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

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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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.

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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

Authors and Affiliations

Authors

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.

Corresponding authors

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

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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)

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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). https://doi.org/10.1038/nature14249

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