Additional file 3: Table S3. of Selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes in hares (Lepus capensis L., 1758) from a steep ecological gradient in North Africa

Pairwise FST values for the three regions as obtained from the different markers. Upper diagonal, FST values (based on haplotype frequencies) as calculated from ATP6, ATP6 synonymous (syn.) positions only, ND2, ND2 syn. positions only, mtHV1, and from the microsatellite data. Lower diagonal, FST values (based on the distance method) as calculated from ATP6, ATP6 syn. positions, ND2, ND2 syn. positions and mtHV1, with 95% CI indicated between parentheses. Significance levels, *: P < 0.05, **: P < 0.01, ***: P < 0.001. (DOCX 13 kb

Additional file 2: Table S2. of Selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes in hares (Lepus capensis L., 1758) from a steep ecological gradient in North Africa

Posterior probabilities (PP) for the currently studied sites following the BEB approach in PAML. These values were reported for single gene analyses and the concatenated genes and for both models allowing positive selection. PP > 0.95 are grey shaded. (DOCX 11 kb

Additional file 1: Table S1. of Selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes in hares (Lepus capensis L., 1758) from a steep ecological gradient in North Africa

Basic sequence statistics for the three mitochondrial regions (ND2, ATP6, mtHV1) as calculated by the software DNASP (Librado & Rozas 2009). S: number of segregating (polymorphic) sites; h: haplotype diversity; π: nucleotide diversity; k: average number of nucleotide differences; D (Tajima 1989); D (Fu & Li 1993); F (Fu & Li 1993), and F (Fu 1997). (DOCX 14 kb

Mitochondrial and nuclear DNA reveals reticulate evolution in hares (<i>Lepus</i> spp., Lagomorpha, Mammalia) from Ethiopia

<div><p>For hares (<i>Lepus</i> spp., Leporidae, Lagomorpha, Mammalia) from Ethiopia no conclusive molecular phylogenetic data are available. To provide a first molecular phylogenetic model for the Abyssinian Hare (<i>Lepus habessinicus</i>), the Ethiopian Hare (<i>L</i>. <i>fagani</i>), and the Ethiopian Highland Hare (<i>L</i>. <i>starcki</i>) and their evolutionary relationships to hares from Africa, Eurasia, and North America, we phylogenetically analysed mitochondrial ATPase subunit 6 (ATP6; n = 153 / 416bp) and nuclear transferrin (TF; n = 155 / 434bp) sequences of phenotypically determined individuals. For the hares from Ethiopia, genotype composition at twelve microsatellite loci (n = 107) was used to explore both interspecific gene pool separation and levels of current hybridization, as has been observed in some other <i>Lepus</i> species. For phylogenetic analyses ATP6 and TF sequences of <i>Lepus</i> species from South and North Africa (<i>L</i>. <i>capensis</i>, <i>L</i>. <i>saxatilis</i>), the Anatolian peninsula and Europe (<i>L</i>. <i>europaeus</i>, <i>L</i>. <i>timidus</i>) were also produced and additional TF sequences of 18 <i>Lepus</i> species retrieved from GenBank were included as well. Median joining networks, neighbour joining, maximum likelihood analyses, as well as Bayesian inference resulted in similar models of evolution of the three species from Ethiopia for the ATP6 and TF sequences, respectively. The Ethiopian species are, however, not monophyletic, with signatures of contemporary uni- and bidirectional mitochondrial introgression and/ or shared ancestral polymorphism. <i>Lepus habessinicus</i> carries mtDNA distinct from South African <i>L</i>. <i>capensis</i> and North African <i>L</i>. <i>capensis</i> sensu lato; that finding is not in line with earlier suggestions of its conspecificity with <i>L</i>. <i>capensis</i>. <i>Lepus starcki</i> has mtDNA distinct from <i>L</i>. <i>capensis</i> and <i>L</i>. <i>europaeus</i>, which is not in line with earlier suggestions to include it either in <i>L</i>. <i>capensis</i> or <i>L</i>. <i>europaeus</i>. <i>Lepus fagani</i> shares mitochondrial haplotypes with the other two species from Ethiopia, despite its distinct phenotypic and microsatellite differences; moreover, it is not represented by a species-specific mitochondrial haplogroup, suggesting considerable mitochondrial capture by the other species from Ethiopia or species from other parts of Africa. Both mitochondrial and nuclear sequences indicate close phylogenetic relationships among all three <i>Lepus</i> species from Ethiopia, with <i>L</i>. <i>fagani</i> being surprisingly tightly connected to <i>L</i>. <i>habessinicus</i>. TF sequences suggest close evolutionary relationships between the three Ethiopian species and Cape hares from South and North Africa; they further suggest that hares from Ethiopia hold a position ancestral to many Eurasian and North American species.</p></div

Genetic differentiation and migration between <i>Lepus</i> species from Ethiopia.

<p>Genetic differentiation and migration between <i>Lepus</i> species from Ethiopia.</p

Hare samples used in this study.

<p>Hare samples used in this study.</p

Median joining network of TF haplotypes.

<p>Haplotypes (pies) are proportional to the total sample number, taxon assignments of single haplotypes (pie slices) represent percentages of taxa per haplotype. Black dots indicate inferred haplotypes, not revealed presently, numbers associated with lines give numbers of substitutions between any two haplotypes/inferred haplotypes, if more than one; single mutational steps between any two haplotypes are not indicated. Evolutionary distances between haplotypes are only roughly in proportional scale. Taxa acronyms: cn–<i>Lepus capensis</i>, North Africa, cs–<i>L</i>. <i>capensis</i>, South Africa, f–<i>L</i>. <i>fagani</i>, h–<i>L</i>. <i>habessinicus</i>, s–<i>L</i>. <i>starcki</i>, x–<i>L</i>. <i>saxatilis</i>, <i>Lsp</i>.–phenotypically undetermined hare specimen, cc–<i>L</i>. <i>capensis</i>, China, co–<i>L</i>. <i>comus</i>, hn–<i>L</i>. <i>hainanus</i>, m–<i>L</i>. <i>mandshuricus</i>, si–<i>L</i>. <i>sinensis</i>, oi–<i>L</i>. <i>oiostolus</i>, y–<i>L</i>. <i>yarkandensis</i>, a–<i>L</i>. <i>arcticus</i>, am–<i>L</i>. <i>americanus</i>, cf–<i>L</i>. <i>californicus</i>, cj–<i>L</i>. <i>castroviejoi</i>, cr–<i>L</i>. <i>corsicanus</i>, e–<i>L</i>. <i>europaeus</i>, g–<i>L</i>. <i>granatensis</i>, o–<i>L</i>. <i>othus</i>, t–<i>L</i>. <i>timidus</i>, tw–<i>L</i>. <i>twonsendii</i>, sf–<i>Sylvilagus floridanus</i>, Oc–<i>Oryctolagus cuniculus</i>.</p

Bayesian dendrogram of mtATP6 haplotypes.

<p>Node support above 50% is given for Bayesian Inference, ML, and NJ analyses, respectively. For details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180137#sec002" target="_blank">Material and methods</a>”.</p

Geographical sample distribution.

<p>Full red circles–<i>Lepus habessinicus</i>, full brown triangles–<i>L</i>. <i>fagani</i>, full blue squares–<i>L</i>. <i>starcki</i>. Open symbols indicate geographical positions of respective holotypes; also given are acronyms of sample localities (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180137#pone.0180137.t001" target="_blank">Table 1</a>).</p

Bayesian dendrogram of TF haplotypes.

<p>Node support above 50% is given for Bayesian Inference, ML, and NJ analyses (for details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180137#sec002" target="_blank">Materials and methods</a>” section). Acronyms of taxa: a–<i>Lepus arcticus</i>, am–<i>L</i>. <i>americanus</i>, c–<i>L</i>. <i>comus</i>, cc–<i>L</i>. <i>capensis</i>, China, cf–<i>L</i>. <i>californicus</i>, cj–<i>L</i>. <i>castroviejoi</i>, cn–<i>L</i>. <i>capensis</i>, North Africa, cr–<i>L</i>. <i>corsicanus</i>, cs–<i>L</i>. <i>capensis</i>, South Africa, e–<i>L</i>. <i>europaeus</i>, f–<i>L</i>. <i>fagani</i>, h–<i>L</i>. <i>habessinicus</i>, hn–<i>L</i>. <i>hainanus</i>, m–<i>L</i>. <i>mandshuricus</i>, o–<i>L</i>. <i>othus</i>, oi–<i>L</i>. <i>oiostolus</i>, s–<i>L</i>. <i>starcki</i>, si–<i>L</i>. <i>sinensis</i>, t–<i>L</i>. <i>timidus</i>, tw–<i>L</i>. <i>townsendii</i>, x–<i>L</i>. <i>saxatilis</i>, y–<i>L</i>. <i>yarkandensis</i>, Oc–<i>Orycotlagus cuniculus</i>, Sf–<i>Sylvilagus floridanus</i>.</p