Breda M. Zimkus

Complex patterns of continental speciation: Molecular phylogenetics and biogeography of sub-Saharan puddle frogs (Phrynobatrachus)

Puddle frogs (Anura: Phrynobatrachidae) are one of the most species-rich sub-Saharan amphibian groups, occupying an extraordinarily diverse range of habitats. We construct the first phylogeny of puddle frogs, utilizing mitochondrial (12S rRNA, valine-tRNA, and 16S rRNA) and nuclear (RAG-1) DNA. Phylogenetic analyses are conducted using separate and combined partitions under maximum parsimony, maximum likelihood, and Bayesian criterion. Monophyly of the Phrynobatrachidae is well supported, and three major clades of Phrynobatrachus are identified. We reconstructed a biogeographic history using habitat preference, elevation, and geographic distribution. Habitat niches appear to be conserved between sister species, with the majority of species favoring forest over savanna habitats and the most recent common ancestor of the Phrynobatrachidae reconstructed as a forest species. Analyses of elevational data identify three independent colonizations of highland regions, one in each of the three major clades. Ancestral reconstructions support an East African origination of puddle frogs. Most species are restricted to one of five sub-Saharan regions and are distributed within the Eastern, Central, and Western zones with far fewer species in Southern Africa. These results elucidate the complex patterns of spatial niche partitioning that have contributed to the diversification of this widely distributed, sub-Saharan genus.

Sky Islands of the Cameroon Volcanic Line: a diversification hot spot for puddle frogs (Phrynobatrachidae: Phrynobatrachus)

The continental highlands of the Cameroon Volcanic Line (CVL) represent biological ‘sky islands’ with high levels of species richness and endemism, providing the ideal opportunity to understand how orogenesis and historical climate change influenced species diversity and distribution in these isolated African highlands. Relationships of puddle frogs (Phrynobatrachus) endemic to the CVL are reconstructed to examine the patterns and timing of puddle frog diversification. Historical distributions were reconstructed using both elevation and geography data. Puddle frogs diversified in the CVL via several dispersal and vicariance events, with most of the locally endemic species distributed across the northern part of the montane forest area in the Bamenda‐Banso Highlands (Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and medium elevation areas connecting these mountains). Two new species, P. jimzimkusi sp. n. and P. njiomock sp. n., are also described based on molecular analyses and morphological examination. We find that these new species are most closely related to one another and P. steindachneri with the ranges of all three species overlapping at Mt. Oku. Phrynobatrachus jimzimkusi sp. n. is distributed in the southern portion of the continental CVL, P. njiomock sp. n. is endemic to Mt. Oku, and P. steindachneri is present in the northeastern part of the montane forest area. Both new species can be distinguished from all other puddle frogs by a combination of morphological characters, including their large size, ventral coloration and secondary sexual characteristics present in males. These results highlight the Bamenda‐Banso Highlands, and specifically emphasize Mt. Oku, as a centre of diversification for puddle frogs, supporting the conservation importance of this region. Our results also provide new insights into the evolutionary processes shaping the CVL ‘sky islands’, demonstrating that lineage diversification in these montane amphibians is significantly older than expected with most species diverging from their closest relative in the Miocene. Whereas climatic changes during the Pliocene and Pleistocene shaped intraspecific diversification, most speciation events were significantly older and cannot be linked to Africas aridification in response to Pleistocene climate fluctuations.

Terrestrialization, Miniaturization and Rates of Diversification in African Puddle Frogs (Anura: Phrynobatrachidae)

Terrestrialization, the evolution of non-aquatic oviposition, and miniaturization, the evolution of tiny adult body size, are recurring trends in amphibian evolution, but the relationships among the traits that characterize these phenomena are not well understood. Furthermore, these traits have been identified as possible “key innovations” that are predicted to increase rates of speciation in those lineages in which they evolve. We examine terrestrialization and miniaturization in sub-Saharan puddle frogs (Phrynobatrachidae) in a phylogenetic context to investigate the relationship between adaptation and diversification through time. We use relative dating techniques to ascertain if character trait shifts are associated with increased diversification rates, and we evaluate the likelihood that a single temporal event can explain the evolution of those traits. Results indicate alternate reproductive modes evolved independently in Phrynobatrachus at least seven times, including terrestrial deposition of eggs and terrestrial, non-feeding larvae. These shifts towards alternate reproductive modes are not linked to a common temporal event. Contrary to the “key innovations” hypothesis, clades that exhibit alternate reproductive modes have lower diversification rates than those that deposit eggs aquatically. Adult habitat, pedal webbing and body size have no effect on diversification rates. Though these traits putatively identified as key innovations for Phrynobatrachus do not seem to be associated with increased speciation rates, they may still provide opportunities to extend into new niches, thus increasing overall diversity.

Light at the end of the tunnel: insights into the molecular systematics of East African puddle frogs (Anura: Phrynobatrachidae)

To investigate intra- and interspecific variability of ‘Little Brown Frogs’ (LBFs) from East Africa, we analysed molecular data from 23 species of Phrynobatrachus using 16S rRNA, a mitochondrial gene considered a universal DNA barcoding marker in amphibians. Conspecific populations, excluding P. natalensis, exhibited low (0–0.5%) sequence divergences, while conspecific populations of P. natalensis exhibited a wide range of differentiation (0.2–6.4%) with a distinct bimodal distribution, affirming that this may be a complex of cryptic species. Sister species yielded a range of differentiation from 1.5–8.3% with median of 2.3%. Results revealed that at least three non-sister populations of miniaturized puddle frogs are currently identified as P. mababiensis, and a new species from Ethiopia was identified. This first step into revising East African Phrynobatrachus systematics illustrates the necessity of rigorous taxonomic study, especially for groups with confusing or highly incomplete taxonomy.

Biodiversity and conservation genetics research in Central Africa: new approaches and avenues for international collaboration

A five-day international workshop was recently convened at the Université des Sciences et Techniques de Masuku in Gabon to enhance international collaboration among Central African, US and European scientists, conservation professionals and policy makers. The overall aims of the workshop were to: (1) discuss emerging priorities in biodiversity and conservation genetics research across Central Africa, and (2) create new networking opportunities among workshop participants. Here we provide a brief overview of the meeting, outline the major recommendations that emerged from it, and provide information on new networking opportunities through the meeting web site.

External morphology and osteology support the placement of Phrynobatrachus nlonakoensis Plath, Herrmann & Böhme, 2006 within the genus Arthroleptis

Abstract Examination of external morphology and osteology of type material of Phrynobatrachus nlonakoensis Plath, Herrmann, and Böhme, 2006 support a recent taxonomic change, transferring the species into the genus Arthroleptis, which was recently made without examination of specimens.

How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation

Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution.

Description of the tadpole of Amietophrynus brauni (Anura: Bufonidae) from the West Usambara Mountains, Tanzania : short communication

Abstract The tadpole of Amietophrynus brauni (Anura: Bufonidae), endemic to the forests of the Eastern Arc Mountains of Tanzania, is barcoded using 16S rRNA and described from the West Usambara Mountains. It differs from tadpoles of A. maculatus by its body proportions, A. garmani and A. xeros by its smaller size, and A. gutturalis by its colour pattern. Body proportions were examined across Gosner stages, revealing that most remain relatively constant across development; however, internarial distance/narial diameter fluctuates dramatically and should not be used in the identification of Amietophrynus tadpoles.

A new species of Phrynobatrachus (Anura: Phrynobatrachidae) from the Eastern Arc Mountians of Tanzania

Phrynobatrachus discogularis sp. nov. (Anura, Phrynobatrachidae) is described from the Eastern Arc Mountains in Tanzania. It occupies upland grasslands and while it morphologically most resembles West and Central African P. gutturosus (Chabanaud, 1921), P. rungwensis (Loveridge, 1932), and P. anotis Schmidt and Inger, 1959, preliminary analysis of mitochondrial 16S rRNA indicates that the new species differs from all other species with published sequence data by a minimum distance of 4.1% and has affinities with P. rungwensis, P. uzungwensis Grandison and Howell, 1983 and P. keniensis Barbour and Loveridge, 1928-all diminutive upland eastern African taxa. The shape and colour of the males external gular apparatus distinguish this species from all other described Phrynobatrachus species. Although geographically either sympatric or closely allopatric with P. uzungwensis, the lack of overlapping morphological characters indicates the two forms are distinct species. The new taxon is compared with other species from the region.

Herpetofauna of Montane Areas of Tanzania. 5. Range Extension of Phrynobatrachus sulfureogularis (Anura, Phrynobatrachidae) from Burundi to the Mahale Mountains of Western Tanzania with a Redescription of the Species

Abstract Two large forest Phrynobatrachus (male, snout–urostyle length 42.2 mm; subadult, snout–urostyle length 27.4 mm) were collected from the Mahale Mountains in Tanzania, exhibiting a large tympanum (55%–76% of the eye diameter), large digital discs, and a bright yellow throat in males. A comparison of these two individuals with all described Phyrnobatrachus species was made, and they were found to fit the description of P. sulfureogularis, a poorly known species only known from the type locality in Burundi. This new population represents a substantial southern range extension or potentially a similar, yet undescribed species.