randomly reduced to equal the number of forest elephants (run multiple times with different sets of savanna elephants); (2) the number of forest and savanna elephant individuals was randomly trimmed to each equal the number of Garamba elephants (run multiple times with different sets of forest and savanna elephants); or (3) the number of STR loci genotyped was increased to 27 for a subset of 35 forest, 9 Garamba, and 142 savanna elephants. Consequently, our results were robust across methodologies and across different resampling schemes of individuals and loci. Geographic distribution of nuclear and mtDNA partitions The mtDNA lineages of African elephants have been studied by a number of researchers who used different designations for mtDNA haplogroups, and who often compared different regions of the mtDNA genome [4,12,13,15,16,36,37]. We were able to combine the results of all of these studies, including that of Johnson et al. (2007) [16] which had included the sequences of Eggert et al. (2002) [15] and Nyakaana et al. (2002) [37] and others, by comparing overlapping fragments of mtDNA across the studies. Given the priority of Debruyne’s (2005) [12] publication (which established with strong bootstrap support the deepest relationships among haplogroups), the current study employs the clade ‘‘S’’ and clade ‘‘F’’ designations of Debruyne (2005) for the two most deeply rooted mtDNA clades. Since mtDNA is non-recombinant, all genes or regions in the mtDNA genome have followed the same evolutionary trajectory [9]; and since some studies had sequenced individual elephants for two or more mtDNA regions [12,13], it was possible to discover the equivalent designations used for the F and S clades across the other elephant mtDNA studies (Figure 3; Figure S1), even across studies that used non-overlapping regions of the mtDNA genome (see Figure S2 for details). For example, Roca et al. (2005) [4] had sequenced a portion of the mtDNA ND5 gene and detected two deep clades, designated I and II (Figure 3). Debruyne (2005) also detected two deep clades, by sequencing the CYTB mtDNA gene, calling them Clades S and F. Sequences from both studies were compared to those of Lei et al. (2008), who had sequenced individual elephants for longer mtDNA regions that overlapped with the sequences both of Debruyne (2005) and of Roca et al. (2005) (Figure S2) [4,12,13]. Using this system, desig- nations for the equivalent clades across studies were identified. The mtDNA clade designations used by all other elephant mtDNA surveys for clades or ‘‘groups’’ corresponding to the F/S clade nomenclature of Debruyne (2005) [12] are shown in Figure 3. In the most recent of these surveys, Johnson et al. (2007) [16], reported the presence of four taxonomic groups of elephants in Africa. They hypothesized that they had uncovered the presence of new genetic groupings of African elephants due to their collection of elephant samples from previously unexamined loca- tions. In our attempt to identify haplogroup synonyms across various studies, we identified the elephants of Debruyne (2005) [12] and Eggert et al. (2002) [15] within the network of Johnson et al. (2007) [16] (Figure 3). We found that the elephant groups of Johnson et al. (2007) were synonymous with five subclades of the basal F and S clades previously identified by Debruyne (2005) [12], with the subclades designated F4 and F5 by Debruyne (2005) [12] consolidated into ‘‘Group II’’ by Johnson et al. (2007) [16] (Figure 3 and Figure S1). Thus, while recognizing the importance of the intensive geographic sampling conducted by Johnson et al. (2007) [16], we consider that the best interpretation of the mtDNA network generated by Johnson et al. (2007) would not be that previously undetected genetic groupings of elephants had been found, but rather that one of their groups corresponded to the mtDNA S clade, while each of the other groups of Johnson et al. (2007) corresponded to subdivisions of the mtDNA F clade Reconciling African Elephant Phylogenies identified previously by Debruyne (2005) [12] and Eggert et al. (2002) [15] (Figure 3). Having established the relationship of mtDNA designations across studies, we combined all of their results, assessing F and S clade mtDNA data on elephants from 81 locations across Africa (Figure 4; Figure S3) [4,12,13,15,16,36,37]. The conclusion drawn from this combined dataset was remarkable: not a single elephant from the African tropical forests carried S clade mtDNA (Figure 4). The combined results surveyed at least 30 tropical forest locations across both Guinean and Congolian forest blocks (Figure 4, Figure S3), yet S clade was not detected at any tropical forest locales. This is especially remarkable given that three independent surveys have each reported, by contrast, that about 20% of Africa’s savanna elephants carry forest elephant (F clade) mtDNA (Figures 3, 5) [4,12,13]. Although S clade was not detected in all savanna locales, in many cases, the apparent absence of S clade at a locale was likely due to an insufficient number of elephants being sampled, since samples from nearby savanna regions did detect S clade (e.g., in northern Botswana; Figure 4). Some previous studies had analyzed mtDNA haplogroups before Debruyne (2005) identified the basal F and S clades [15,37]; others did not consider whether their mtDNA haplogroups comprised subsets of the F or S clade [16]. Thus the pattern we are demonstrating for the distribution of mtDNA S clade has not been reported previously with this degree of geographic sampling. Our approach to mtDNA phylogeography (Figure 4) produced another notable result: the presence of S clade mtDNA closely corresponds to the range boundaries traditionally ascribed to the savanna elephant species based on nuclear DNA (Figure 1), morphology and habitat [1,2,3,4,6,7,13,24,25,26,32,33,38,39]. This helps to reconcile the apparent conflict previously reported between phylogeographic patterns of mtDNA in African elephants and the very different geographic patterns in morphology and nuclear genotypes [12]. For mtDNA the presence or absence of S clade corresponded to the geographic pattern previously reported using nuclear and morphological markers [3,6]. A contingency table was analyzed, comparing the presence (vs. lack of detection) of S clade mtDNA to the type of habitat at a locale (tropical forest vs. other or mixed habitats). Since 30 tropical forest locales lacked elephants carrying S-clade mtDNA, while S clade was detected in 34 of 51 non-tropical-forest samples, the association was found to be extremely significant (Fisher’s exact test, two-tailed p,1024). Genetic studies of African elephants have detected mito-nuclear discordance [4], in which patterns inferred using mtDNA markers do not correspond to those found using nuclear markers in the same forest and savanna elephants [4,11,12,13]. We therefore compared the partitioning of elephant nuclear genotypes by the program STRUCTURE with the pattern of mtDNA present in the same elephant individuals and locales [4]. In Figure 5, each African elephant locale is plotted with the x-axis showing the proportion of mtDNA haplotypes that belong to the ‘‘S clade’’ or savanna clade of mtDNA [4], while the y-axis shows the pro- portion of the nuclear genotypes assigned to the savanna elephant cluster by STRUCTURE (see Figure 1). Although savanna elephant locales all had an overwhelmingly high proportion of savanna elephant nuclear genotypes, the proportion of savanna clade mtDNA (S clade) carried by the elephants at savanna locales can range from 100% down to 0% (Figure 5) [4]. In fact, the proportion of savanna elephant nuclear genotypes carried by savanna elephants appeared to have no relationship to the pro- portion of S clade savanna mtDNA present in the population (Figure 5). By contrast, while forest elephant locales showed little or no nuclear genetic contribution from savanna elephants, they also carry no S clade mtDNA (Figure 5) [4]. Previous studies have PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20642