A new species of Atelopus (Amphibia: Bufonidae) from eastern Panama.

A new species of the genus Atelopus, Atelopus fronterizo sp. nov., from eastern Panama is described herein based on molecular, morphological, and bioacoustic evidence. The new species can be distinguished from its congeners occurring in the region by a combination of the following characters: (1) phalangeal reduction in thumb; (2) SVL (females only) (35.1-50.1; n=13), HW/SVL (0.23-0.34; n=59), EYND/HW (0.27-0.39; n=60), TIBL/SVL (0.41-0.56; n=58), and HAL/SVL (0.22-0.28; n=49); (3) dorsal color pattern with green or yellow background and extensive dark olive blotches forming transversal bands or mottling; (4) advertisement call duration 176-235 ms with 19-34 pulses, average pulse rate 131.69 pulses/s, and dominant frequency 2 422.50-2 606.50 Hz. The new species is nested within the Central American clade of Atelopus. The minimum Kimura-2-parameter (K2P) genetic divergence between Atelopus fronterizo sp. nov. and its most phylogenetically similar congeners ( A. certus and A. glyphus) is >2.6% for 16S and >4.9% for COI (Table 1). The phylogenetic relationship is strongly supported by ultrafast bootstrap values for the maximum-likelihood trees of both genetic markers (16S, 96; COI, 100, Figure 1A). Bayesian analysis of the concatenated sequences resulted in a tree with similar topology and high posterior probability support (0.99; Supplementary Figure S1). In addition, haplotype networks inferred from COI and 16S (Supplementary Figure S2) showed a well-separated clade containing the new species (two for COI, four for 16S). The number of mutational steps between haplotypes for the new species samples is very low (1-4 in 16S; one in COI), and the minimum number of mutational steps from the nearest species is nine for 16S (distance to A. certus) and 28 for COI (distance to A. glyphus).

A reassessment of the diversity of green toads (Bufotes) in the circum-Aegean region.

The dynamic biogeography of glacial refugia may cause complex patterns of genetic admixture between parapatric taxa, which in turn can mislead their systematics, diversity, and distributions. We investigated this issue for green toads (Bufotes) inhabiting the circum-Aegean region, a biodiversity hotspot of the Eastern Mediterranean. A previous phylogeographic study based on mitochondrial and microsatellite loci identified the hybrid zone between the European (viridis) and Anatolian (sitibundus) lineages of B. viridis all over the Balkan Peninsula, but subsequent range-wide genomic analyses (>1000 SNPs) located this transition in Turkey, a thousand kilometers eastwards. In order to clarify the diversity and taxonomy of the circum-Aegean populations, we reconciled these conflicting findings by integrating previous data with pure sitibundus individuals. Our results confirmed that the viridis/sitibundus hybrid zone extends in Western Anatolia, but that southeastern European populations feature cytonuclear discordances and a high and structured microsatellite diversity. This remarkable situation may stem from a massive geographic displacement of the hybrid zone during the last glacial fluctuations, an underappreciated yet seemingly common feature among the herpetofauna of the region. Our study thus contributes to the rising view that mitochondrial DNA can be a poor predictor of current phylogeographic structure, hence the need for genomic data, especially for narrowly distributed taxa. Finally, the analyses unambiguously support the distinction of a micro-endemic clade of green toads unique to some Cyclades islands, for which we provide a formal taxonomic description.

Variation in Bufadienolide Composition of Parotoid Gland Secretion From Three Taxa of Japanese Toads.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Two New Ansonia from Mountains of Borneo (Anura, Bufonidae).

Two lineages of stream toads in the genus Ansonia from Malaysian Borneo have long been suspected to be specifically distinct on the basis of molecular data. We assessed the taxonomic status of these lineages using morphological and additional genetic data. In mtDNA phylogeny, each lineage-one from Bario, Kelabit Highlands of Sarawak, the other from Mt. Mulu of Sarawak and the Crocker Range of Sabah-is separated from other congeners by large genetic distances, comparable with those observed between heterospecific species in the genus. These lineages are also morphologically distinguishable from other species, and are considered to represent valid, independently evolving species. We therefore describe them as A. kelabitensis sp. nov. and A. kanak sp. nov.

Fifteen shades of green: The evolution of Bufotes toads revisited.

The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (<6My), above which species barely admix and eventually evolve different mating calls (6-10My), or can successfully cross-breed through allopolyploidization (>15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians.

To split or not to split? Multilocus phylogeny and molecular species delimitation of southeast Asian toads (family: Bufonidae).

BACKGROUND: Recent studies have demonstrated that Bayesian species delimitation based on the multispecies coalescent model can produce inaccurate results by misinterpreting population splits as species divergences. An approach based on the genealogical divergence index (gdi) was shown to be a viable alternative, especially for delimiting allopatric populations where gene flow is low. We implemented these analyses to assess species boundaries in Southeast Asian toads, a group that is understudied and characterized by numerous unresolved species complexes. RESULTS: Multilocus phylogenetic analyses showed that deep evolutionary relationships including the genera Sigalegalephrynus, Ghatophryne, Parapelophryne, Leptophryne, Pseudobufo, Rentapia, and Phrynoides remain unresolved. Comparison of genetic divergences revealed that intraspecific divergences among allopatric populations of Pelophyrne signata (Borneo vs. Peninsular Malaysia), Ingerophrynus parvus (Peninsular Malaysia vs. Myanmar), and Leptophryne borbonica (Peninsular Malaysia, Java, Borneo, and Sumatra) are consistent with interspecific divergences of other Southeast Asian bufonid taxa. Conversely, interspecific divergences between Pelophryne guentheri/P. api, Ansonia latiffi/A. leptopus, and I. gollum/I. divergens were low (< 3%) and consistent with intraspecific divergences of other closely related taxa. The BPP analysis produced variable results depending on prior settings and priors estimated from empirical data produced the best results that were also congruent with the gdi analysis. CONCLUSIONS: This study showed that the evolutionary history of Southeast Asian toads is difficult to resolve and numerous relationships remain ambiguous. Although some results from the species delimitation analyses were inconclusive, they were nevertheless efficacious at identifying potential new species and taxonomic incompatibilities for future in-depth investigation. We also demonstrated the sensitivity of BPP to different priors and that careful selection priors based on empirical data can greatly improve the analysis. Finally, the gdi can be a robust tool to complement other species delimitation methods.

How stabilizing selection and nongenetic inheritance combine to shape the evolution of phenotypic plasticity.

Relatively little is known about whether and how nongenetic inheritance interacts with selection to impact the evolution of phenotypic plasticity. Here, we empirically evaluated how stabilizing selection and a common form of nongenetic inheritance-maternal environmental effects-jointly influence the evolution of phenotypic plasticity in natural populations of spadefoot toads. We compared populations that previous fieldwork has shown to have evolved conspicuous plasticity in resource-use phenotypes ("resource polyphenism") with those that, owing to stabilizing selection favouring a narrower range of such phenotypes, appear to have lost this plasticity. We show that: (a) this apparent loss of plasticity in nature reflects a condition-dependent maternal effect and not a genetic loss of plasticity, that is "genetic assimilation," and (b) this plasticity is not costly. By shielding noncostly plasticity from selection, nongenetic inheritance generally, and maternal effects specifically, can preclude genetic assimilation from occurring and consequently impede adaptive (genetic) evolution.

Phylogeography of Aegean green toads (Bufo viridis subgroup): continental hybrid swarm vs. insular diversification with discovery of a new island endemic.

BACKGROUND: Debated aspects in speciation research concern the amount of gene flow between incipient species under secondary contact and the modes by which post-zygotic isolation accumulates. Secondary contact zones of allopatric lineages, involving varying levels of divergence, provide natural settings for comparative studies, for which the Aegean (Eastern Mediterranean) geography offers unique scenarios. In Palearctic green toads (Bufo viridis subgroup or Bufotes), Plio-Pleistocene (~ 2.6 Mya) diverged species show a sharp transition without contemporary gene flow, while younger lineages, diverged in the Lower-Pleistocene (~ 1.9 Mya), admix over tens of kilometers. Here, we conducted a fine-scale multilocus phylogeographic analysis of continental and insular green toads from the Aegean, where a third pair of taxa, involving Mid-Pleistocene diverged (~ 1.5 Mya) mitochondrial lineages, earlier tentatively named viridis and variabilis, (co-)occurs. RESULTS: We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids. CONCLUSIONS: The genetic signatures of insular Aegean toads appear governed by bathymetry and Quaternary sea level changes, resulting in long-term isolation (Central Cyclades: Naxos) and recent land-bridges (coastal islands). Conversely, Crete has been isolated since the end of the Messinian salinity crisis (5.3 My) and Cretan populations thus likely result from human-mediated colonization, at least since Antiquity, from Peloponnese and Anatolia. Comparisons of green toad hybrid zones support the idea that post-zygotic hybrid incompatibilities accumulate gradually over the genome. In this radiation, only one million years of divergence separate a scenario of complete reproductive isolation, from a secondary contact resulting in near panmixia.

Two New Species of Ansonia from Thailand (Anura: Bufonidae).

The taxonomic status of two populations of stream toads of the genus Ansonia from Thailand, previously suspected to be specifically distinct on the basis of molecular data, are assessed using morphological and additional genetic data. In mtDNA phylogeny, each of the two Thai lineages, one from Pilok Subdistrict, Kanchanaburi, and another from Phuket Island, are separated from other congenerics from the Thai-Malay Peninsula by large genetic distances, comparable with those observed between other species in the genus. Although each of the two lineages is superficially similar to other species morphologically, they are distinguishable in several morphological traits and are considered to represent valid, independently evolving species. We therefore describe them as A. pilokensis sp. nov. and A. phuketensis sp. nov.

A New Species of Pelophryne from Western Sarawak, Malaysian Borneo (Anura, Bufonidae).

A new small, semi-arboreal toad of the genus Pelophryne is described from western Sarawak, Malaysian Borneo, on the basis of molecular and morphological evidence. Of the two morphotypes recognized in the genus, the new species belongs to the one in which the tips of the fingers are expanded into truncate discs. Among the species in the morphotype, the new species is most similar to P. murudensis, but differs from it by body size, relative hindlimb length, and dorsal coloration. The new species is currently known only from a limited area on Gunung (= Mt.) Penrissen, and future measures of its habitat conservation are necessary.

Hybrid zone formation and contrasting outcomes of secondary contact over transects in common toads.

Much progress in speciation research stems from documenting patterns of morphological and genetic variation in hybrid zones. Contrasting patterns of marker introgression in different sections of the contact can provide valuable insights on the relative importance of various evolutionary mechanisms maintaining species differences in the face of hybridization and gene flow and on hybrid zone temporal and spatial dynamics. We studied species interactions in the common toads Bufo bufo and B. spinosus in France and northwestern Italy using morphological and molecular data from the mitochondrial and nuclear genomes in an extensive survey, including two independent transects west and east of the Alps. At both, we found sharp, coincident and concordant nuclear genetic transitions. However, morphological clines were wider or absent and mtDNA introgression was asymmetric. We discuss alternative, nonexclusive hypotheses about evolutionary processes generating these patterns, including drift, selection, long-distance dispersal and spatial shifts in hybrid zone location and structure. The distribution of intraspecific mtDNA lineages supports a scenario in which B. bufo held a local refugium during the last glacial maximum. Present-day genetic profiles are best explained by an advance of B. spinosus from a nearby Iberian refugium, largely superseding the local B. bufo population, followed by an advance of B. bufo from the Balkans, with prongs north and south of the Alps, driving B. spinosus southwards. A pendulum moving hybrid zone, first northwards and then southwards, explains the wide areas of introgression at either side of the current position of the contact zones.

Phytogeographic patterns and cryptic diversity in an aposematic toad from NW Argentina.

The Yungas Redbelly Toad, Melanophryniscus rubriventris, is patchily distributed in Argentina, confined to the upland portion (1000-2000m above sea level) of the montane forests of northern and central regions of Salta, and in central-eastern and south-eastern Jujuy. This species is known for its striking aposematic color variation across its geographic distribution, and was once treated as a complex of three subspecies based on distinctive color patterns. Here we assess the geographical genetic variation within M. rubriventris and quantify divergence in color and pattern among individuals sampled from Northwestern Argentina. We compare multi-gene phylogeography of M. rubriventris to patterns of dorsal and ventral coloration to test whether evolutionary affinities predict variation in warning color. Our results reveal two well-supported species lineages: one confined to the extreme northern portion of our sampling area, and the other extending over most of the Argentine portion of the species' range, within which there are two populations. However, these well-supported evolutionary relationships do not mirror the marked variation in warning coloration. This discordance between DNA genealogy and warning color variation may reflect selection brought about by differences in local predation pressures, potentially coupled with effects of sexual selection and thermoregulation.

When new human-modified habitats favour the expansion of an amphibian pioneer species: Evolutionary history of the natterjack toad (Bufo calamita) in a coal basin.

Human activities affect microevolutionary dynamics by inducing environmental changes. In particular, land cover conversion and loss of native habitats decrease genetic diversity and jeopardize the adaptive ability of populations. Nonetheless, new anthropogenic habitats can also promote the successful establishment of emblematic pioneer species. We investigated this issue by examining the population genetic features and evolutionary history of the natterjack toad (Bufo [Epidalea] calamita) in northern France, where populations can be found in native coastal habitats and coalfield habitats shaped by European industrial history, along with an additional set of European populations located outside this focal area. We predicted contrasting patterns of genetic structure, with newly settled coalfield populations departing from migration-drift equilibrium. As expected, coalfield populations showed a mosaic of genetically divergent populations with short-range patterns of gene flow, and native coastal populations indicated an equilibrium state with an isolation-by-distance pattern suggestive of postglacial range expansion. However, coalfield populations exhibited (i) high levels of genetic diversity, (ii) no evidence of local inbreeding or reduced effective population size and (iii) multiple maternal mitochondrial lineages, a genetic footprint depicting independent colonization events. Furthermore, approximate Bayesian computations suggested several evolutionary trajectories from ancient isolation in glacial refugia during the Pleistocene, with biogeographical signatures of recent expansion probably confounded by human-mediated mixing of different lineages. From an evolutionary and conservation perspective, this study highlights the ecological value of industrial areas, provided that ongoing regional gene flow is ensured within the existing lineage boundaries.

Phylogenetic relationships of diurnal, phytotelm-breeding Melanophryniscus (Anura: Bufonidae) based on mitogenomic data.

Melanophryniscus is a bufonid frog genus with a broad geographic distribution over southeastern South America. In recent years, several new species of Melanophryniscus have been discovered in southern Brazil showing a distinctive life-history strategy for the genus - breeding in phytotelmata - as well as a strong association with high-altitude regions. In this study, we use mitogenomic data to infer the phylogenetic relationships among diurnal, phytotelm-breeding Melanophryniscus and to determine the timing of their divergence. We obtained the mitochondrial genomes (not including the control region) for eight individuals of Melanophryniscus representing all three described species (M. alipioi, M. milanoi, and M. xanthostomus), as well as some recently-discovered and potentially new species. Gene order was conserved in all species and corresponded to the general order found in bufonids. Although the phylogenetic relationships among the studied species was poorly supported, dating confirmed that they diverged during the Pleistocene, suggesting that phytotelm breeding could have arisen during drier periods in the glacial/interglacial cycles due to a decrease in the availability of permanent streams or ephemeral/temporary streams or ponds in which Melanophryniscus species commonly breed.

Parotoid, radial, and tibial macroglands of the frog Odontophrynus cultripes: Differences and similarities with toads.

Anuran integument is characterized by the presence of glands, some of which are responsible for toxin production. In some species these glands accumulate in parts of the body strategically located against predators, forming structures known as macroglands. This is the case for parotoid macroglands, on the dorsum of the head, tibial macroglands, on the rear limbs, and radial macroglands, on the forelimbs of toads and some other anurans. The toad Rhinella jimi, for example, simultaneously displays all three types of macroglands, which is unusual even among bufonids. Interestingly, considering the phylogenetic distance, the frog Odontophrynus cultripes (Odontophrynidae) also presents these three macroglandular types. In this study we analyze the morphology of O. cultripes macroglands and the chemical composition of their poison using an interdisciplinary approach. In this species, the parotoid, tibial, and radial macroglands consist of aggregates of elongated and juxtaposed poison glands, arranged in a honeycomb style, very similar to that of toads. Comparative analysis of these three macrogland types shows significant differences in both the morphology of secretory granules and biochemical composition. The present work on O. cultripes contributes to the evidence that amphibians, or at least anurans, share a basic design for all cutaneous glandular accumulations. The determinant factor for macroglandular formation may be the selective pressure for defense against predators.

A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae).

The horned toad assemblage, genus Megophrys sensu lato, currently includes three groups previously recognized as the genera Atympanophrys, Xenophrys and Megophrys sensu stricto. The taxonomic status and species composition of the three groups remain controversial due to conflicting phenotypic analyses and insufficient phylogenetic reconstruction; likewise, the position of the monotypic Borneophrys remains uncertain with respect to the horned toads. Further, the diversity of the horned toads remains poorly understood, especially for widespread species. Herein, we evaluate species-level diversity based on 45 of the 57 described species from throughout southern China, Southeast Asia and the Himalayas using Bayesian inference trees and the Generalized Mixed Yule Coalescent (GMYC) approach. We estimate the phylogeny using both mitochondrial and nuclear DNA data. Analyses reveal statistically significant mito-nuclear discordance. All analyses resolve paraphyly for horned toads involving multiple strongly supported clades. These clades correspond with geography. We resurrect the genera Atympanophrys and Xenophrys from the synonymy of Megophrys to eliminate paraphyly of Megophrys s.l. and to account for the morphological, molecular and biogeographic differences among these groups, but we also provide an alternative option. Our study suggests that Borneophrys is junior synonym of Megophrys sensu stricto. We provide an estimation of timeframe for the horned toads. The mitochondrial and nuclear trees indicate the presence of many putative undescribed species. Widespread species, such as Xenophrys major and X. minor, likely have dramatically underestimated diversity. The integration of morphological and molecular evidence can validate this discovery. Montane forest dynamics appear to play a significant role in driving diversification of horned toads.

Comparative study of host response to chytridiomycosis in a susceptible and a resistant toad species.

In the past century, recently emerged infectious diseases have become major drivers of species decline and extinction. The fungal disease chytridiomycosis has devastated many amphibian populations and exacerbated the amphibian conservation crisis. Biologists are beginning to understand what host traits contribute to disease susceptibility, but more work is needed to determine why some species succumb to chytridiomycosis while others do not. We conducted an integrative laboratory experiment to examine how two toad species respond to infection with the pathogen Batrachochytrium dendrobatidis in a controlled environment. We selected two toad species thought to differ in susceptibility - Bufo marinus (an invasive and putatively resistant species) and Bufo boreas (an endangered and putatively susceptible species). We measured infection intensity, body weight, histological changes and genomewide gene expression using a custom assay developed from transcriptome sequencing. Our results confirmed that the two species differ in susceptibility with the more susceptible species, B. boreas, showing higher infection intensities, loss in body weight, more dramatic histological changes and larger perturbations in gene expression. We found key differences in skin expression responses in multiple pathways including upregulation of skin integrity-related genes in the resistant B. marinus. Together, our results show intrinsic differences in host response between related species, which are likely to be important in explaining variation in response to a deadly emerging pathogen in wild populations. Our study also underscores the importance of understanding differences among host species to better predict disease outcomes and reveal generalities about host response to emerging infectious diseases of wildlife.

Development of Nuclear Microsatellite Loci and Mitochondrial Single Nucleotide Polymorphisms for the Natterjack Toad, Bufo (Epidalea) calamita (Bufonidae), Using Next Generation Sequencing and Competitive Allele Specific PCR (KASPar).

Amphibians are undergoing a major decline worldwide and the steady increase in the number of threatened species in this particular taxa highlights the need for conservation genetics studies using high-quality molecular markers. The natterjack toad, Bufo (Epidalea) calamita, is a vulnerable pioneering species confined to specialized habitats in Western Europe. To provide efficient and cost-effective genetic resources for conservation biologists, we developed and characterized 22 new nuclear microsatellite markers using next-generation sequencing. We also used sequence data acquired from Sanger sequencing to develop the first mitochondrial markers for KASPar assay genotyping. Genetic polymorphism was then analyzed for 95 toads sampled from 5 populations in France. For polymorphic microsatellite loci, number of alleles and expected heterozygosity ranged from 2 to 14 and from 0.035 to 0.720, respectively. No significant departures from panmixia were observed (mean multilocus F IS = -0.015) and population differentiation was substantial (mean multilocus F ST = 0.222, P < 0.001). From a set of 18 mitochondrial SNPs located in the 16S and D-loop region, we further developed a fast and cost-effective SNP genotyping method based on competitive allele-specific PCR amplification (KASPar). The combination of allelic states for these mitochondrial DNA SNP markers yielded 10 different haplotypes, ranging from 2 to 5 within populations. Populations were highly differentiated (G ST = 0.407, P < 0.001). These new genetic resources will facilitate future parentage, population genetics and phylogeographical studies and will be useful for both evolutionary and conservation concerns, especially for the set-up of management strategies and the definition of distinct evolutionary significant units.

Designation and description of a neotype of Sclerophrys maculata (Hallowell, 1854), and reinstatement of S. pusilla (Mertens, 1937) (Amphibia: Anura: Bufonidae).

Molecular analysis indicates that African material previously referred to Amietophrynus maculatus (Hallowell, 1854; now Sclerophrys maculata), is divisible into two distinct clades: a Western Clade from Cameroon westwards and an Eastern Clade from Central African Republic eastwards, and Uganda southwards to South Africa, extending to Angola-Namibia. Preliminary morphological and bioacoustic data support this division. The two clades are recognised here as two separate species. The Western species retains the name S. maculata, with Hallowell's designated type locality of Liberia. The Eastern Clade retains the name published by Mertens (1937), S. pusilla. It is noted that a type specimen of S. maculata cannot be traced and is presumed lost; the so-called syntypes in the Philadelphia Academy of Natural Sciences are not the material described by Hallowell. None of these have been designated as a neotype, consequently a specimen from Liberia in the collection of the Natural History Museum, London, is designated here as the neotype of S. maculata.

The phylogenetic relationships of a new Stream Toad of the genus Ansonia Stoliczka, 1870 (Anura: Bufonidae) from a montane region in Peninsular Malaysia.

Twelve species of Ansonia occur on the Thai-Malay peninsula, of which, five from Peninsular Malaysia, form a monophyletic group. One of these, A. jeetsukumarani, is endemic to the Titiwangsa Mountain Range, in which, we discovered a new population of Ansonia that is not A. jeetsukumarani or even its closest relative. Based on morphology, color pattern, and molecular phylogenetic analyses using the mitochondrial genes 12s and 16s rRNA, we have determined that this new species, A. smeagol sp. nov., forms the sister lineage to an upland, monophyletic group composed of A. jeetsukumarani, A. lumut, A. malayana, and A. penangensis. We have noted similar biogeographic patterns in other taxa from the Titiwangsa Mountain Range in a number of upland lineages in Peninsular Malaysia. We hypothesize that the phylogeographic structure of these upland populations is a result of stochastic processes stemming from interaction of climate-driven forest dynamics and life histories.