Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16–5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains1,3,4,5,6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12–10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information.
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All the mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD013838. Generated ancient protein consensus sequences for G. blacki can be found in Supplementary Data 1. Full thermal age calculations can be found in Supplementary Data 2.
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E.C. and F.W. are supported by VILLUM FONDEN (17649) and by the European Commission through a Marie Skłodowska-Curie (MSCA) Individual Fellowship (795569). T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), NIHM grant U01 MH106874, Howard Hughes International Early Career grant, Obra Social ‘La Caixa’ and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). E.C., J.C., J.V.O., D.S. and P.G. are supported by the Marie Skłodowska-Curie European Training Network (ETN) TEMPERA, a project funded by the European Union’s EU Framework Program for Research and Innovation Horizon 2020 under grant agreement no. 722606. M.J.C. and M.M. are supported by the Danish National Research Foundation award PROTEIOS (DNRF128). Work at the Novo Nordisk Foundation Center for Protein Research is funded in part by a donation from the Novo Nordisk Foundation (NNF14CC0001). Research at Chuifeng Cave is made possible by support from the National Natural Science Foundation of China (41572023) and by a grant from the Bagui Scholar of Guangxi. M.K. was supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship (KU 3467/1-1) and the Postdoctoral Junior Leader Fellowship Programme from ‘la Caixa’ Banking Foundation (LCF/BQ/PR19/11700002). M.E.A. is supported by the Independent Research Fund Denmark (7027-00147B). We thank E. Willerslev for critical reading of the manuscript, scientific support and guidance.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Benedikt Kessler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Landscape outside Chuifeng Cave. b, Elevated altitude of Chuifeng Cave (arrow points to the entrance). Photo credit: W.W.
a, Main entrance of Chuifeng Cave. b, Well-preserved deposits before excavation. c, The stratigraphic profile (1.3 m in height) of area D. d, W.W. excavating in area D. e, Excavated channel. f, g, In situ G. blacki teeth (scale bars, 3 cm). Photo credit: W.W.
a, HCl extract. b, TFA extract. Note differences in maximum total ion currents on the y axes. Each extract was analysed only once.
a, Summed and normalized peptide intensities for each combination of peptide length and number of acidic residues (aspartic acid, glutamic acid, deamidated asparagine, deamidated glutamine). Circle sizes are proportional to the percentage of the total intensity, for each combination of peptide length and number of acidic residues. b, Summed and normalized intensities by peptide length. c, Summed and normalized peptide intensities across peptide hydrophobicity (GRAVY index values calculated using the R package Peptides, scale ‘KyteDoolittle’). Insets show peptide count distribution across peptide hydrophobicity. d, Extraction performance for various data categories. Values scaled to one and compared to the best-performing extraction method for each category independently. SAPs refer to those SAPs informative within Hominoidea. e, Proportional Venn diagram of unique peptide sequences identified in the two demineralization methods. All comparisons based on MaxQuant LFQ data only. N, number of peptides.
a, Comparison of peptide lengths, showing an identical distribution for the TFA (n = 305) and HCl (n = 191) extractions (two-sided t-test, t394 = −0.599, P = 0.5495). b, Comparison of asparagine deamidation. c, Comparison of glutamine deamidation. b, c, Violin plots describe the distribution of bootstrap replicates (n = 1,000) of intensity-based peptide deamidation32. The number of peptides used for the calculation are shown at the bottom. For some proteins, only deamidated asparagines or glutamines were observed (for example, AMBN), while DCD is included as an example of a non-deamidated contaminant. All comparisons based on MaxQuant data only. For a–c, box plots define the range of the data (whiskers extending to 1.5× the interquartile range), outliers (beyond 1.5× the interquartile range), 25th and 75th percentiles (boxes) and medians (dots).
a, Pairwise distances between groups of selected Hominoids and Pongo estimated using the concatenated protein alignments and the phangorn R package. n shows number of pairwise comparisons. b, Maximum-likelihood tree computed on a distance matrix using pml R function. Support values were obtained from 1,000 bootstrap replicates. c, Rooted phylogenetic tree obtained using MrBayes. For each bipartition, we show the posterior probability (0–1) obtained from the Bayesian approach and the support values obtained from 100 non-parametric bootstrap replicates in a PHyML maximum-likelihood (0–100) tree. PHyML and MrBayes recover the same topology. b and c are based on the same concatenated alignment of the five proteins retrieved from Gigantopithecus, and resulted in the same tree topology.
Extended Data Fig. 7 Bayesian phylogenetic tree of G. blacki including 162 modern genomes of great apes and a single hylobatid (N. leucogenys).
Tree obtained from the concatenated alignment using MrBayes. M. mulatta was used as an outgroup. The internal nodes corresponding to the Gorilla, Pan and Homo clades are collapsed for visualization purposes. The number of individuals in each of these nodes is indicated in parentheses.
All AHSG-specific peptides, identified by PEAKS and MaxQuant, derive from a single sequence region bridging cystatin domains 1 and 2. The surviving sequence region is evolutionarily conserved across Catarrhini. It contains a regular repeat of acidic amino acid residues (aspartic acid, D, on positions 133, 137 and 141) that enable binding of basic calcium phosphate (residues highlighted in green), similarly to a conserved region just N-terminal (glutamic acid, E, on position 111, and aspartic acid, D, on position 113 and 115). At the bottom, a fragment ion alignment is given of MaxQuant-identified AHSG peptides. The serine is phosphorylated in all matching spectra. The majority-based amino acid consensus sequence for the alignment of Catarrhini is shown at the top for amino acid positions 100–149 (amino acid coordinates following UniProt accession P02765 [FETUA_HUMAN]).
Supplementary Information containing SGDP human individual accessions, 3 supplementary tables, and 12 supplementary figures.
File in .fasta format containing recovered Gigantopithecus protein sequences. Sequence coverage obtained after combining the results from MaxQuant and PEAKS analysis.
Excel file containing all numerical values used for the calculation of the thermal ages presented in Fig. 1.
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Welker, F., Ramos-Madrigal, J., Kuhlwilm, M. et al. Enamel proteome shows that Gigantopithecus was an early diverging pongine. Nature 576, 262–265 (2019). https://doi.org/10.1038/s41586-019-1728-8
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