Reconciling African Elephant Phylogenies Figure 5. Partitioning of forest and savanna elephant nuclear genotypes and mtDNA haplotypes at each African locale. For each location, the x-axis shows the proportion of mtDNA from the S clade or savanna clade of mtDNA [4]. The y-axis shows the proportion of the total population that corresponds to cluster 2 (savanna elephant nuclear genotype) of the STRUCTURE analysis. Savanna elephant locales (orange) all have a very high proportion of savanna elephant nuclear genotypes. By contrast, the proportion of savanna elephant S clade mtDNA varies from 100% down to 0% among the savanna locales, with no effect on nuclear genotypes. Remarkably, the proportion of S clade mtDNA varies greatly on a local level (e.g., CH-Chobe and SA-Savuti are geographically adjacent locales), calling into question attempts to infer the population structure of African elephants using only mtDNA. Forest elephant (green) locales show little or no nuclear genetic contribution from savanna elephants. None of the forest elephants carry a savanna elephant or S clade mtDNA [4,29], in this limited number of sampling locales (see Figure 4 for a more extensive survey of mtDNA data). The dashed line shows the ‘‘expected’’ relationship, if an assumption is made that the proportions of forest and savanna elephant nuclear genotypes in a population should reflect the proportion of F and S clade mtDNA haplotypes, respectively, in the population [12,16]. This ‘‘expected’’ relationship forms the basis for statements that forest and savanna elephants currently have a geographically large hybrid zone with large numbers of hybrids present across Africa [12,16]. Our current data suggests that such inferences are not valid since mtDNA patterns do not reflect population genotypes. Locale names are abbreviated as in Figure 1; we show the only mixed habitat locale (GR-Garamba) in blue. doi:10.1371/journal.pone.0020642.g005 elephants from the Congolian forest block (Figures 1 and 2), even when greater numbers of genetic clusters (K) were considered in the STRUCTURE analysis (not shown); and this individual has previously been found to have nuclear sequences identical to that of some Central African forest elephants (though distinct from that of any savanna elephants) [32]. Of course, a single sample would be insufficient to reach conclusions about the affinities of elephants in West Africa. We therefore considered an alternative if indirect approach to assessing the genetic similarity between West and Central African elephants. There has been one previous study that examined a large number of nuclear genetic markers among elephants in both West and Central Africa, which reported on the use of these markers in geographic assignment tests of elephants [26], but without additional analyses of genetic structure. These and other previous genetic assignment tests have been able to identify indi- viduals as being Asian, African savanna, or African forest elephants, with 100% success [7,26]. The success of these assign- ment methods was seen as supporting the division of African elephants into forest and savanna elephant species, since members of one species were never incorrectly assigned to the other species [7,26]. The assignment tests reflect the nearly complete lack of gene flow between forest and savanna elephants that has also been inferred using other nuclear loci [4,6]. Since lack of gene flow between populations would be an indication that speciation has occurred, we re-examined this previously published data [26], comparing assignment success between West and Central African elephant locations to determine whether this would provide support for the hypothesis that they comprise distinct species. Figure 6 shows a map with all locations used in the prior assignment study [26], including many locales outside of those shown in Figure 1, and including a substantial number of sites in both West and Central Africa. As an indirect measure of genetic similarity, we plotted the misassignment of elephants between one location and another, which is an indication of genetic similarity between elephants at two locales. Lines connecting the locales indicate cases where at least one elephant of known provenance was assigned to the wrong locale. What is striking is that among the hundreds of elephants assigned to a location using nuclear genotypes [7,26], not a single savanna elephant has been assigned to a forest location [26], even in cases where forest and savanna locales were geographically much closer between than within species. However there were many mis-assignments between two savanna or between two central forest locales (Figure 6). Given the complete success in assignment of elephants as either savanna (orange) or forest (green) elephants, the substantial degree of PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20642