Mitogenomics of historical type specimens clarifies the taxonomy of Ethiopian Ptychadena Boulenger, 1917 (Anura, Ptychadenidae).

The taxonomy of the Ptychadenaneumanni species complex, a radiation of grass frogs inhabiting the Ethiopian highlands, has puzzled scientists for decades because of the morphological resemblance among its members. Whilst molecular phylogenetic methods allowed the discovery of several species in recent years, assigning pre-existing and new names to clades was challenged by the unavailability of molecular data for century-old type specimens. We used Illumina short reads to sequence the mitochondrial DNA of type specimens in this group, as well as ddRAD-seq analyses to resolve taxonomic uncertainties surrounding the P.neumanni species complex. The phylogenetic reconstruction revealed recurrent confusion between Ptychadenaerlangeri (Ahl, 1924) and P.neumanni (Ahl, 1924) in the literature. The phylogeny also established that P.largeni Perret, 1994 represents a junior synonym of P.erlangeri (Ahl, 1924) and distinguished between two small species, P.nana Perret, 1994, restricted to the Arussi Plateau, and P.robeensis Goutte, Reyes-Velasco, Freilich, Kassie & Boissinot, 2021, which inhabits the Bale Mountains. The phylogenetic analyses of mitochondrial DNA from type specimens also corroborate the validity of seven recently described species within the group. Our study shows how modern molecular tools applied to historical type specimens can help resolve long-standing taxonomic issues in cryptic species complexes.

Taxonomic revision of grass frogs (Ptychadenidae, Ptychadena) endemic to the Ethiopian highlands.

Frogs of the genus Ptychadena that inhabit the Ethiopian highlands serve as a model system to understand biogeography, diversification, and adaptations to high elevations. Despite recent studies focusing on the systematics of this group, the taxonomy of the Ptychadena neumanni species complex remains only partially resolved, owing largely to the morphological resemblance of its members. Here, the taxonomy of this historically problematic group of frogs is revised by integrating morphological and molecular analyses on both century-old type specimens and more recently collected material. Based on these multiple lines of evidence, the P. neumanni species complex is shown to be more speciose than previously thought and four new species are described. With the aim of clarifying and stabilizing the taxonomy of the group, six species are also re-described and morphological and acoustic identification keys are provided. This study also establishes species distribution maps and reveals important differences in range size between the members of the P. neumanni complex, calling for adapted conservation measures across the Ethiopian highlands.

Diversification of African tree frogs (genus Leptopelis) in the highlands of Ethiopia.

The frog genus Leptopelis is composed of ~50 species that occur across sub-Saharan Africa. The majority of these frogs are typically arboreal; however, a few species have evolved a fossorial lifestyle. Most species inhabit lowland forests, but a few species have adapted to high elevations. Five species of Leptopelis occupy the Ethiopian highlands and provide a good opportunity to study the evolutionary transition from an arboreal to a fossorial lifestyle, as well as the diversification in this biodiversity hot spot. We sequenced 14 nuclear and three mitochondrial genes, and generated thousands of SNPs from ddRAD sequencing to study the evolutionary relationships of Ethiopian Leptopelis. The five species of highland Leptopelis form a monophyletic group, which diversified during the late Miocene and Pliocene. We found strong population structure in the fossorial species L. gramineus, with levels of genetic differentiation between populations similar to those found between arboreal species. This could indicate that L. gramineus is a complex of cryptic species. We propose that after the original colonization of the Ethiopian highlands by the ancestor of the L. gramineus group, episodes of vicariance fragmented the ancestral populations of this group. We also report the re-evolution of arboreality in L. susanae, which evolved from a fossorial ancestor, a rare ecological switch in frogs that had previously been reported only once.

Revisiting the phylogeography, demography and taxonomy of the frog genus Ptychadena in the Ethiopian highlands with the use of genome-wide SNP data.

Understanding the diversification of biological lineages is central to evolutionary studies. To properly study the process of speciation, it is necessary to link micro-evolutionary studies with macro-evolutionary mechanisms. Micro-evolutionary studies require proper sampling across a taxon's range to adequately infer genetic diversity. Here we use the grass frogs of the genus Ptychadena from the Ethiopian highlands as a model to study the process of lineage diversification in this unique biodiversity hotspot. We used thousands of genome-wide SNPs obtained from double digest restriction site associated DNA sequencing (ddRAD-seq) in populations of the Ptychadena neumanni species complex from the Ethiopian highlands in order to infer their phylogenetic relationships and genetic structure, as well as to study their demographic history. Our genome-wide phylogenetic study supports the existence of approximately 13 lineages clustered into 3 species groups. Our phylogenetic and phylogeographic reconstructions suggest that those endemic lineages diversified in allopatry, and subsequently specialized to different habitats and elevations. Demographic analyses point to a continuous decrease in the population size across the majority of lineages and populations during the Pleistocene, which is consistent with a continuous period of aridification that East Africa experienced since the Pliocene. We discuss the taxonomic implications of our analyses and, in particular, we warn against the recent practice to solely use Bayesian species delimitation methods when proposing taxonomic changes.

Comparative Phylogeography of Ethiopian anurans: impact of the Great Rift Valley and Pleistocene climate change.

BACKGROUND: The Ethiopian highlands are a biodiversity hotspot, split by the Great Rift Valley into two distinct systems of plateaus and mountains. The Rift Valley is currently hot and dry and acts as a barrier to gene flow for highland-adapted species. It is however unlikely that the conditions in the Rift were inhospitable to highland species during the entire Pleistocene. To assess the significance of the Ethiopian Rift as a biogeographic barrier as well as the impact Pleistocene climatic changes have had on the evolution of Ethiopian organisms, we performed phylogeographic analyses and developed present and past niche models on seven anuran species with different elevational and ecological preferences. RESULTS: We found that highland species on the east and the west sides of the Rift are genetically differentiated and have not experienced any detectable gene flow for at least 0.4 my. In contrast, species found at elevations lower than 2500 m do not show any population structure. We also determined that highland species have lower effective population sizes than lowland species, which have experienced a large, yet gradual, demographic expansion, starting approximately half a million year ago. CONCLUSIONS: The pattern we report here is consistent with the increasingly warmer and drier conditions of the Pleistocene in East Africa, which resulted in the expansion of savanna, the fragmentation of forests and the shrinking of highland habitats. Climatic niche models indicated that the Rift is currently non suitable for most of the studied species, but it could have been a more permeable barrier during the Last Glacial Maximum. However, considering the strong genetic structure of highland species, we hypothesize that the barrier mechanisms at the Rift are not only climatic but also topographical.

Hiding in the highlands: evolution of a frog species complex of the genus Ptychadena in the Ethiopian highlands.

The Ethiopian highlands are a biodiversity hotspot characterized by a high level of endemism, particularly in amphibians. Frogs of the genus Ptychadena have experienced an evolutionary radiation in these highlands. Thus, this group provides an excellent opportunity to study the process of speciation in this important biogeographic area. We sequenced two mitochondrial (16S and COI) and four nuclear (Rag-1, CXCR4, NCX1 and Tyr) genes in a sample of 236 frogs from 49 Ethiopian localities. Phylogenetic analyses of the mitochondrial genes revealed the presence of eight divergent mitochondrial lineages. We uncovered a near perfect concordance between these lineages and genetic clusters based on nuclear sequences. A Bayesian species delimitation analysis confirmed that these eight lineages correspond to eight genetically isolated populations which may represent eight species. Some of these species have already been recognized due to their distinct morphology (P. cooperi, P. nana and P. erlangeri) but we determined that the species P. neumanni is a complex of 5 cryptic species, thus increasing substantially the number of species in this genus and for this country. We resolved the phylogeny of Ethiopian highland Ptychadena using a species tree approach and determined that Ptychadena species group on the phylogeny according to their habitat preference. We propose that the diversity of Ethiopian Ptychadena results from an early phase of specialization to distinct elevations followed by a phase of ecological diversification within each elevational range. We estimated that the early phase of diversification of Ethiopian Ptychadena probably occurred in the late Miocene and the most recent speciation events in the late-Pliocene or Pleistocene.