From Gondwana to the Yellow Sea, evolutionary diversifications of true toads Bufo sp. in the Eastern Palearctic and a revisit of species boundaries for Asian lineages.

Taxa with vast distribution ranges often display unresolved phylogeographic structures and unclear taxonomic boundaries resulting in hidden diversity. This hypothesis-driven study reveals the evolutionary history of Bufonidae, covering the phylogeographic patterns found in Holarctic bufonids from the West Gondwana to the phylogenetic taxonomy of Asiatic true toads in the Eastern Palearctic. We used an integrative approach relying on fossilized birth-death calibrations, population dynamics, gene-flow, species distribution, and species delimitation modeling to resolve the biogeography of the clade and highlight cryptic lineages. We verified the near-simultaneous Miocene radiations within Western and Eastern Palearctic Bufo, c. 14.49-10.00 Mya, temporally matching with the maximum dust outflows in Central Asian deserts. Contrary to earlier studies, we demonstrated that the combined impacts of long dispersal and ice-age refugia equally contributed to the current genetic structure of Bufo in East Asia. Our findings reveal a climate-driven adaptation in septentrional Eastern Asian Bufo, explaining its range shifts toward northern latitudes. We resolve species boundaries within the Eastern Palearctic Bufo, and redefine the taxonomic and conservation units of the northeastern species: B. sachalinensis and its subspecies.

The east Asian Asiatic toad (also known by the latin name Bufo gargarizans) lives in a wide range of habitats across East Asia including forests, meadows and cultivated land. However, it remains unclear how these toads evolved and became so widespread ­ partly because it has proved difficult for researchers to clearly define the species and what distinguishes it from other closely-related species of toads (collectively known as Bufo toads). Othman et al. combined several bioinformatics techniques to study Asiatic toads and 38 other species of bufonid toads from across the globe. This approach found that Bufo toads first emerged in eastern Asia between 14 and 10 million years ago. This coincides with a point in time when large swathes of land in central Asia turned from adequate to sustain toad populations into desert, suggesting the change in climate prompted the toads to migrate eastwards from central Asia. The Bufo toads then divided into two groups of species: one in mainland East Asia and the other in Japan. Furthermore, the study revealed there is more genetic diversity ­ that is, more variety in the DNA of individuals ­ in Asiatic toads than previously thought. The findings also help to define several other species of Bufo toads more clearly and describe a new toad species restricted to the Korean Peninsula, northeastern China and the Russian Primorye region: the Sakhalin toad (Bufo sachalinensis). This work demonstrates that a large-scale study of many species across the globe can be used to understand how the species evolved and more clearly distinguish one species from another. The findings of Othman et al. will be of interest to both professional and citizen scientists interested in the natural history of Asia. Furthermore, as several species of Bufo toads are in decline in the wild, they provide evidence that may aid future efforts to conserve them.

Efficacy of environmental DNA to detect and quantify stream tadpoles of Odorrana splendida.

Environmental DNA (eDNA) can be used to detect and estimate the density of rare or secretive species, especially in aquatic systems. However, the efficacy of eDNA method has not been validated in lotic systems. We examined the efficacy of the eDNA method to detect and estimate abundance and biomass of a stream-dwelling frog species, Odorrana splendida. We conducted eight field surveys over 2 years and obtained 53 water samples from 10 streams with known distribution of O. splendida tadpoles. The eDNA method accurately detected the presence of O. splendida in 79.2% of survey samples. The amount of O. splendida eDNA (copies s-1) in the water samples fluctuated seasonally and each site showed different peaks during different seasons. The relationship between the abundance or biomass of tadpoles and the amount of eDNA was significantly positive, but was not strong, probably because of a large difference in the relationship patterns among streams. In lotic systems, water flow might prevent even distribution of eDNA and thus make it difficult to obtain eDNA reflecting its total amount in the water. Sampling a larger amount of water or higher number of subsamples might more accurately reflect the presence and absolute amount of eDNA in water.

Spinal arthritis in cane toads across the Australian landscape.

Loss of fitness can be a consequence of selection for rapid dispersal ability in invasive species. Increased prevalence of spinal arthritis may occur in cane toad populations at the invasion front as a cost of increased invasiveness, but our knowledge of the ecological drivers of this condition is lacking. We aimed to determine the factors explaining the prevalence of spinal arthritis in populations across the Australian landscape. We studied populations across a gradient of invasion histories. We collected 2415 toads over five years and determined the presence and size of spondylosis for each individual. We examined the effect of host size, leg length and invasion history on the prevalence of spondylosis. Host size was a significant predictor of spondylosis across populations. Contrary to our expectation, the overall prevalence of spondylosis was not positively related to invasion history and did not correlate with toad relative leg length. Rather than invasion age, the latitude at which populations were sampled provided an alternate explanation for the prevalence of spondylosis in cane toad populations and suggested that the incidence of this condition did not increase as a physiological cost of invasion, but is instead related to physical variables, such as climate.

Rapid differentiation of sexual signals in invasive toads: call variation among populations.

Advertisement calls tend to differ among populations, based on morphological and environmental factors, or simply geographic distance, in many taxa. Invasive cane toads (Rhinella marina) were introduced to Australia in 1935 and their distribution has expanded at increasing rates over time. Rapid evolution occurred in morphological and behavioural characters that accelerate dispersal, but the effects of rapid expansion on sexual signals have not been examined. We collected advertisement calls from four populations of different ages since invasion, and analysed the geographic differentiation of seven call parameters. Our comparisons indicate that the calls of R. marina differ among Australian populations. The signal variation was not simply clinal with respect to population age, climate, or morphological differentiation. We suggest that selection on signalling among populations has been idiosyncratic and may reflect local female preferences or adaptation to environmental factors that are not clinal such as energy availability.

Cell Density Effects of Frog Skin Bacteria on Their Capacity to Inhibit Growth of the Chytrid Fungus, Batrachochytrium dendrobatidis.

Bacterial symbionts on frog skin can reduce the growth of the chytrid fungus Batrachochytrium dendrobatidis (Bd) through production of inhibitory metabolites. Bacteria can be effective at increasing the resistance of amphibians to chytridiomycosis when added to amphibian skin, and isolates can be screened for production of metabolites that inhibit Bd growth in vitro. However, some bacteria use density-dependent mechanism such as quorum sensing to regulate metabolite production. It is therefore important to consider cell density effects when evaluating bacteria as possible candidates for bioaugmentation. The aim of our study was to evaluate how the density of cutaneous bacteria affects their inhibition of Bd growth in vitro. We sampled cutaneous bacteria isolated from three frog species in the tropical rainforests of northern Queensland, Australia, and selected ten isolates that were inhibitory to Bd in standardised pilot trials. We grew each isolate in liquid culture at a range of initial dilutions, sub-sampled each dilution at a series of times during the first 48 h of growth and measured spectrophotometric absorbance values, cell counts and Bd-inhibitory activity of cell-free supernatants at each time point. The challenge assay results clearly demonstrated that the inhibitory effects of most isolates were density dependent, with relatively low variation among isolates in the minimum cell density needed to inhibit Bd growth. We suggest the use of minimum cell densities and fast-growing candidate isolates to maximise bioaugmentation efforts.