The phylogenetic relationships between hominins of the Early Pleistocene epoch in Eurasia, such as Homo antecessor, and hominins that appear later in the fossil record during the Middle Pleistocene epoch, such as Homo sapiens, are highly debated1,2,3,4,5. For the oldest remains, the molecular study of these relationships is hindered by the degradation of ancient DNA. However, recent research has demonstrated that the analysis of ancient proteins can address this challenge6,7,8. Here we present the dental enamel proteomes of H. antecessor from Atapuerca (Spain)9,10 and Homo erectus from Dmanisi (Georgia)1, two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal and divergence. We provide evidence that H. antecessor is a close sister lineage to subsequent Middle and Late Pleistocene hominins, including modern humans, Neanderthals and Denisovans. This placement implies that the modern-like face of H. antecessor—that is, similar to that of modern humans—may have a considerably deep ancestry in the genus Homo, and that the cranial morphology of Neanderthals represents a derived form. By recovering AMELY-specific peptide sequences, we also conclude that the H. antecessor molar fragment from Atapuerca that we analysed belonged to a male individual. Finally, these H. antecessor and H. erectus fossils preserve evidence of enamel proteome phosphorylation and proteolytic digestion that occurred in vivo during tooth formation. Our results provide important insights into the evolutionary relationships between H. antecessor and other hominin groups, and pave the way for future studies using enamel proteomes to investigate hominin biology across the existence of the genus Homo.
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Mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD014342. Generated ancient protein consensus sequences used for phylogenetic analysis for H. antecessor (Atapuerca) and H. erectus (Dmanisi) hominins can be found in the Supplementary Data 2, which is formatted as a .fasta file. Full protein sequence alignments used during phylogenetic analysis can be accessed via Figshare (https://doi.org/10.6084/m9.figshare.9927074). Amino acid racemization data are available online through the NOAA database. The wiNNer model can be accessed on GitHub (https://github.com/cox-labs/wiNNer.git).
Gabunia, L. et al. Earliest Pleistocene hominid cranial remains from Dmanisi, Republic of Georgia: taxonomy, geological setting, and age. Science 288, 1019–1025 (2000).
Zhu, Z. et al. Hominin occupation of the Chinese Loess Plateau since about 2.1 million years ago. Nature 559, 608–612 (2018).
Stringer, C. The origin and evolution of Homo sapiens. Phil. Trans. R. Soc. Lond. B 371, 20150237 (2016).
Hublin, J. J. The origin of Neandertals. Proc. Natl Acad. Sci. USA 106, 16022–16027 (2009).
Rightmire, G. Human evolution in the Middle Pleistocene: the role of Homo heidelbergensis. Evol. Anthropol. 6, 218–227 (1998).
Cappellini, E. et al. Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny. Nature 574, 103–107 (2019).
Chen, F. et al. A late Middle Pleistocene Denisovan mandible from the Tibetan Plateau. Nature 569, 409–412 (2019).
Welker, F. et al. Enamel proteome shows that Gigantopithecus was an early diverging pongine. Nature 576, 262–265 (2019).
Bermúdez de Castro, J. M. et al. A hominid from the lower Pleistocene of Atapuerca, Spain: possible ancestor to Neandertals and modern humans. Science 276, 1392–1395 (1997).
Carbonell, E. et al. Lower Pleistocene hominids and artifacts from Atapuerca-TD6 (Spain). Science 269, 826–830 (1995).
Duval, M. et al. The first direct ESR dating of a hominin tooth from Atapuerca Gran Dolina TD-6 (Spain) supports the antiquity of Homo antecessor. Quat. Geochronol. 47, 120–137 (2018).
Freidline, S. E., Gunz, P., Harvati, K. & Hublin, J.-J. Evaluating developmental shape changes in Homo antecessor subadult facial morphology. J. Hum. Evol. 65, 404–423 (2013).
Lacruz, R. S. et al. Facial morphogenesis of the earliest Europeans. PLoS One 8, e65199 (2013).
Ferring, R. et al. Earliest human occupations at Dmanisi (Georgian Caucasus) dated to 1.85–1.78 Ma. Proc. Natl Acad. Sci. USA 108, 10432–10436 (2011).
Lordkipanidze, D. et al. A complete skull from Dmanisi, Georgia, and the evolutionary biology of early Homo. Science 342, 326–331 (2013).
Stewart, N. A., Gerlach, R. F., Gowland, R. L., Gron, K. J. & Montgomery, J. Sex determination of human remains from peptides in tooth enamel. Proc. Natl Acad. Sci. USA 114, 13649–13654 (2017).
Tiwary, S. et al. High-quality MS/MS spectrum prediction for data-dependent and data-independent acquisition data analysis. Nat. Methods 16, 519–525 (2019).
Castiblanco, G. A. et al. Identification of proteins from human permanent erupted enamel. Eur. J. Oral Sci. 123, 390–395 (2015).
Asaka, T. et al. Type XVII collagen is a key player in tooth enamel formation. Am. J. Pathol. 174, 91–100 (2009).
Porto, I. M., Laure, H. J., de Sousa, F. B., Rosa, J. C. & Gerlach, R. F. New techniques for the recovery of small amounts of mature enamel proteins. J. Archaeol. Sci. 38, 3596–3604 (2011).
Gasse, B., Chiari, Y., Silvent, J., Davit-Béal, T. & Sire, J.-Y. Amelotin: an enamel matrix protein that experienced distinct evolutionary histories in amphibians, sauropsids and mammals. BMC Evol. Biol. 15, 47 (2015).
Demarchi, B. et al. Protein sequences bound to mineral surfaces persist into deep time. eLife 5, e17092 (2016).
Tagliabracci, V. S. et al. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization. Science 336, 1150–1153 (2012).
Hu, J. C. C., Yamakoshi, Y., Yamakoshi, F., Krebsbach, P. H. & Simmer, J. P. Proteomics and genetics of dental enamel. Cells Tissues Organs 181, 219–231 (2005).
Glimcher, M. J., Cohen-Solal, L., Kossiva, D. & de Ricqles, A. Biochemical analyses of fossil enamel and dentin. Paleobiology 16, 219–232 (1990).
Wagner, G. A. et al. Radiometric dating of the type-site for Homo heidelbergensis at Mauer, Germany. Proc. Natl Acad. Sci. USA 107, 19726–19730 (2010).
Martinón-Torres, M. et al. Dental evidence on the hominin dispersals during the Pleistocene. Proc. Natl Acad. Sci. USA 104, 13279–13282 (2007).
Bermúdez de Castro, J. M., Martinón-Torres, M., Arsuaga, J. L. & Carbonell, E. Twentieth anniversary of Homo antecessor (1997–2017): a review. Evol. Anthropol. 26, 157–171 (2017).
Gómez-Robles, A., Bermúdez de Castro, J. M., Arsuaga, J.-L., Carbonell, E. & Polly, P. D. No known hominin species matches the expected dental morphology of the last common ancestor of Neanderthals and modern humans. Proc. Natl Acad. Sci. USA 110, 18196–18201 (2013).
Meyer, M. et al. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins. Nature 531, 504–507 (2016).
Prüfer, K. et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature 505, 43–49 (2014).
Lacruz, R. S. et al. The evolutionary history of the human face. Nat. Ecol. Evol. 3, 726–736 (2019).
Mallick, S. et al. The Simons Genome Diversity Project: 300 genomes from 142 diverse populations. Nature 538, 201–206 (2016).
The 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).
Welker, F. et al. Middle Pleistocene protein sequences from the rhinoceros genus Stephanorhinus and the phylogeny of extant and extinct Middle/Late Pleistocene Rhinocerotidae. PeerJ 5, e3033 (2017).
Hill, R. C. et al. Preserved proteins from extinct Bison latifrons identified by tandem mass spectrometry; hydroxylysine glycosides are a common feature of ancient collagen. Mol. Cell. Proteomics 14, 1946–1958 (2015).
Wadsworth, C. & Buckley, M. Proteome degradation in fossils: investigating the longevity of protein survival in ancient bone. Rapid Commun. Mass Spectrom. 28, 605–615 (2014).
Orlando, L. et al. Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74–78 (2013).
Penkman, K. E. H., Kaufman, D. S., Maddy, D. & Collins, M. J. Closed-system behaviour of the intra-crystalline fraction of amino acids in mollusc shells. Quat. Geochronol. 3, 2–25 (2008).
Dickinson, M., Lister, A. M. & Penkman, K. E. H. A new method for enamel amino acid racemization dating: a closed system approach. Quat. Geochronol. 50, 29–46 (2019).
Hill, R. L. Hydrolysis of proteins. Adv. Protein Chem. 20, 37–107 (1965).
Mackie, M. et al. Palaeoproteomic profiling of conservation layers on a 14th century italian wall painting. Angew. Chem. Int. Ed. Engl. 57, 7369–7374 (2018).
Castellano, S. et al. Patterns of coding variation in the complete exomes of three Neandertals. Proc. Natl Acad. Sci. USA 111, 6666–6671 (2014).
de Manuel, M. et al. Chimpanzee genomic diversity reveals ancient admixture with bonobos. Science 354, 477–481 (2016).
Nater, A. et al. Morphometric, behavioral, and genomic evidence for a new orangutan species. Curr. Biol. 27, 3487–3498.e10 (2017).
Prado-Martinez, J. et al. Great ape genetic diversity and population history. Nature 499, 471–475 (2013).
Hanson-Smith, V. & Johnson, A. PhyloBot: a web portal for automated phylogenetics, ancestral sequence reconstruction, and exploration of mutational trajectories. PLOS Comput. Biol. 12, e1004976 (2016).
Welker, F. Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment. BMC Evol. Biol. 18, 23 (2018).
Hendy, J. et al. A guide to ancient protein studies. Nat. Ecol. Evol. 2, 791–799 (2018).
Zhang, J. et al. PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol. Cell. Proteomics 11, M111.010587 (2012).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Chun, Y. H. P. et al. Cleavage site specificity of MMP-20 for secretory-stage ameloblastin. J. Dent. Res. 89, 785–790 (2010).
Yamakoshi, Y., Hu, J. C. C., Fukae, M., Yamakoshi, F. & Simmer, J. P. How do enamelysin and kallikrein 4 process the 32-kDa enamelin? Eur. J. Oral Sci. 114 (Suppl 1), 45–51, 93–95, 379–380 (2006).
Iwata, T. et al. Processing of ameloblastin by MMP-20. J. Dent. Res. 86, 153–157 (2007).
Nagano, T. et al. Mmp-20 and Klk4 cleavage site preferences for amelogenin sequences. J. Dent. Res. 88, 823–828 (2009).
Fukae, M. et al. Primary structure of the porcine 89-kDa enamelin. Adv. Dent. Res. 10, 111–118 (1996).
Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J. & Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 6, 786–787 (2009).
Prüfer, K. et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017).
Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222–226 (2012).
Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).
Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).
Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012).
Bouckaert, R. et al. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLOS Comput. Biol. 15, e1006650 (2019).
Miller, M. A., Pfeiffer, W. & Schwartz, T. in Gateway Computing Environments Workshop (GCE) 1–8 (New Orleans, 2010).
Besenbacher, S., Hvilsom, C., Marques-Bonet, T., Mailund, T. & Schierup, M. H. Direct estimation of mutations in great apes reconciles phylogenetic dating. Nat. Ecol. Evol. 3, 286–292 (2019).
Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67, 901–904 (2018).
F.W. is supported by a Marie Skłodowska Curie Individual Fellowship (no. 795569). E. Cappellini was supported by VILLUM FONDEN (no. 17649). E.W. is supported by the Lundbeck Foundation, the Danish National Research Foundation, the Novo Nordisk Foundation, the Carlsberg Foundation, KU2016 and the Wellcome Trust. Without the effort of the members of the Atapuerca research team during fieldwork, this work would have not been possible; we make a special mention of J. Rosell, who supervises the excavation of the TD6 level. The research of the Atapuerca project has been supported by the Dirección General de Investigación of the Ministerio de Ciencia, Innovación y Universidades (grant numbers PGC2018-093925-B-C31, C32, and C33); field seasons are supported by the Consejería de Cultura y Turismo of the Junta de Castilla y León and the Fundación Atapuerca. We acknowledge The Leakey Foundation through the personal support of G. Getty (2013) and D. Crook (2014–2016, 2018, and 2019) to M.M.-T., as well as F.W. (2017). Restoration and conservation work on the material have been carried out by P. Fernández-Colón and E. Lacasa from the Conservation and Restoration Area of CENIEH-ICTS and L. López-Polín from IPHES. The picture of the specimen ATD6-92 was made by M. Modesto-Mata. E. Cappellini, J.C., J.V.O. and P. Gutenbrunner are supported by the Marie Skłodowska-Curie European Training Network (ETN) TEMPERA, a project funded by the European Union’s Framework Program for Research and Innovation Horizon 2020 (grant agreement no. 722606). Amino acid analyses were undertaken thanks to the Leverhulme Trust (PLP-2012-116) and NERC (NE/K500987/1). T.M.-B. is supported by BFU2017-86471-P (MINECO/FEDER, UE), U01 MH106874 grant, Howard Hughes International Early Career, 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). C.L.-F. is supported by a FEDER-MINECO grant (PGC2018-095931-B-100). M.K. was supported by the Postdoctoral Junior Leader Fellowship Programme from ‘la Caixa’ Banking Foundation (LCF/BQ/PR19/11700002). M.M. is 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 (grant number NNF14CC0001). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium, which is supported by grant PT17/0019 of the PE I+D+i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We acknowledge support from the Spanish Ministry of Science, Innovation and Universities, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208, and ‘Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya’ (2017SGR595). D.L. and A.M. are supported by the John Templeton Foundation (no. 52935) and by the Shota Rustaveli National Science Foundation of Georgia (no. FR-18-27262). We thank M. L. Schjellerup Jørkov for providing specimen Ø1952.
The authors declare no competing interests.
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Extended data figures and tables
a, Geographic location of Gran Dolina and Dmanisi. Base map was generated using public domain data from www.naturalearthdata.com. b, Summarized stratigraphic profile of Gran Dolina, including the location of hominin fossils in layers ‘Pep’ and ‘Jordi’ of sublevel TD6.2.
a, ATD6-92 in buccal view. The fragment represents a portion of a permanent lower left first or second molar. b, D4163 in occlusal view. The specimen is a fragmented right upper first molar. Scale bar differs between a and b.
a, b, The extent of intracrystalline racemization in enamel for the free amino acid (FAA) (x axis) fraction and the total hydrolysable amino acids (THAA) (y axis) fraction for aspartic acid plus asparagine (here denoted Asx) (a), and glutamic acid plus glutamine (here denoted Glx) (b), demonstrates endogenous amino acids breaking down within a closed system. The hominin value is displayed in relation to values for enamel samples from other fauna from Dmanisi6 (blue squares) and a range of previously obtained Pleistocene and Pliocene Proboscidea from the UK40 (grey diamonds). Fauna are shown for comparison, but different rates in their protein breakdown mean that they will show different extents of racemization. The x and y axis are on different scales.
Extended Data Fig. 4 Sequence coverage for five enamel-specific proteins across Pleistocene samples and recent human controls.
For each protein, the bars span protein positions covered, with positions remapped to the human reference proteome. The top row indicates the position of a selection of known MMP20 and KLK4 cleavage products of the enamel-specific proteins AMELX55, AMBN52 and ENAM56. Several in vivo proteolytic degradation fragments of ENAM share the same N terminus, but have unknown C termini53. Dotted line for AMBN indicates a putative cleavage product based on known MMP20 (squares) and KLK4 (circles) in vivo cleavage positions. For AMTN, serines (S) at positions 115 and 116 (indicated by asterisks) are conserved among vertebrates and involved in mineral-binding21. Additional cleavage products as well as MMP20 and KLK4 cleavage sites are known in all enamel-specific proteins. SK33916 and Ø1952 are two recent human control samples (Methods). AA, amino acids; Steph., Stephanorhinus6; TRAP, tyrosine-rich amelogenin polypeptide.
a, Mass spectrum of an AMELY-specific peptide from the recent human control Ø1952. b, Mass spectrum of the same AMELY-specific peptide from H. antecessor. c, Alignment of a selection of AMELY- and AMELX-specific peptide fragment ion series deriving from H. antecessor. The alignment stretches along human AMELX isoform 1, positions 37 to 52 only (Uniprot accession numbers Q99217 (AMELX), Q99218 (AMELY)). See Supplementary Fig. 5 for another example of an AMELY-specific MS2 spectrum.
a, b, Glutamine (Q) and asparagine (N) deamidation of enamel-specific proteins from H. antecessor (Atapuerca) (a), and H. erectus (Dmanisi) (b). Values are based on 1,000 bootstrap replications of protein deamidation. c, Relationship between mean asparagine (N) and glutamine (Q) deamidation for all proteins in both the Atapuerca and Dmanisi hominin datasets. Error bars represent 95% confidence interval window of 1,000 bootstrap replications of protein deamidation. Dashed line is x = y. d, Peptide length distribution of H. antecessor (Atapuerca), H. erectus (Dmanisi), four previously published enamel proteomes6,8,16 and one additional human Medieval control sample (Ø1952). For a, b and d, the number of peptides (n) is given for each violin plot. The box plots within the violin plots define the range of the data (whiskers extend to 1.5× the interquartile range), outliers (black dots, beyond 1.5× the interquartile range), 25th and 75th percentiles (boxes), and medians (centre lines). P values of two-sided t-tests conducted between sample pairs are indicated. No independent replication of these experiments was performed.
Extended Data Fig. 7 Survival of in vivo MMP20 and KLK4 cleavage sites in the Atapuerca enamel proteome.
a, Experimentally observed cleavage matrices for ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMELX and AMELY) (Methods). Fold differences are colour-coded by comparing observed PSM cleavage frequencies to a random cleavage matrix for each protein separately7. b, Fold differences for all observed cleavage pairs per protein. Red filled circles represent MMP20, KLK4 and signal peptide cleavage sites mentioned in the literature53,54,55,56. Red open circles indicate cleavage sites located up to two amino acid positions away from such sites. c, PSM coverage for each protein. The signal peptide (thick horizontal bar labelled ‘sig’), known MMP20 and KLK4 cleavage sites (vertical bars), and O- and N-linked glycosylation sites (asterisks) are also indicated. For AMELX, peptide positions for all three known isoforms were remapped to the coordinates of isoform 3, which represents the longest isoform (UniProt accession Q99217-3). The x and y axes differ between the three panels of c.
Nomascus leucogenys and M. mulatta were used as outgroups.
Supplementary Information containing supplementary information on the archaeological sites of Gran Dolina, Atapuerca, and Dmanisi, amino acid racemization, proteomic data extraction, data generation, and data analysis, as well as phylogenetic analysis of recovered ancient hominin proteomes. The Supplementary Information contains 11 SI tables and 16 SI figures.
Supplementary information file containing annotated MS/MS spectra of phylogenetic interest.
File in .fasta format containing recovered Homo antecessor (Atapuerca) and Homo erectus (Dmanisi) protein sequences. Sequence coverage obtained after MaxQuant and PEAKS analysis is combined.
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Welker, F., Ramos-Madrigal, J., Gutenbrunner, P. et al. The dental proteome of Homo antecessor. Nature 580, 235–238 (2020). https://doi.org/10.1038/s41586-020-2153-8
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