Reconciling Apparent Conflicts between Mitochondrial and Nuclear Phylogenies in African Elephants Yasuko Ishida1., Taras K. Oleksyk2*., Nicholas J. Georgiadis3, Victor A. David4, Kai Zhao 1 , Robert M. Stephens5, Sergios-Orestis Kolokotronis6, Alfred L. Roca1,7* 1 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 2 Department of Biology, University of Puerto Rico at Mayagu¨ ez, Mayagu¨ ez, Puerto Rico, 3 Bole and Klingenstein Foundation, Cody, Wyoming, United States of America, 4 Laboratory of Genomic Diversity, National Cancer Institute at Frederick, Frederick, Maryland, United States of America, 5 Advanced Biomedical Computing Center, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, United States of America, 6 Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, New York, United States of America, 7 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America Abstract Conservation strategies for African elephants would be advanced by resolution of conflicting claims that they comprise one, two, three or four taxonomic groups, and by development of genetic markers that establish more incisively the provenance of confiscated ivory. We addressed these related issues by genotyping 555 elephants from across Africa with microsatellite markers, developing a method to identify those loci most effective at geographic assignment of elephants (or their ivory), and conducting novel analyses of continent-wide datasets of mitochondrial DNA. Results showed that nuclear genetic diversity was partitioned into two clusters, corresponding to African forest elephants (99.5% Cluster-1) and African savanna elephants (99.4% Cluster-2). Hybrid individuals were rare. In a comparison of basal forest ‘‘F’’ and savanna ‘‘S’’ mtDNA clade distributions to nuclear DNA partitions, forest elephant nuclear genotypes occurred only in populations in which S clade mtDNA was absent, suggesting that nuclear partitioning corresponds to the presence or absence of S clade mtDNA. We reanalyzed African elephant mtDNA sequences from 81 locales spanning the continent and discovered that S clade mtDNA was completely absent among elephants at all 30 sampled tropical forest locales. The distribution of savanna nuclear DNA and S clade mtDNA corresponded closely to range boundaries traditionally ascribed to the savanna elephant species based on habitat and morphology. Further, a reanalysis of nuclear genetic assignment results suggested that West African elephants do not comprise a distinct third species. Finally, we show that some DNA markers will be more useful than others for determining the geographic origins of illegal ivory. These findings resolve the apparent incongruence between mtDNA and nuclear genetic patterns that has confounded the taxonomy of African elephants, affirm the limitations of using mtDNA patterns to infer elephant systematics or population structure, and strongly support the existence of two elephant species in Africa. Citation: Ishida Y, Oleksyk TK, Georgiadis NJ, David VA, Zhao K, et al. (2011) Reconciling Apparent Conflicts between Mitochondrial and Nuclear Phylogenies in African Elephants. PLoS ONE 6(6): e20642. doi:10.1371/journal.pone.0020642 Editor: William J. Murphy, Texas A&M University, United States of America Received February 23, 2011; Accepted May 6, 2011; Published June 8, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: Funding was provided by the U. S. Fish and Wildlife Service African Elephant Conservation Fund Grant no. AFE-0458-98210-8-G743. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: roca@illinois.edu (ALR); taras.oleksyk@upr.edu (TKO) . These authors contributed equally to this work. Introduction Central to the successful management of endangered taxa is determination of whether their populations comprise one or more species. Molecular methods can play a major role in establishing the systematics of endangered taxa. Where more than one species is revealed, a separate conservation strategy would be justified for each. African elephants (genus Loxodonta) occupying tropical forest habitats were long considered to comprise a single species with elephants in other African habitats. Recent studies have renewed debate about their taxonomy, after both morphological and nuclear DNA analyses suggested that African elephants form two distinct species separated by a relatively narrow hybrid zone [1]. Skull measurements from 295 elephants of known provenance suggested that forest and savanna elephants fall into two mor- phologically distinct species [2,3]. Nuclear DNA analyses using both slower-evolving nuclear sequences [4,5,6] and more rapidly evolving microsatellite loci [7] have provided concordant evidence that forest and savanna elephants are distinct species [4,6,8,9] that are as divergent genetically as Asian elephants are from mammoths [5]. Few morphological intermediates [3,10] and nuclear genetic hybrids [4,6,7] between forest and savanna elephants have been detected, primarily in a zone of mixed forest-savanna habitat that surrounds the tropical forests of Africa [1]. The deeper relationships present among mtDNA lineages have also been determined in African elephants [4,11,12,13]. While two highly divergent mtDNA clades are present, a non-monophyletic pattern was revealed that did not cleanly separate forest and savanna elephants. Studies basing their conclusions primarily or completely on mtDNA data have inferred conflicting conclusions about African elephant systematics. Debruyne (2005) has stated that ‘‘the only defendable attitude assumes that Africa harbors a PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20642