Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates


No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as ‘South American native ungulates’. To Charles Darwin1,2, who first collected their remains, they included perhaps the ‘strangest animal[s] ever discovered’. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago3, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria4,5,6. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies7,8,9. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed5,6, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates4,6 from ‘condylarths’, a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Samples used in this investigation.
Figure 2: Relationship of Toxodon (Notoungulata) and Macrauchenia (Litopterna) to other placental mammals.

Accession codes

Data deposits

Raw MS/MS and PEAKS search files have been deposited to the ProteomeXchange with identifier PXD001411. Generated COL1 species consensus sequences will be available in the UniProt Knowledgebase under the accession numbers C0HJN3–C0HJP8.


  1. Owen, R. in The Zoology of the Voyage of H. M. S. Beagle, under the Command of Captain Fitzroy, during the Years 1832 to 1836 (ed. Darwin, C. ) Part I, Numbers I–IV (Smith Elder. 1838–40).

  2. Darwin, C. Journal of Researches into the Geology and Natural History of the Various Countries Visited by H.M.S. Beagle: Under the Command of Captain FitzRoy, R.N. from 1832 to 1836 (Henry Colburn, 1839)

    Google Scholar 

  3. Husson, D. et al. Astronomical calibration of the Maastrichtian (Late Cretaceous). Earth Planet. Sci. Lett. 305, 328–340 (2011)

    Article  ADS  CAS  Google Scholar 

  4. De Muizon, C. & Cifelli, R. L. The “condylarths” (archaic Ungulata, Mammalia) from the early Palaeocene of Tiupampa (Bolivia): implications on the origin of the South American ungulates. Geodiversitas 22, 1–150 (2000)

    Google Scholar 

  5. Agnolin, F. L. & Chimento, N. R. Afrotherian affinities for endemic South American “ungulates”. Mamm. Biol. 76, 101–108 (2011)

    Article  Google Scholar 

  6. O’Leary, M. A. et al. The placental mammal ancestor and the post-K-Pg radiation of placentals. Science 339, 662–667 (2013)

    Article  ADS  PubMed  CAS  Google Scholar 

  7. Dos Reis, M. et al. Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny. Proc. R. Soc. B 279, 3491–3500 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  8. Song, S., Liu, L., Edwards, S. V. & Wu, S. Resolving conflict in eutherian mammal phylogeny using phylogenomics and the multispecies coalescent model. Proc. Natl Acad. Sci. USA 109, 14942–14947 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Meredith, R. W. et al. Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification. Science 334, 521–524 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  10. McKenna, M. C., Bell, S. K. & Simpson, G. G. Classification of Mammals above the Species Level (Columbia Univ. Press, 1997)

    Google Scholar 

  11. Simpson, G. G. The beginning of the age of mammals in South America. Part 2. Bull. Am. Mus. Nat. Hist. 137, 1–259 (1967)

    Google Scholar 

  12. Patterson, B. & Pascual, R. The fossil mammal fauna of South America. Q. Rev. Biol. 43, 409–451 (1968)

    Article  Google Scholar 

  13. Cifelli, R. L. in Mammal Phylogeny (eds Szalay, F. S., Novacek, M. J. & McKenna, M. C. ) 195–216 (Springer, 1993)

    Book  Google Scholar 

  14. Horovitz, I. Eutherian mammal systematics and the origins of South American ungulates as based on postcranial osteology. Bull. Carnegie Mus. Nat. Hist. 63–79 (2004)

    Article  Google Scholar 

  15. Asher, R. J. & Lehmann, T. Dental eruption in afrotherian mammals. BMC Biol. 6, 14 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  16. Sánchez-Villagra, M. R., Narita, Y. & Kuratani, S. Thoracolumbar vertebral number: the first skeletal synapomorphy for afrotherian mammals. Syst. Biodivers. 5, 1–7 (2007)

    Article  Google Scholar 

  17. Van Bocxlaer, I., Roelants, K., Biju, S. D., Nagaraju, J. & Bossuyt, F. Late Cretaceous vicariance in Gondwanan amphibians. PLoS ONE 1, e74 (2006)

    Article  ADS  PubMed  Google Scholar 

  18. Murphy, W. J., Pringle, T. H., Crider, T. A., Springer, M. S. & Miller, W. Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res. 17, 413–421 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Billet, G. & Martin, T. No evidence for an afrotherian-like delayed dental eruption in South American notoungulates. Naturwissenschaften 98, 509–517 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Kramarz, A. & Bond, M. Critical revision of the alleged delayed dental eruption in South American “ungulates”. Mamm. Biol. 79, 170–175 (2014)

    Article  Google Scholar 

  21. Van Doorn, N. L. in Encyclopedia of Global Archaeology 7998–8000 (Springer, 2014)

    Book  Google Scholar 

  22. Buckley, M. & Collins, M. J. Collagen survival and its use for species identification in Holocene-lower Pleistocene bone fragments from British archaeological and paleontological sites. Antiqua 1, e1 (2011)

    Article  Google Scholar 

  23. Hamza, V. M. & Vieira, F. P. in Climate Change - Geophysical Foundations and Ecological Effects (eds Blanco, J. & Kheradmand, H. ) Ch. 6, 113–136 (Intech, 2011)

    Google Scholar 

  24. 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)

    Article  CAS  PubMed  ADS  Google Scholar 

  25. Asara, J. M., Schweitzer, M. H., Freimark, L. M., Phillips, M. & Cantley, L. C. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry. Science 316, 280–285 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Wilf, P., Rubén Cúneo, N., Escapa, I. H., Pol, D. & Woodburne, M. O. Splendid and seldom isolated: the paleobiogeography of Patagonia. Annu. Rev. Earth Planet. Sci. 41, 561–603 (2013)

    Article  ADS  CAS  Google Scholar 

  27. Van Valen, L. M. Paleocene dinosaurs or Cretaceous ungulates in South America? Evol. Monogr. 10, 1–79 (1988)

    Google Scholar 

  28. Vizcaino, M., Mikolajewicz, U., Jungclaus, J. & Schurgers, G. Climate modification by future ice sheet changes and consequences for ice sheet mass balance. Clim. Dyn. 34, 301–324 (2010)

    Article  Google Scholar 

  29. Allentoft, M. E. et al. The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils. Proc. R. Soc. B 279, 4724–4733 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. US. Central Intelligence Agency. The World Factbook 2013–14 (Central Intelligence Agency, 2013)

  31. Ma, B. et al. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2337–2342 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  32. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Drummond, A. J. et al. Geneious v4.7. (Geneious, 2010)

  34. Orlando, L. et al. Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74–78 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

  35. Buckley, M. A molecular phylogeny of Plesiorycteropus reassigns the extinct mammalian order ‘Bibymalagasia’. PLoS ONE 8, e59614 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. Terajima, M. et al. Glycosylation and cross-linking in bone type I collagen. J. Biol. Chem. (2014)

  37. Hudson, D. M., Weis, M. & Eyre, D. R. Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens. PLoS ONE 6, e19336 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hudson, D. M., Werther, R., Weis, M., Wu, J.-J. & Eyre, D. R. Evolutionary origins of C-terminal (GPP)n 3-hydroxyproline formation in vertebrate tendon collagen. PLoS ONE 9, e93467 (2014)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  39. Schweitzer, M. H. et al. Analyses of soft tissue from Tyrannosaurus rex suggest the presence of protein. Science 316, 277–280 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  40. Buckley, M. et al. Comment on “Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry”. Science 319, 33 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Van Doorn, N. L., Wilson, J., Hollund, H., Soressi, M. & Collins, M. J. Site-specific deamidation of glutamine: a new marker of bone collagen deterioration. Rapid Commun. Mass Spectrom. 26, 2319–2327 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Rohland, N. & Hofreiter, M. Comparison and optimization of ancient DNA extraction. Biotechniques 42, 343–352 (2007)

    Article  CAS  PubMed  Google Scholar 

  43. Leonard, J. A. et al. Animal DNA in PCR reagents plagues ancient DNA research. J. Archaeol. Sci. 34, 1361–1366 (2007)

    Article  Google Scholar 

  44. Brace, S. et al. Population history of the Hispaniolan hutia Plagiodontia aedium (Rodentia: Capromyidae): testing the model of ancient differentiation on a geotectonically complex Caribbean island. Mol. Ecol. 21, 2239–2253 (2012)

    Article  PubMed  Google Scholar 

  45. Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. 2010, (2010)

  46. Dabney, J. & Meyer, M. Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques 52, 87–94 (2012)

    Article  CAS  PubMed  Google Scholar 

  47. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011)

    Article  Google Scholar 

  48. Zuckerkandl, E., Jones, R. T. & Pauling, L. A comparison of animal hemoglobins by tryptic peptide pattern analysis. Proc. Natl Acad. Sci. USA 46, 1349–1360 (1960)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sarich, V. M. & Wilson, A. C. Rates of albumin evolution in primates. Proc. Natl Acad. Sci. USA 58, 142–148 (1967)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lanfear, R., Calcott, B., Ho, S. Y. W. & Guindon, S. Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol. Biol. Evol. 29, 1695–1701 (2012)

    Article  CAS  PubMed  Google Scholar 

  51. 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)

    Article  PubMed  PubMed Central  Google Scholar 

  52. Rambaut, A., Drummond, A. J. & Suchard, M. Tracer v.1. 6. (2013)

  53. Swofford, D. L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) v.4.0b10 (Sinauer Associates, 2003)

    Google Scholar 

  54. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Drummond, A. J., Suchard, M. A., Xie, D. & Rambaut, A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Benton, M. J., Donoghue, P. C. J. & Asher, R. J. in The Timetree of Life (eds Hedges, B. S. & Kumar, S. ) 35–86 (Oxford Univ. Press, 2009)

    Google Scholar 

  57. Rambaut, A. & Drummond, A. BEAUTi v.1. 4.2. Bayesian evolutionary analysis utility. (2007)

  58. Humphrey, W., Dalke, A. & Schulten, K. VMD – Visual Molecular Dynamics. J. Mol. Graph. 14, 33–38 (1996)

    Article  CAS  Google Scholar 

Download references


We thank the Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires (MACN), the Museo de La Plata (MLP), and the Natural History Museum of Denmark, Copenhagen (ZMK), for allowing us to sample fossil specimens in their collections for this project. The American Museum of Natural History and the Copenhagen Zoo provided samples of extant mammals suitable for collagen extraction. Mogens Andersen and Kristian Gregersen of ZMK provided information on specimens in their care. This work was partly supported by SYNTAX award “Barcode of Death”, European Research Council (ERC) Advanced Award CodeX, ERC Consolidator Award GeneFlow, SYNTHESYS FP7 grant agreement 226506, Engineering and Physical Sciences Research Council NE/G012237/1 and National Science Foundation OPP 1142052. J.T.-O. and D.A.A. are members of the York Centre of Excellence in Mass Spectrometry, created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative.

Author information

Authors and Affiliations



R.D.E.M., I.B., and M.J.C. conceived the project and coordinated the writing of the paper with F.W. and J.A.T., with all authors participating. J.N.G., A.K., M.R., E.C., and R.D.E.M. collected fossil and extant mammal samples for protein extraction. M.W., S.B., I.B., J.A.T., J.B., and M.H. conducted DNA analyses. F.W., M.W., P.A., S.K., C.B., C.K., D.A., J.T.-O., R.F., B.K., P.K., J.A.E., E.C., L.O., and M.J.C. performed protein analyses and interpretation of results. J.A.T., I.B., F.W., and M.W. conducted the phylogenetic analyses and constructed trees. S.T.T., J.N.G., M.R., D.M.P., and R.D.E.M. provided the historical, systematic, and palaeontological framework for this study. J.-J.H., E.W., and J.S. provided technical information. Final editing and manuscript preparation was coordinated by M.J.C., R.D.E.M., and I.B.

Corresponding authors

Correspondence to Frido Welker, Matthew J. Collins, Ian Barnes or Ross D. E. MacPhee.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Examples of MALDI–TOF–MS and MS/MS product ion spectra.

a, MALDI–TOF–MS ZooMS spectra for Toxodon (upper) and Macrauchenia (lower) were used to screen for samples for the best collagen preservation. b, PEAKS alignment of matching product ion spectra for Macrauchenia MLP 96-V-10-19 (specimen sample number MLP2012.12) highlighting peptides aligning to the sequence GPNGEAGSAGPTGPPGLR. c, d, Annotated PEAKS report of product ion spectra for the same peptide sequence detailed in b for Toxodon (c) and Macrauchenia (d), detailing differences between both genera (gsT and gsA, highlighted) and shared substitutions compared with Equus (gpA for Equus, gpT for Toxodon and Macrauchenia). Note in b that both deamidation (N→D) and variable hydroxylation (P→h) were detected in different peptides covering this region of the sequence.

Extended Data Figure 2 Collagen type I substitution variability for placental mammals (genomic and proteomic data) compared with the dasyurid marsupial Sarcophilus harrisii (Tasmanian devil) as outgroup.

Substitution variability scores range between 0 and 1 and incorporate sequence coverage for a given number of species over a 15-amino-acid moving average (95% standard deviation in lighter tone). Top, along-chain variation in genomic sequence variability (upper red) is similar to proteomic sequence variability (lower blue) both for COL1α1 and for COL1α2 chains. Bottom, molecular surface rendering (via VMD58) of the collagen unit cell taken from coordinates given in Protein Data Bank accession number 3HR2. Colours represent genomic (left) and proteomic (right) sequence variability throughout the structure.

Extended Data Figure 3 Comparison of levels of deamidation for samples in this study with ref. 22 (diamonds).

The Macrauchenia sample was 14C dead, consistent with observed levels of deamidation, which are lower than either Toxodon dated to 12,000 years ago or Equus sp. (Tapalqué; not dated). Dotted lines indicate error ranges on Gln estimation for samples that were not dated or were undateable. The measurement approach used in this study—frequency of deamidation in positions represented in at least seven MS/MS spectra—is different from the approach used in ref. 22, so the absolute values may not be directly comparable.

Extended Data Figure 4 Bayesian constraint tree based on phylogeny published in figure 1 in ref. 6.

See Methods and Supplementary Information section 3.2 for further details and discussion.

Extended Data Figure 5 Maximum clade credibility phylogeny from BEAST molecular dating analysis.

Branch lengths are measured in millions of years; scale axis indicates intervals of 100 Ma. Node labels show 95% highest probability densities for molecular dates (in millions of years). Fossil constraints are provided in Supplementary Table 3. Vertical dashed line indicates Cretaceous/Palaeogene boundary.

Extended Data Table 1 Toxodon and Macrauchenia specimens used in this study
Extended Data Table 2 Comparative run statistics combining multiple runs

Supplementary information

Supplementary Information

This file contains Supplementary Materials, Supplementary Tables 1-5, a Supplementary Discussion and additional references. (PDF 608 kb)

Supplementary Information

This fie contains the MS/MS spectra for residues where Toxodon and Macrauchenia differ. (PDF 12189 kb)

Supplementary Data

This file contains the Amino Acid Sequence data. (TXT 166 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Welker, F., Collins, M., Thomas, J. et al. Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates. Nature 522, 81–84 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing