The smallest of its kind: Description of a new cryptic Amnirana species (Amphibia, Anura, Ranidae) from West African rainforests.

The distribution of the White-Lipped Frog Amnirana albolabris was long assumed to extend from eastern-central to western African rainforests. However, recent molecular phylogenetic analyses revealed that this taxon likely represents several undescribed species. Because the name-bearing types were collected in Gabon, the distantly related West African populations clearly represent an undescribed species that partly occurs in sympatry with Amnirana fonensis. Based on an integrative taxonomic approach, including molecular, morphological, and acoustic data, we describe the 'albolabris' populations from the Upper Guinean Forest Zone as a new species, and redescribe the morphologically similar A. fonensis on the bases of a larger series of genotyped individuals, including the first known females. We also provide new biological information for A. fonensis, including their advertisement call, habitat, and reproductive data. The new species is sister to A. fonensis and the two species differ by 5.8% in the mitochondrial 16S ribosomal RNA gene. Morphologically, males of the new species are smaller and have relatively smaller humeral glands. Females of the new species seem to have shorter legs than A. fonensis. In comparison to A. fonensis, the advertisement call of the new species has a higher dominant frequency and more pronounced frequency modulation. The two species differ in their distribution and habitat preferences, as revealed by environmental niche modelling. Whereas the new species is restricted to the Upper Guinean forests and thus is a true lowland-rainforest inhabitant, A. fonensis lives predominantly in Guinean montane forests in the forest-savanna mosaic zone.

Systematics of the Central African Spiny Reed Frog Afrixalus laevis (Anura: Hyperoliidae), with the description of two new species from the Albertine Rift.

The geographically widespread species Afrixalus laevis (Anura: Hyperoliidae) currently has a disjunct distribution in western Central Africa (Cameroon, Equatorial Guinea, Gabon, and possibly adjacent countries) and the area in and near the Albertine Rift in eastern Democratic Republic of the Congo and neighboring countries. At least two herpetologists have previously suggested that these disjunct populations represent distinct species, and herein, we utilize an integrative taxonomic approach with molecular and morphological data to reconcile the taxonomy of these spiny reed frogs. We sequenced 1554 base pairs of the 16S and RAG1 genes from 34 samples of A. laevis and one sample of A. orophilus (sympatric with eastern populations of A. laevis), and combined these data with previously sequenced GenBank Afrixalus samples via the bioinformatics toolkit SuperCRUNCH. Phylogenetic trees, dated phylogenetic analyses, and species-delimitation analyses were generated with RAxML, BEAST, and BPP, respectively. Eleven mensural characters were taken from multiple specimens of A. laevis and A. orophilus, and compared with paired t-tests and analyses of covariance. These combined results suggested populations of A. laevis in western Central Africa (Cameroon and Bioko Island, Equatorial Guinea) represent one species, whereas populations from the Albertine Rift and nearby forests represent two undescribed taxa that are sister to A. dorsimaculatus. The two new species (A. lacustris sp. nov. and A. phantasma sp. nov.) are distinguished by our phylogenetic and species-delimitation analyses, significant differences in several mensural characters, qualitative morphological differences, and by their non-overlapping elevational distribution.

A global reptile assessment highlights shared conservation needs of tetrapods.

Comprehensive assessments of species' extinction risks have documented the extinction crisis1 and underpinned strategies for reducing those risks2. Global assessments reveal that, among tetrapods, 40.7% of amphibians, 25.4% of mammals and 13.6% of birds are threatened with extinction3. Because global assessments have been lacking, reptiles have been omitted from conservation-prioritization analyses that encompass other tetrapods4-7. Reptiles are unusually diverse in arid regions, suggesting that they may have different conservation needs6. Here we provide a comprehensive extinction-risk assessment of reptiles and show that at least 1,829 out of 10,196 species (21.1%) are threatened-confirming a previous extrapolation8 and representing 15.6 billion years of phylogenetic diversity. Reptiles are threatened by the same major factors that threaten other tetrapods-agriculture, logging, urban development and invasive species-although the threat posed by climate change remains uncertain. Reptiles inhabiting forests, where these threats are strongest, are more threatened than those in arid habitats, contrary to our prediction. Birds, mammals and amphibians are unexpectedly good surrogates for the conservation of reptiles, although threatened reptiles with the smallest ranges tend to be isolated from other threatened tetrapods. Although some reptiles-including most species of crocodiles and turtles-require urgent, targeted action to prevent extinctions, efforts to protect other tetrapods, such as habitat preservation and control of trade and invasive species, will probably also benefit many reptiles.

Qualitative and Quantitative Analysis of Tryptamines in the Poison of Incilius alvarius (Amphibia: Bufonidae).

Rising numbers of psychoactive tryptamine derivatives have become available on the drug market over the last decade, making these naturally occurring or synthetically manufactured compounds highly relevant for forensic analyses. One of these compounds is 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a constituent of the dried poison of Incilius alvarius (Colorado River toad), which has a history of ritual and/or recreational use. Still, comprehensive and validated qualitative as well as quantitative analytical data on the psychoactive components of this poison are scarce. In this study, samples of the poison of Incilius alvarius were collected from live toads in the Sonoran Desert, Arizona (USA), and analyzed with a set of complementary methods. Acetone/water (70/30, v/v) proved to be the solvent of choice for the extraction of tryptamine derivatives. Trace compounds were enriched, and overload with 5-MeO-DMT was prevented by chromatographic separation of 5-MeO-DMT prior to qualitative analyses. The method for quantification was validated. Attenuated total reflection-Fourier transform infrared spectroscopy was suitable to identify 5-MeO-DMT as the main tryptamine in samples of the poison. The combined evaluation of analytical data gained from gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-quadrupole time-of-flight high-resolution MS (HPLC-qToF-HRMS) and HPLC-MS-MS confirmed the presence of 5-MeO-DMT, 5-MeO-N-methyltryptamine, 5-MeO-tryptamine, 5-MeO-tryptophol, 2-(5-methoxy-1H-indol-3-yl)-acetic-acid (5-MIAA), 5-HO-N-methyltryptamine, bufotenin, DMT and tryptophan. For the first time, 5-MeO-tryptamine and two positional isomers of hydroxylated MeO-DMT were detected in the poison of Incilius alvarius. The tryptamine present in the highest concentrations was 5-MeO-DMT (mean ± SD: 410,000 ± 30,000 µg/g). Mean concentrations of 5-MeO-tryptamine (490 ± 260 µg/g), 5-HO-N-methyltryptamine (270 ± 120 µg/g), bufotenin (2,800 ± 1,900 µg/g) and DMT (250 ± 80 µg/g) showed a relatively high variability between individual samples. The comprehensive analytical reference data of Incilius alvarius poison presented here might prove useful for forensic chemists.

Giant Tree Frog diversification in West and Central Africa: Isolation by physical barriers, climate, and reproductive traits.

Secondary sympatry amongst sister lineages is strongly associated with genetic and ecological divergence. This pattern suggests that for closely related species to coexist in secondary sympatry, they must accumulate differences in traits that mediate ecological and/or reproductive isolation. Here, we characterized inter- and intraspecific divergence in three giant tree frog species whose distributions stretch across West and Central Africa. Using genome-wide single-nucleotide polymorphism data, we demonstrated that species-level divergence coincides temporally and geographically with a period of large-scale forest fragmentation during the late Pliocene. Our environmental niche models further supported a dynamic history of climatic suitability and stability, and indicated that all three species occupy distinct environmental niches. We found modest morphological differentiation amongst the species with significant divergence in tympanum diameter and male advertisement call. In addition, we confirmed that two species occur in secondary sympatry in Central Africa but found no evidence of hybridization. These patterns support the hypothesis that cycles of genetic exchange and isolation across West and Central Africa have contributed to globally significant biodiversity. Furthermore, divergence in both ecology and reproductive traits appear to have played important roles in maintaining distinct lineages. At the intraspecific level, we found that climatic refugia, precipitation gradients, marine incursions, and potentially riverine barriers generated phylogeographic structure throughout the Pleistocene and into the Holocene. Further studies examining phenotypic divergence and secondary contact amongst these geographically structured populations may demonstrate how smaller scale and more recent biogeographic barriers contribute to regional diversification.

La sympatrie secondaire parmi les espèces sœurs est fortement associée à la divergence génétique et écologique. Ce modèle suggère que pour que des espèces étroitement liées coexistent en sympatrie secondaire, elles doivent accumuler des différences dans les traits qui contribuent à l'isolement écologique ou reproductif. Ici, nous avons caractérisé la divergence inter- et intra-spécifique chez trois espèces de grenouilles arboricoles géantes dont les distributions s'étendent à travers l'Afrique de l'Ouest et Centrale. Avec des données génétiques, nous avons démontré que la divergence au niveau des espèces coïncide temporellement et géographiquement avec une période de fragmentation forestière à la fin du Pliocène. Nos modèles de niches environnementales ont soutenu une histoire dynamique de stabilité climatique, et ont indiqué que les trois espèces occupent des niches environnementales distinctes. Nous avons trouvé une différenciation morphologique modeste parmi les trois espèces mais une divergence significative dans le diamètre du tympan et les cris des mâles. De plus, nous avons confirmé que deux espèces sont présentes en sympatrie secondaire en Afrique Centrale mais n'avons trouvé aucune preuve d'hybridation. Ces résultats soutiennent l'hypothèse que les cycles d'échange génétique et d'isolement à travers l'Afrique de l'Ouest et Centrale ont contribué à une profonde concentration de biodiversité dans la région. De plus, la divergence des traits écologiques et reproducteurs semble avoir joué un rôle important dans le maintien de lignées distinctes. Au niveau intra-spécifique, nous avons constaté que les refuges climatiques, les gradients de précipitation, les incursions marines et potentiellement les barrières fluviales ont généré une structure phylogéographique pendant le Pléistocène et jusqu'à l'Holocène. Des études examinant la divergence phénotypique et le contact secondaire entre ces populations géographiquement structurées pourraient démontrer comment des barrières biogéographiques à échelle plus petite et plus récentes contribuent à la diversification régionale.

Rivers, not refugia, drove diversification in arboreal, sub-Saharan African snakes.

The relative roles of rivers versus refugia in shaping the high levels of species diversity in tropical rainforests have been widely debated for decades. Only recently has it become possible to take an integrative approach to test predictions derived from these hypotheses using genomic sequencing and paleo-species distribution modeling. Herein, we tested the predictions of the classic river, refuge, and river-refuge hypotheses on diversification in the arboreal sub-Saharan African snake genus Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo-distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our results revealed significant population genetic structure within both Toxicodryas species, corresponding geographically to river barriers and divergence times from the mid-Miocene to Pliocene. Our demographic analyses supported the interpretation that rivers are indications of strong barriers to gene flow among populations since their divergence. Additionally, we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river-refuge hypotheses in favor of the river-barrier hypothesis. Based on conservative interpretations of our species delimitation analyses with the Sanger and ddRAD data sets, two new cryptic species are identified from east-central Africa. This study highlights the complexity of diversification dynamics in the African tropics and the advantages of integrative approaches to studying speciation in tropical regions.

Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens.

Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single-stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step-by-step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies.

Night stalkers from above: A monograph of Toxicodryas/ tree snakes (Squamata: Colubridae) with descriptions of two new cryptic species from Central Africa.

The genus Toxicodryas, historically included with the renowned Australasian cat-eyed snakes of the colubrid genus Boiga, currently includes two widespread species (T. blandingii and T. pulverulenta) in western, central, and eastern Africa. We leverage findings from a recent phylogenomic and historical demographic analysis of this genus (based on 2848-4471 Rad-seq loci from across the genome), with robust sampling from throughout the ranges of both species, to define two additional taxonomic units, with species boundaries corresponding to river barriers. Additional morphometric data from scores of examined museum specimens and literature records bolster the recognition of these two new cryptic species. We hypothesize that T. blandingii occurs west of the confluence of the Congo and Ubangi rivers, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of ventral scale counts in both sexes, additional significant differences in several scale counts, and lower venom toxicity. Toxicodryas pulverulenta occurs west of the Niger Delta in West Africa, whereas a cryptic new species that is found east of this biogeographic barrier has significantly higher numbers of subcaudal scale counts in both sexes. A review of published information regarding morphological variation, ecology, natural history, habitat, and venom is summarized for these four Toxicodryas species.

Time-for-space substitution in N-mixture models for estimating population trends: a simulation-based evaluation.

N-mixture models usually rely on a meta-population design, in which repeated counts of individuals in multiple sampling locations are obtained over time. The time-for-space substitution (TSS) in N-mixture models allows to estimate population abundance and trend of a single population, without spatial replication. This application could be of great interest in ecological studies and conservation programs; however, its reliability has only been evaluated on a single case study. Here we perform a simulation-based evaluation of this particular application of N-mixture modelling. We generated count data, under 144 simulated scenarios, from a single population surveyed several times per year and subject to different dynamics. We compared simulated abundance and trend values with TSS estimates. TSS estimates are overall in good agreement with real abundance. Trend and abundance estimation is mainly affected by detection probability and population size. After evaluating the reliability of TSS, both against real world data, and simulations, we suggest that this particular application of N-mixture model could be reliable for monitoring abundance in single populations of rare or difficult to study species, in particular in cases of species with very narrow geographic ranges, or known only for few localities.

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary, multi-scale approach.

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear-cutting. Retention of habitat trees and deadwood has since become common practice also in continuous-cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade-offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple-use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven-aged and selectively harvested continuous-cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi-functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio-economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.

Retention as an integrated biodiversity conservation approach for continuous-cover forestry in Europe.

Retention forestry implies that biological legacies like dead and living trees are deliberately selected and retained beyond harvesting cycles to benefit biodiversity and ecosystem functioning. This model has been applied for several decades in even-aged, clearcutting (CC) systems but less so in uneven-aged, continuous-cover forestry (CCF). We provide an overview of retention in CCF in temperate regions of Europe, currently largely focused on habitat trees and dead wood. The relevance of current meta-analyses and many other studies on retention in CC is limited since they emphasize larger patches in open surroundings. Therefore, we reflect here on the ecological foundations and socio-economic frameworks of retention approaches in CCF, and highlight several areas with development potential for the future. Conclusions from this perspective paper, based on both research and current practice on several continents, although highlighting Europe, are also relevant to other temperate regions of the world using continuous-cover forest management approaches.

Evolutionary history of burrowing asps (Lamprophiidae: Atractaspidinae) with emphasis on fang evolution and prey selection.

Atractaspidines are poorly studied, fossorial snakes that are found throughout Africa and western Asia, including the Middle East. We employed concatenated gene-tree analyses and divergence dating approaches to investigate evolutionary relationships and biogeographic patterns of atractaspidines with a multi-locus data set consisting of three mitochondrial (16S, cyt b, and ND4) and two nuclear genes (c-mos and RAG1). We sampled 91 individuals from both atractaspidine genera (Atractaspis and Homoroselaps). Additionally, we used ancestral-state reconstructions to investigate fang and diet evolution within Atractaspidinae and its sister lineage (Aparallactinae). Our results indicated that current classification of atractaspidines underestimates diversity within the group. Diversification occurred predominantly between the Miocene and Pliocene. Ancestral-state reconstructions suggest that snake dentition in these taxa might be highly plastic within relatively short periods of time to facilitate adaptations to dynamic foraging and life-history strategies.

Divergent trends in functional and phylogenetic structure in reptile communities across Africa.

Despite extensive research on ecological community compositions, general patterns across large-scale environmental gradients have remained unclear. A widely used explanatory model is the stress dominance hypothesis (SDH), predicting that the relative influence of environmental filtering is greater in stressful habitats while competition is more important in benign environments. Here, we test the SDH using African squamates as a model system to facilitate general predictions on community structures amidst changing global environments. For the first time we investigate changes in functional, phylogenetic and species diversity across continental, environmental gradients within a multidimensional, phylogenetically informed approach. Results suggest that phylogenetic patterns of African squamates were likely shaped by clade-specific biogeographic histories, whereas functional structure reflects SDH predictions. We further detected significant structuring at both local and regional spatial scales, emphasizing the impact of regional-historical processes on local assemblages, and the need for broad conceptual frameworks to detect general patterns of community composition.

The next generation of site-based long-term ecological monitoring: Linking essential biodiversity variables and ecosystem integrity.

Global change effects on biodiversity and human wellbeing call for improved long-term environmental data as a basis for science, policy and decision making, including increased interoperability, multifunctionality, and harmonization. Based on the example of two global initiatives, the International Long-Term Ecological Research (ILTER) network and the Group on Earth Observations Biodiversity Observation Network (GEO BON), we propose merging the frameworks behind these initiatives, namely ecosystem integrity and essential biodiversity variables, to serve as an improved guideline for future site-based long-term research and monitoring in terrestrial, freshwater and coastal ecosystems. We derive a list of specific recommendations of what and how to measure at a monitoring site and call for an integration of sites into co-located site networks across individual monitoring initiatives, and centered on ecosystems. This facilitates the generation of linked comprehensive ecosystem monitoring data, supports synergies in the use of costly infrastructures, fosters cross-initiative research and provides a template for collaboration beyond the ILTER and GEO BON communities.

Phylogeny and biogeography of the African burrowing snake subfamily Aparallactinae (Squamata: Lamprophiidae).

Members of the snake subfamily Aparallactinae occur in various habitats throughout sub-Saharan Africa. The monophyly of aparallactine snakes is well established, but relationships within the subfamily are poorly known. We sampled 158 individuals from six of eight aparallactine genera in sub-Saharan Africa. We employed concatenated gene-tree analyses, divergence dating approaches, and ancestral-area reconstructions to infer phylogenies and biogeographic patterns with a multi-locus data set consisting of three mitochondrial (16S, cyt b, and ND4) and two nuclear genes (c-mos and RAG1). As a result, we uncover several cryptic lineages and elevate a lineage of Polemon to full species status. Diversification occurred predominantly during the Miocene, with a few speciation events occurring subsequently in the Pliocene and Pleistocene. Biogeographic analyses suggested that the Zambezian biogeographic region, comprising grasslands and woodlands, facilitated radiations, vicariance, and dispersal for many aparallactines. Moreover, the geographic distributions of many forest species were fragmented during xeric and cooler conditions, which likely led to diversification events. Biogeographic patterns of aparallactine snakes are consistent with previous studies of other sub-Saharan herpetofauna.

Diversity and biogeography of frogs in the genus Amnirana (Anura: Ranidae) across sub-Saharan Africa.

Frogs in the genus Amnirana (family Ranidae) are widely distributed across sub-Saharan Africa and present a model system for exploring the relationship between diversification and geography across the continent. Using multiple loci from the mitochondrial (16S) and nuclear genomes (DISP2, FICD, KIAA2013, REV3L), we generated a strongly supported species-level phylogeny that provides insights into the continental biogeography of African species of Amnirana, which form a monophyletic group within the genus. Species delimitation analyses suggest that there may be as many as seven additional species of Amnirana in Africa. The biogeographic history of Amnirana is marked by several dispersal and vicariance events, including dispersal from the Lower Guinean Forest into the Congo Basin. In addition, phylogeographic patterns within two widespread species, A. albolabris and A. galamensis, reveal undescribed cryptic diversity. Populations assigned to A. albolabris in western Africa are more closely related to A. fonensis and require recognition as a distinct species. Our analyses reveal that the Lower and Upper Guinean Forest regions served as important centers of interspecific and intraspecific diversifications for Amnirana.

Terrestrial reproduction as an adaptation to steep terrain in African toads.

How evolutionary novelties evolve is a major question in evolutionary biology. It is widely accepted that changes in environmental conditions shift the position of selective optima, and advancements in phylogenetic comparative approaches allow the rigorous testing of such correlated transitions. A longstanding question in vertebrate biology has been the evolution of terrestrial life histories in amphibians and here, by investigating African bufonids, we test whether terrestrial modes of reproduction have evolved as adaptations to particular abiotic habitat parameters. We reconstruct and date the most complete species-level molecular phylogeny and estimate ancestral states for reproductive modes. By correlating continuous habitat measurements from remote sensing data and locality records with life-history transitions, we discover that terrestrial modes of reproduction, including viviparity evolved multiple times in this group, most often directly from fully aquatic modes. Terrestrial modes of reproduction are strongly correlated with steep terrain and low availability of accumulated water sources. Evolutionary transitions to terrestrial modes of reproduction occurred synchronously with or after transitions in habitat, and we, therefore, interpret terrestrial breeding as an adaptation to these abiotic conditions, rather than an exaptation that facilitated the colonization of montane habitats.

Leapfrogging into new territory: How Mascarene ridged frogs diversified across Africa and Madagascar to maintain their ecological niche.

The Mascarene ridged frog, Ptychadena mascareniensis, is a species complex that includes numerous lineages occurring mostly in humid savannas and open forests of mainland Africa, Madagascar, the Seychelles, and the Mascarene Islands. Sampling across this broad distribution presents an opportunity to examine the genetic differentiation within this complex and to investigate how the evolution of bioclimatic niches may have shaped current biogeographic patterns. Using model-based phylogenetic methods and molecular-clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the group based on mitochondrial 16S rRNA and cytochrome b (cytb) genes and the nuclear RAG1 gene from 173 individuals. Haplotype networks were reconstructed and species boundaries were investigated using three species-delimitation approaches: Bayesian generalized mixed Yule-coalescent model (bGMYC), the Poisson Tree Process model (PTP) and a cluster algorithm (SpeciesIdentifier). Estimates of similarity in bioclimatic niche were calculated from species-distribution models (maxent) and multivariate statistics (Principal Component Analysis, Discriminant Function Analysis). Ancestral-area reconstructions were performed on the phylogeny using probabilistic approaches implemented in BioGeoBEARS. We detected high levels of genetic differentiation yielding ten distinct lineages or operational taxonomic units, and Central Africa was found to be a diversity hotspot for these frogs. Most speciation events took place throughout the Miocene, including "out-of-Africa" overseas dispersal events to Madagascar in the East and to São Tomé in the West. Bioclimatic niche was remarkably well conserved, with most species tolerating similar temperature and rainfall conditions common to the Central African region. The P. mascareniensis complex provides insights into how bioclimatic niche shaped the current biogeographic patterns with niche conservatism being exhibited by the Central African radiation and niche divergence shaping populations in West Africa and Madagascar. Central Africa, including the Albertine Rift region, has been an important center of diversification for this species complex.

Two new Phrynobatrachus species (Amphibia: Anura: Phrynobatrachidae) from the Republic of the Congo.

We describe two new species of puddle frogs, genus Phrynobatrachus, from the south-western Republic of the Congo. One of them, P. horsti sp. nov., occurs also in neighbouring Gabon and is morphologically most similar to the Cameroonian P. ruthbeateae. It differs from the latter species by smaller males with longer thighs and shanks. The new species comprises various colour morphs but always has less conspicuous black borders between flanks and belly than P. ruthbeateae. The distinct and large black axillary blotch of P. ruthbeateae is either much smaller in P. horsti sp. nov., or broken into numerous irregularly shaped smaller dots. Similarly, a black transversal line at the anterior ventral border of thighs and the black face mask is less distinct and irregularly delimitated in P. horsti sp. nov. when compared to P. ruthbeateae. The mean genetic difference in the sampled region of the 16S rRNA gene between P. horsti sp. nov. and 40 other western African congeners range from 3.66-18.10%. The second new species, P. mayokoensis sp. nov., differs from all other known congeners by the combination of a compact and warty body, the absence of a spiny eyelid tubercle and pedal webbing, a conspicuous black triangle on throat and anterior part of the belly, and a distinct large red blotch on the anterior-proximal surface of the thighs. It exhibited a mean genetic difference in the 16S rRNA to 40 other western African congeners ranging from 1.34-16.98%. The genetically most similar sequence stems from a GenBank entry of a Gabonese frog, determined as P. ogoensis. A comparison of the new species with P. ogoensis syntypes confirmed their specific distinctiveness, most convincingly underlined by the absence of pedal webbing in the new species and the pronounced pedal webbing in P. ogoensis. The GenBank entry thus most likely is based on a misidentification and P. mayokoensis sp. nov. may also occur in neighbouring Gabon. The discovery of the two new frog species is further evidence of the huge gap in our knowledge concerning the species richness in the Guineo-Congolian rainforests.