Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiatherian mammals: Application to the tribe Bovini (Cetartiodactyla, Bovidae) Alexandre Hassanin a,b,⇑ , Junghwa An a,b , Anne Ropiquet c , Trung Thanh Nguyen a , Arnaud Couloux d a Muséum national d’Histoire naturelle (MNHN), Département Systématique et Evolution, UMR 7205 – Origine, Structure et Evolution de la Biodiversité, 75005 Paris, France b MNHN, UMS 2700, Service de Systématique Moléculaire, 75005 Paris, France c Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, Western Cape, South Africa d Genoscope, Centre National de Séquençage, 91057 Evry, France article info Article history: Received 15 May 2012 Revised 27 September 2012 Accepted 1 November 2012 Available online xxxx Keywords: Laurasiatheria Bovinae Nuclear introns Phylogeny Taxonomy Mitochondrial introgression Indels abstract Mitochondrial sequences are widely used for species identification and for studying phylogenetic rela- tionships among closely related species or populations of the same species. However, many studies of mammals have shown that the maternal history of the mitochondrial genome can be discordant with the true evolutionary history of the taxa. In such cases, the analyses of multiple nuclear genes can be more powerful for deciphering interspecific relationships. Here, we designed primers for amplifying 13 new exon-primed intron-crossing (EPIC) autosomal loci for studying shallow phylogeny and taxonomy of Laurasiatherian mammals. Three criteria were used for the selection of the markers: gene orthology, a PCR product length between 600 and 1200 nucleotides, and different chromosomal locations in the bovine genome. Positive PCRs were obtained from different species representing the orders Carnivora, Cetartiodactyla, Chiroptera, Perissodactyla and Pholidota. The newly developed markers were analyzed in a phylogenetic study of the tribe Bovini (the group con- taining domestic and wild cattle, bison, yak, African buffalo, Asian buffalo, and saola) based on 17 taxa and 18 nuclear genes, representing a total alignment of 13,095 nucleotides. The phylogenetic results were compared to those obtained from analyses of the complete mitochondrial genome and Y chromosomal genes. Our analyses support a basal divergence of the saola (Pseudoryx) and a sister-group relationship between yak and bison. These results contrast with recent molecular studies but are in better agreement with morphology. The comparison of pairwise nucleotide distances shows that our nuDNA dataset pro- vides a good signal for identifying taxonomic levels, such as species, genera, subtribes, tribes and subfam- ilies, whereas the mtDNA genome fails because of mtDNA introgression and higher levels of homoplasy. Accordingly, we conclude that the genus Bison should be regarded as a synonym of Bos, with the Euro- pean bison relegated to a subspecies rank within Bos bison. We compared our molecular dating estimates to the fossil record in order to propose a biogeographic scenario for the evolution of Bovini during the Neogene. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction In animals, mitochondrial DNA (mtDNA) sequences are com- monly used for species identification (DNA barcoding) or for study- ing phylogenetic relationships among closely related species (molecular systematics) or among populations of the same species (phylogeography and population genetics). Its utility is primarily explained by three major characteristics of the mitochondrial gen- ome. First, its evolutionary rates are substantially faster than the nuclear genome (Brown et al., 1979; Saccone et al., 2006), which ensures obtaining sufficient nucleotide variation for comparing closely related organisms. Second, its simple and conserved struc- ture, with two rRNA genes and 13 protein-coding genes, which lack introns and are separated by well-conserved tRNA genes (Boore, 1999), has facilitated the development of primers for PCR amplifi- cation of popular genes, such as the cytochrome b (Cytb) and the first subunit of the cytochrome c oxidase (CO1). Third, there are many mtDNA molecules per cell, which provides DNA extracts of sufficient quantity and quality from all kinds of tissues, including those collected from late Pleistocene remains (e.g., Reich et al., 2010). 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.11.003 ⇑ Corresponding author at: Muséum national d’Histoire naturelle (MNHN), Département Systématique et Evolution, UMR 7205 – Origine, Structure et Evolution de la Biodiversité, 75005 Paris, France. Fax: +33 1 40 79 30 63. E-mail address: hassanin@mnhn.fr (A. Hassanin). Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Please cite this article in press as: Hassanin, A., et al. Combining multiple autosomal introns for studying shallow phylogeny and taxonomy of Laurasiathe- rian mammals: Application to the tribe Bovini (Cetartiodactyla, Bovidae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.003