Abstract

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.

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Accessions

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.

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Acknowledgements

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

Affiliations

  1. BioArCh, University of York, York YO10 5DD, UK

    • Frido Welker
    • , Matthew J. Collins
    • , Jessica A. Thomas
    • , Marc Wadsley
    • , Keri Rowsell
    •  & Michael Hofreiter
  2. Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany

    • Frido Welker
    •  & Jean-Jacques Hublin
  3. Department of Earth Sciences, Natural History Museum, London SW7 5BD, UK

    • Selina Brace
    •  & Ian Barnes
  4. Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5–7, 1350 Copenhagen K, Denmark

    • Enrico Cappellini
    • , Eske Willerslev
    •  & Ludovic Orlando
  5. Institute of Zoology, Zoological Society of London, London NW1 4RY, UK

    • Samuel T. Turvey
  6. CONICET- División Paleontología de Vertebrados, Museo de La Plata. Facultad de Ciencias Naturales y Museo de La Plata, Universidad Nacional de La Plata. Paseo del Bosque s/n, B1900FWA, La Plata, Argentina

    • Marcelo Reguero
    •  & Javier N. Gelfo
  7. Sección Paleontología de Vertebrados. Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, 470 Angel Gallardo Av., C1405DJR, Buenos Aires, Argentina

    • Alejandro Kramarz
  8. Institute of Anthropology, Johannes Gutenberg-University, Anselm-Franz-von-Bentzel-Weg 7, D-55128 Mainz, Germany

    • Joachim Burger
  9. Department of Chemistry, University of York, York YO10 5DD, UK

    • Jane Thomas-Oates
  10. Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, UK

    • David A. Ashford
    •  & Peter D. Ashton
  11. Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA

    • Duncan M. Porter
  12. Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK

    • Benedikt Kessler
    •  & Roman Fischer
  13. Applications Development, Bruker Daltonik GmbH, 28359 Bremen, Germany

    • Carsten Baessmann
    •  & Stephanie Kaspar
  14. Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark

    • Jesper V. Olsen
    •  & Christian D. Kelstrup
  15. Department of Materials Science and Metallurgy, University of Cambridge, Cambridge CB3 0FS, UK

    • Patrick Kiley
    •  & James A. Elliott
  16. Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland

    • Victoria Mullin
  17. Institute for Biochemistry and Biology, Karl-Liebknecht-Strasse 24–25, 14476 Potsdam OT Golm, Germany

    • Michael Hofreiter
  18. Department of Mammalogy, American Museum of Natural History, New York, New York 10024, USA

    • Ross D. E. MacPhee

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Materials, Supplementary Tables 1-5, a Supplementary Discussion and additional references.

  2. 2.

    Supplementary Information

    This fie contains the MS/MS spectra for residues where Toxodon and Macrauchenia differ.

Text files

  1. 1.

    Supplementary Data

    This file contains the Amino Acid Sequence data.

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DOI

https://doi.org/10.1038/nature14249

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