in Eurasia (eds Dereviano, A.P., Shunov, M. V.), 81–89 (Institute of Archaeology and Ethnography SB AS, 2011). 25.þt ichards, M. P., aravani, I., Pettitt, P. & Miracle, P. Isotope and faunal evidence for high levels of freshwater sh consumption by Late Glacial humans at the Late Upper Palaeolithic site of Šandalja II, Istria, Croatia. J. Archaeol. Sci. 61, 204–212 (2015). 26.þt Schoeninger, M. J., Deniro, M. J. & Tauber, H. 15N/14N ratios of bone collagen reflect marine and terrestrial components of prehistoric human diet. Am. J. Phys. Anthropol. 60, 252 (1983). 27.þt Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. P. Natl. Acad. Sci. USA 110(39), 15758–15763 (2013). 28.þt Gansauge, M.-T., Meyer, M. & Single-stranded D. N. A. library preparation for the sequencing of ancient or damaged DNA. Nat. Protoc. 8(4), 737–748 (2013). 29.þt Maricic T., Whitten, M. & Pääbo, S. Multiplexed DNA sequence capture of mitochondrial genomes using PC products PLoS One 5(11), e14004 (2010). 30.þt Briggs, A. W. et al. Patterns of damage in genomic DNA sequences from a Neandertal. P. Natl. Acad. Sci. USA 104(37), 14616–14621 (2007). 31.þt rause, J. et al. A complete mtDNA genome of an early modern human from osteni, ussia. Curr. Biol. 20(3), 231–236 (2010). 32.þt Sawyer, S., rause, J., Guschansi, ., Savolainen, V. & Pääbo, S. Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA. PLoS One 7(3), e34131 (2012). 33.þt Soglund, P. et al. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. P. Natl. Acad. Sci. USA 111(6), 2229–2234 (2014). 34.þt Meyer, M. et al. A mitochondrial genome sequence of a hominin from Sima de los Huesos. Nature 505(7483), 403–406 (2014). 35.þt Briggs, A. W. et al. Targeted retrieval and analysis of ve Neandertal mtDNA genomes. Science 325, 318–321 (2009). 36.þt orlevi, P. et al. educing microbial and human contamination in DNA extractions from ancient bones and teeth. BioTechniques 58, 87–93 (2015). 37.þt Meyer, M. & ircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harbor Protocols 2010(6), pdb.prot5448 (2010). 38.þt ircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids es. 40(1), e3 (2012). 39.þt Dabney, J. & Meyer, M. Length and GC-biases during sequencing library amplication: A comparison of various polymerase-buer systems with ancient and modern DNA sequencing libraries. BioTechniques 52, 87–94 (2012). 40.þt Fu, Q. et al. DNA analysis of an early modern human from Tianyuan Cave, China. P. Natl. Acad. Sci. USA 110(6), 2223–2227 (2013). 41.þt enaud, G., Stenzel, U. & elso, J. leeHom: adaptor trimming and merging for Illumina sequencing reads. Nucleic Acids es. 42(18), e141 (2014). 42.þt Li, H. & Durbin, . Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26(5), 589–595 (2010). 43.þt Green, . E. et al. A Dra Sequence of the Neandertal Genome. Science 328(5979), 710–722 (2010). 44.þt Ermini, L. et al. Complete mitochondrial genome sequence of the Tyrolean Iceman. Curr. Biol. 18, 1687–1693 (2008). 45.þt Gilbert, M. T. P. et al. Paleo-Esimo mtDNA Genome eveals Matrilineal Discontinuity in Greenland. Science 320(5884), 1787–1789 (2008). 46.þt Fu, Q. et al. A revised timescale for human evolution based on ancient mitochondrial genomes. Curr. Biol . 23(7), 553–559 (2013). 47.þt Fu, Q. et al. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514, 445–449 (2014). 48.þt Green, . E. et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134(3), 416–426 (2008).

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8 49.þt Gansauge M.-T. & Meyer, M. Selective enrichment of damaged DNA molecules for ancient genome sequencing. Genome es. 24, 1543–1549 (2014). 50.þt Horai, S., Hayasaa, ., ondo, ., Tsugane, . & Taahata, N. ecent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. P. Natl. Acad. Sci. USA 92, 532–536 (1995). 51.þt atoh, . & Standley, D. M. MAFFT multiple sequence alignment soware version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). 52.þt Felsenstein, J. Condence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791 (1985). 53.þt Tamura, ., Stecher, G., Peterson, D., Filipsi, A. & umar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).…‘™Ž‡†‰‡‡–•is project forms part of PalaeoChron, an Advanced Investigator’s grant awarded to Prof. omas Higham by the European Research Council (ERC: grant 324139). Signicant funding was also provided by the Royal Society, from a grant awarded to Dr. Michael Buckley. is study was also supported by the Russian Science Foundation (RSCF), under project No. 14-50-00036 and the Presidential Innovation Fund of the Max Planck Society. We are grateful to each of these organisations. is project would not have been possible without the use of facilities at the University of Oxford, the University of Manchester, the Max Planck Institute of Evolutionary Anthropology, the University of Craneld, and, particularly, the Institute of Archeology and Ethnography in Novosibirsk. We would also like to thank the sta of the Oxford Radiocarbon Accelerator Unit, and express our gratitude to Vladimir Vaneev and Maksim Kozlikin for their aid in accessing samples in Novosibirsk. Mitochondrial DNA analysis was undertaken at the Max Planck Institute for Evolutionary Anthropology. We thank Mateja Hajdinjak, Barbara Höber, Birgit Nickel, Anna Schmidt, and Antje Weihmann for help in the laboratory; Gabriel Renaud and Udo Stenzel for data processing; and Janet Kelso and Kay Prüfer for helpful input.—–Š‘”‘–”‹„—–‹‘•T.H. and K.D. designed the project and, with M.B, supervised its progress throughout; S.B, M.B. and N.P undertook the ZooMS analysis; V.S., S.P. and M.M. carried out the mtDNA analysis; S.B., D.C. and T.H undertook the radiocarbon analysis; F.B. carried out the CT Scan; M.S. and A.D. excavated the samples analysed in this project; S.B. and M.B. were responsible for interpretation of data and wrote the paper.††‹–‹‘ƒŽfˆ‘”ƒ–‹‘Accession code: e mitochondrial genome sequence of specimen DC1227 (Denisova 11) was deposited in GenBank (accession number KU131206). Supplementary information accompanies this paper at http://www.nature.com/srep Competing nancial interests: e authors declare no competing nancial interests. How to cite this article: Brown, S. et al. Identication of a new hominin bone from Denisova Cave, Siberia using collagen ngerprinting and mitochondrial DNA analysis. Sci. Rep. 6, 23559; doi: 10.1038/srep23559 (2016). is work is licensed under a Creative Commons Attribution 4.0 International License. e images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/


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