For conservation of endangered species, basic ecological information is indispensable. Environmental DNA (eDNA) is increasingly used to estimate the presence/absence of aquatic organisms such as frog species. However, the efficiency of detecting eDNA may depend on the ecological characteristics of the target frog species. Here, we adopted an eDNA approach for detecting 3 endangered Japanese species (Babina subaspera, Odorrana splendida, and O. amamiensis). We compared eDNA detection with a traditional acoustic survey method in 3 different seasons. Detection of species was not always consistent between the 2 methods, probably because acoustic surveys target only calling adult males but eDNA detects individuals of both sexes and at any life-history stage in water. Moreover, the differences in detection patterns between the 2 methods among species may be correlated with different characteristics of mating behavior (e.g., calling volume) and life history of tadpoles (e.g., migration ability). The optimal season for detection by eDNA method also differed between the 3 species, probably because of differences in breeding season and larval characteristics. We show that results obtained from eDNA detection methods and traditional field observation techniques, including acoustic surveys, are not always consistent with each other but rather depend on the ecological characteristics of the respective target species. It is therefore necessary to consider the ecological traits of frog species, such as breeding season and seasonal shifts in tadpole abundance, when applying eDNA methods for distribution surveys.
Taxa with vast distribution ranges often display unresolved phylogeographic structures and unclear taxonomic boundaries resulting into 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 fossilised birth-death calibrations, population dynamic, gene-flow, species distribution and species delimitation modelling 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, explained its range shifts towards 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.
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.
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.
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