Evolution, Systematics, and Phylogeography of Pleistocene Horses in the New World: A Molecular Perspective Jaco Weinstock 1* , Eske Willerslev 1¤a , Andrei Sher 2 , Wenfei Tong 3 , Simon Y.W. Ho 1 , Dan Rubenstein 3 , John Storer 4 , James Burns 5 , Larry Martin 6 , Claudio Bravi 7 , Alfredo Prieto 8 , Duane Froese 9 , Eric Scott 10 , Lai Xulong 11 , Alan Cooper 1¤b* 1 Ancient Biomolecules Centre, Department of Zoology, University of Oxford, United Kingdom, 2 Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia, 3 Department of Ecology and Evolutionary Biology, Princeton University, United States of America, 4 Government of the Yukon, Cultural Services Branch, Whitehorse, Canada, 5 Quaternary Paleontology Program, Provincial Museum of Alberta, Edmonton, Canada, 6 Natural History Museum, University of Kansas, Lawrence, Kansas, United States of America, 7 Instituto Multidisciplinario de Biologia Celular (IMBICE), La Plata, Argentina, 8 Instituto de la Patagonia, Universidad de Magallanes, Punta Arenas, Chile, 9 Department of Earth and Atmospheric Science, University of Alberta, Canada, 10 San Bernardino County Museum, Redlands, California, United States of America, 11 China University of Geosciences, Wuhan, China The rich fossil record of horses has made them a classic example of evolutionary processes. However, while the overall picture of equid evolution is well known, the details are surprisingly poorly understood, especially for the later Pliocene and Pleistocene, c. 3 million to 0.01 million years (Ma) ago, and nowhere more so than in the Americas. There is no consensus on the number of equid species or even the number of lineages that existed in these continents. Likewise, the origin of the endemic South American genus Hippidion is unresolved, as is the phylogenetic position of the ‘‘stilt-legged’’ horses of North America. Using ancient DNA sequences, we show that, in contrast to current models based on morphology and a recent genetic study, Hippidion was phylogenetically close to the caballine (true) horses, with origins considerably more recent than the currently accepted date of c. 10 Ma. Furthermore, we show that stilt- legged horses, commonly regarded as Old World migrants related to the hemionid asses of Asia, were in fact an endemic North American lineage. Finally, our data suggest that there were fewer horse species in late Pleistocene North America than have been named on morphological grounds. Both caballine and stilt-legged lineages may each have comprised a single, wide-ranging species. Citation: Weinstock J, Willerslev E, Sher A, Tong W, Ho SYW, et al. (2005) Evolution, systematics, and phylogeography of Pleistocene horses in the new world: A molecular perspective. PLoS Biol 3(8): e241. Introduction Nowhere is the later evolution of horses more problematic than in the Americas, where more than 50 species of Pleistocene equids have been named, most of them during the 19th and early 20th centuries. While recent paleonto- logical studies suggest that this number should be drastically revised [1,2], no consensus has been reached about the number of valid species or their phylogenetic relationships. This has limited an in-depth investigation of the biogeog- raphy, evolution, and extinction of American Pleistocene equids and their phylogenetic relationships with synchronous Eurasian forms. A particularly problematic example is the so- called North American or New World ‘‘stilt-legged’’ horses (NWSL) found in Mid- and Late Pleistocene deposits in the north and west of North America [1–3]. The NWSL are often found with a second equid form commonly regarded as closely related to the Eurasian caballines, a group that includes the domestic horse (Equus caballus) and the extant wild Przewalskii horse. The stilt-legged forms have been taxonomically assigned to a number of species, but all have gracile limbs similar to the extant Asian asses (hemionids— e.g., onager, E. hemionus; and kiang, E. kiang, Figure 1), leading to suggestions that they are closely related or even result from a dispersal of Eurasian hemionids via the exposed Bering Strait during the last glacial period [4–9]. Alternatively, the NWSL have been regarded as North American endemics [10,11]. Likewise, the systematic position and origins of the South American Pleistocene genus Hippidion need to be re-eval- uated. This form appeared in South America approximately 2.5 million years (Ma) ago, i.e., shortly after the formation of the Isthmus of Panama enabled the movement of animals between the continents, an event known as the Great American Biotic Interchange [12]. Hippidion is considered to be a descendant of the pliohippines, a primitive group of Received February 14, 2005; Accepted May 6, 2005; Published June 28, 2005 DOI: 10.1371/journal.pbio.0030241 Copyright: Ó 2005 Weinstock et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: bp, base pairs; HVR, hypervariable region; Ma, million years; MCMC, Markov chain Monte Carlo; MRCA, most recent common ancestor; NWSL, New World ‘‘stilt-legged’’ horses. Academic Editor: David Hillis, University of Texas, United States of America *To whom correspondence should be addressed. E-mail: jacobo.weinstock@zoo. ox.ac.uk (JW); alan.cooper@adelaide.edu.au (AC) ¤a Current address: Ancient DNA and Evolution Group, Niels Bohr Institute, University of Copenhagen, Denmark ¤b Current address: School of Earth and Environmental Sciences, University of Adelaide, Australia PLoS Biology | www.plosbiology.org August 2005 | Volume 3 | Issue 8 | e241 0001 Open access, freely available online P L o S BIOLOGY