completely absent among elephants across the traditional range of the forest elephant. Previous discrepancies between mtDNA patterns and those of nuclear loci or morphology could thus begin to be resolved by considering the distribution of mtDNA haplo- groups in populations rather than individuals [12]. The pattern we found lends support to a hypothesis that the source of nuclear alleles present in savanna but not forest elephants was the population in which the mtDNA S clade evolved [1,4,5,25]. The integrity of two species was maintained despite a historically shifting hybrid zone Some previous studies have made the (often unstated) assumption that the presence of both F (forest elephant-derived) and S (savanna elephant-derived) mitochondrial DNA clades at a locale is an indication that the population of elephants at that locale must comprise a mixture of forest and savanna elephant genotypes [12,16]. By contrast, we find that the mtDNA is an incongruent locus, not representative of the overall genetic makeup of the populations (Figure 5). In savanna locales where F clade mtDNA haplotypes are carried by a majority of the elephants the savanna elephant populations nonetheless have little or no discernible forest elephant contribution to nuclear genotypes (Figure 5). Other than carrying F clade mtDNA, these elephants proved not to be hybrids (Figures 1, 2) [57], with most savanna individuals carrying F clade mtDNA nonetheless displaying ca. 100% assignment of their genotypes into the savanna elephant partition in STRUCTURE analyses (Figures 2, 5). Even in Garamba, which demonstrates the greatest degree of hybridization among our sampled locales, the majority of individuals partitioned completely as forest elephants (Figure 2). This might be consistent with hybridization being a response to recent destruction of forests in Garamba [34], which may have permitted migration of savanna elephants into previously forested habitat and recent nuclear intro- gression [58]. Even in Garamba, a panmictic blend of forest and savanna elephants, in which all individuals would display a mix of forest and savanna genotypes, is not evident (Figure 2). One previous hypothesis, based on interpretations of mtDNA patterns, that ‘‘the hybrid zone between africana and cyclotis is not fairly ‘narrow’,’’ [12] is not supported by the current data, especially given the overlap of sampling locations (Cameroon, Zimbabwe) between the current and previous studies. Isolation from savanna elephant gene flow appears to be complete among forest elephants, since in the current survey not a single forest elephant was found to carry S clade mtDNA across 30 forest locales in West or Central Africa. Previous studies had failed to detect, among a smaller survey of forest elephants, even a single Y-chromosome or X-chromosome haplotype derived from savanna elephants [4]. Although F clade mtDNA has crossed the species barrier and is often present in savanna elephants (Figures 3, 4, 5), the reverse pattern has not occurred to any detectable degree in our numerically large and geographically extensive current reanalysis of previously sequenced [4,12,13,15,16,36,37] forest elephants. Given the presence of hybrid individuals where the two types of elephant currently meet, and given the extensive historic hybrid zone suggested by the geographic expanse of F clade mtDNA [1,4,12], the complete absence of any detectable savanna elephant nuclear or mtDNA haplotype introgression into elephant populations in the tropical forests strongly suggests that isolating mechanisms between species [1,25,46] have prevented savanna or hybrid elephants from successfully contributing to the forest elephant gene pool. The situation is more complicated among savanna elephants. Forest elephant derived F clade mtDNA has a very broad geographic range that extends long distances away from current Reconciling African Elephant Phylogenies forest habitats. F clade mtDNA is carried by ca. 20% of savanna elephants [4,12,13], as far north as Mali, east into Tanzania, and is carried by a majority of elephants in some locations as far south as Botswana and Zimbabwe [4,12,13,15,16]. Given the lack of any nuclear genetic evidence for hybridization in these regions (Figure 5), the presence of F clade mtDNA in these locales is likely an indication that the hybrid zone separating forest and savanna elephants has shifted [58] as climate and the distribution of forest habitats shifted during the geological history of Africa [4,59,60,61,62]. While at any point in time the hybrid zone may have remained narrow even as habitats shifted, nonetheless the very large geographic distribution of F clade [1,4,12] suggests that the hybrid zone shifted across a vast expanse of the African continent, suggesting that many generations of hybridization generated the current mtDNA pattern. Nonetheless, the genetic integrity of forest and savanna elephants has remained intact (Figure 5). This would also be consistent with the presence of species isolation mechanisms restricting nuclear gene flow between forest and savanna elephants [1,25,46]. Maintenance of genetic integrity and isolation in amidst such a hybrid zone would define species even under the biological species concept [46,47,48], as has been noted previously [1]. The historical reproductive success of female hybrids can be inferred by the presence of F clade mtDNA, derived from forest elephants, in these savanna elephant herds (Figures 3, 5) [1,4,12,13,25,33]; while the failure of male hybrids to reproduce was inferred from the complete or nearly complete lack of forest elephant contribution to the nuclear genetic makeup of the savanna populations carrying forest-derived mtDNA (Figure 5) [1,4,13,33]. The mito-nuclear pattern observed in savanna elephants can only result if male forest-savanna elephant hybrids are (relatively) reproductively unsuccessful while hybrid females repeatedly backcross to savanna males [1,4,25]. These inferences would be consistent with Haldane’s rule [63] sensu lato, a com- monly observed phenomenon detected following hybridization between distant taxa, in which the deleterious effects of hybri- dization tend to have greater impact on the heterogametic sex (i.e. males in the case of mammals), since the mito-nuclear patterns suggest that hybrid females have been reproductively successful but hybrid males have not [4,46,63]. The inferred backcrossing of hybrid females to non-hybrid savanna males would over gene- rations dilute out forest elephant typical nuclear alleles in the savanna population, hence removing the relationship between the apparent population structure revealed by examination of mtDNA patterns, and the true population structure that remains after a population is stripped of the presence of forest elephant-derived nuclear alleles [1,4,13]. The mtDNA patterns would be rendered misleading for inferring the overall population structure or taxo- nomic status of Africa’s elephants (Figures 1, 2, 5) [4,56,57]. The taxonomic status of West Africa’s elephants Although cautious in their interpretation [15], Eggert and colleagues have proposed the hypotheses that the elephants ‘‘of West Africa belong to a newly recognized and yet to be formally named species’’ [14], and that ‘‘West African populations are now genetically distinct from other forest and savanna elephants and have been on a different trajectory for more than 2 Myr’’ [15], based in part on the phylogeography of mtDNA control region haplotypes. Debruyne (2005) [36] has questioned this hypothesis, after finding West African elephants interspersed with Central African elephants on a phylogenetic reconstruction based on their mtDNA. It may also be relevant that markers that are 100% effective at assigning African savanna, Asian, and African forest elephant species, are nonetheless found to mis-assign many PLoS ONE | www.plosone.org 12 June 2011 | Volume 6 | Issue 6 | e20642