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Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse

Nature volume 499, pages 74–78 (2013)Cite this article

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Abstract

The rich fossil record of equids has made them a model for evolutionary processes1. Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560–780 thousand years before present (kyr bp)2,3. Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr bp), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski’s horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr bp), twice the conventionally accepted time to the most recent common ancestor of the genus Equus4,5. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski’s and domestic horse populations diverged 38–72 kyr bp, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski’s horse investigated. This supports the contention that Przewalski’s horses represent the last surviving wild horse population6. We find similar levels of genetic variation among Przewalski’s and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski’s horse. Such regions could correspond to loci selected early during domestication.

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Figure 1: The early Middle Pleistocene horse metapodial from Thistle Creek (TC).
Figure 2: Amino acid, protein and DNA preservation of the Thistle Creek horse bone.
Figure 3: Horse phylogenetic relationships and population divergence times.
Figure 4: Horse demographic history.

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Accession codes

Accessions

Sequence Read Archive

Data deposits

All sequence data have been submitted to Sequence Read Archive under accession number SRA082086 and are available for download, together with final BAM and VCF files, de novo donkey scaffolds, and proteomic data at http://geogenetics.ku.dk/publications/middle-pleistocene-omics.

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Acknowledgements

We thank T. Brand, the laboratory technicians at the Danish National High-throughput DNA Sequencing Centre and the Illumina sequencing platform at SciLifeLab-Uppsala for technical assistance; J. Clausen for help with the donkey samples; S. Rasmussen for computational assistance; J. N. MacLeod and T. Kalbfleisch for discussions involving the re-sequencing of the horse reference genome; and S. Sawyer for providing published ancient horse data. This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU); the Danish National Research Foundation; the Novo Nordisk Foundation; the Lundbeck Foundation (R52-A5062); a Marie-Curie Career Integration grant (FP7 CIG-293845); the National Science Foundation ARC-0909456; National Science Foundation DBI-0906041; the Searle Scholars Program; King Saud University Distinguished Scientist Fellowship Program (DSFP); Natural Science and Engineering Research Council of Canada; the US National Science Foundation DMR-0923096; and a grant RC2 HG005598 from the National Human Genetics Research Institute (NHGRI). A.G. was supported by a Marie-Curie Intra-European Fellowship (FP7 IEF-299176). M.F. was supported by EMBO Long-Term Post-doctoral Fellowship (ALTF 229-2011). A.-S.M. was supported by a fellowship from the Swiss National Science Foundation (SNSF). Mi.S. was supported by the Lundbeck foundation (R82-5062).

Author information

Author notes

  1. Damian Szklarczyk & Jakob Vinther

    Present address: Present addresses: Bioinformatics Group, Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (D.S.); Departments of Earth Sciences and Biological Sciences, University of Bristol BS8 1UG, UK (Ja.V.).,

  2. Ludovic Orlando, Aurélien Ginolhac and Guojie Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5–7, 1350 Copenhagen K, Denmark,

    Ludovic Orlando, Aurélien Ginolhac, Mikkel Schubert, Enrico Cappellini, Julia T. Vilstrup, Maanasa Raghavan, Thorfinn Korneliussen, Anna-Sapfo Malaspinas, Jesper Stenderup, Amhed M. V. Velazquez, Morten Rasmussen, Andaine Seguin-Orlando, Cecilie Mortensen, Kim Magnussen, Kristian Gregersen, Anders Krogh, M. Thomas P. Gilbert, Kurt Kjær & Eske Willerslev

  2. Shenzhen Key Laboratory of Transomics Biotechnologies, BGI-Shenzhen, Shenzhen 518083, China,

    Guojie Zhang, Xiaoli Wang, Jiumeng Min & Jun Wang

  3. Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta T6G 2E3, Canada,

    Duane Froese

  4. Department of Biology, The Bioinformatics Centre, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark,

    Anders Albrechtsen, Ida Moltke & Anders Krogh

  5. Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, California 95064, USA,

    Mathias Stiller, James Cahill & Beth Shapiro

  6. Department of Systems Biology, Center for Biological Sequence Analysis, Technical University of Denmark, DK-2800 Lyngby, Denmark

    Bent Petersen, Josef Vogt, Søren Brunak & Thomas Sicheritz-Ponten

  7. Department of Human Genetics, The University of Chicago, Chicago, Illinois 60637, USA,

    Ida Moltke

  8. Department of Biology, Emory University, Atlanta, Georgia 30322, USA,

    Philip L. F. Johnson

  9. Department of Integrative Biology, University of California, Berkeley, California 94720, USA,

    Matteo Fumagalli

  10. Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark,

    Damian Szklarczyk, Christian D. Kelstrup, Lars Juhl Jensen & Jesper V. Olsen

  11. Jackson School of Geosciences, The University of Texas at Austin, 1 University Road, Austin, Texas 78712, USA,

    Jakob Vinther

  12. Texas Materials Institute, The University of Texas at Austin, Austin, Texas 78712, USA,

    Andrei Dolocan

  13. Department of Tourism and Culture, Government of Yukon, Yukon Palaeontology Program, PO Box 2703 L2A, Whitehorse, Yukon Territory Y1A 2C6, Canada,

    Grant D. Zazula

  14. Danish National High-throughput DNA Sequencing Centre, University of Copenhagen, Øster Farimagsgade 2D, 1353 Copenhagen K, Denmark,

    Andaine Seguin-Orlando, Cecilie Mortensen & Kim Magnussen

  15. NABsys Inc, 60 Clifford Street, Providence, Rhode Island 02903, USA,

    John F. Thompson

  16. Archeology, University of Southampton, Avenue Campus, Highfield, Southampton SO17 1BF, UK,

    Jacobo Weinstock

  17. Zoological Museum, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark

    Kristian Gregersen

  18. Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Box 8146 Dep, N-0033 Oslo, Norway,

    Knut H. Røed

  19. Département histoire de la Terre, UMR 5143 du CNRS, paléobiodiversité et paléoenvironnements, MNHN, CP 38, 8, rue Buffon, 75005 Paris, France,

    Véra Eisenmann

  20. Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, SE-751 23 Uppsala, Sweden

    Carl J. Rubin & Leif Andersson

  21. Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853, USA,

    Donald C. Miller & Douglas F. Antczak

  22. Center for Zoo and Wild Animal Health, Copenhagen Zoo, 2000 Frederiksberg, Denmark,

    Mads F. Bertelsen

  23. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2970 Hørsholm, Denmark

    Søren Brunak & Thomas Sicheritz-Ponten

  24. Zoology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

    Khaled A. S. Al-Rasheid

  25. San Diego Zoo’s Institute for Conservation Research, Escondido, California 92027, USA,

    Oliver Ryder

  26. Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark,

    John Mundy & Jun Wang

  27. Department of Biology, The University of York, Wentworth Way, Heslington, York YO10 5DD, UK,

    Michael Hofreiter

  28. Departments of Integrative Biology and Statistics, University of California, Berkeley, Berkeley, California 94720, USA,

    Rasmus Nielsen

  29. King Abdulaziz University, Jeddah 21589, Saudi Arabia

    Jun Wang

  30. Macau University of Science and Technology, Avenida Wai long, Taipa, Macau 999078, China,

    Jun Wang

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Contributions

L.O. and E.W. initially conceived and headed the project; G.Z. and Ju.W. headed research at BGI; L.O. and E.W. designed the experimental research project set-up, with input from B.S. and R.N.; D.F. and G.D.Z. provided the Thistle Creek specimen, stratigraphic context and morphological information, with input from K.K.; K.H.R., B.S., K.G., D.C.M., D.F.A., K.A.S.A.-R. and M.F.B. provided samples; L.O., J.T.V., Ma.R., M.H., C.M. and J.S. did ancient and modern DNA extractions and constructed Illumina DNA libraries for shotgun sequencing; Ja.W. did the independent replication in Oxford; Ma.S. did ancient DNA extractions and generated target enrichment sequence data; Ji.M. and X.W. did Illumina libraries on donkey extracts; K.M., C.M. and A.S.-O. performed Illumina sequencing for the Middle Pleistocene and the 43-kyr-old horse genomes, the five domestic horse genomes and the Przewalski’s horse genome at Copenhagen, with input from Mo.R.; Ji.M. and X.W. performed Illumina sequencing for the Middle Pleistocene and the donkey genomes at BGI; J.F.T. headed true Single DNA Molecule Sequencing of the Middle Pleistocene genome; A.G., B.P. and Mi.S. did the mapping analyses and generated genome alignments, with input from L.O. and A.K.; Jo.V. and T.S.-P. did the metagenomic analyses, with input from A.G., B.P., S.B. and L.O.; Jo.V. and T.S.-P. did the ab initio prediction of the donkey genes and the identification of the Y chromosome scaffolds, with input from A.G. and Mi.S.; L.O., A.G. and P.L.F.J. did the damage analyses, with input from I.M.; A.G. did the functional SNP assignment; A.M.V.V. and L.O. did the PCA analyses, with input from O.R.; B.S. did the phylogenetic and Bayesian skyline reconstructions on mitochondrial data; Mi.S. did the phylogenetic and divergence dating based on nuclear data, with input from L.O.; A.G. did the PSMC analyses using data generated by C.J.R. and L.A.; L.O. and A.G. did the population divergence analyses, with input from J.C., R.N. and M.F.; L.O., A.G. and T.K. did the selection scans, with input from A.-S.M. and R.N.; A.A., I.M. and M.F. did the admixture analyses, with input from R.N.; L.O. and A.G. did the analysis of paralogues and structural variation; Ja.V. and A.D. did the amino-acid composition analyses; E.C., C.D.K., D.S., L.J.J. and J.V.O. did the proteomic analyses, with input from M.T.P.G. and A.M.V.V.; L.O. and V.E. performed the morphological analyses, with input from D.F. and G.D.Z.; L.O. and E.W. wrote the manuscript, with critical input from M.H., B.S., Jo.M. and all remaining authors.

Corresponding authors

Correspondence to Ludovic Orlando, Jun Wang or Eske Willerslev.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text and Data, Supplementary Figures, Supplementary Tables and additional references (see Contents for details). This file was updated on 3 July 2013 to correctly display figure S1.3 (PDF 20068 kb)

Supplementary Figures

This file contains Supplementary Figures S6.8-S6.38, which show DNA fragmentation and nucleotide misincorporation patterns for mitochondrial reads from other ancient samples analyzed in this study. (PDF 2191 kb)

Supplementary Tables

This zipped file contains Supplementary Tables 4.2, 4.3, 4.4, 5.9, 11.3, 11.4, 11.7 and 12.8. (ZIP 10146 kb)

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Orlando, L., Ginolhac, A., Zhang, G. et al. Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74–78 (2013). https://doi.org/10.1038/nature12323

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