The phylogeography of Middle Eastern tree frogs in Israel.

Western Palearctic treefrogs of the genus Hyla provide an example of a morphologically and ecologically cryptic group. Up to three distinct Hyla species have been proposed as resident in Israel and this number has consistently been subject to taxonomical debates. Here, we analyzed 16S rRNA and COI gene fragments of 658 individuals sampled at 47 pools in nine regions across Israel and the West Bank in order to resolve the taxonomic status of Hyla frogs. We generated both Bayesian and Maximum Likelihood phylogenies, and constructed time-calibrated trees to provide an evolutionary and historical context of sequence variations. We further applied SAMOVA as well as Monmonier's maximum-difference algorithm to study the genetic structure among populations and to identify potential zones acting as barriers to gene flow across locations. Our results revealed two distinct haplogroups for each gene fragment, with 95% CI divergence times dated from 8.9-17.1 Mya (16S) and 7.1-23.6 Mya (COI), respectively. SAMOVA and barrier analyses partitioned the populations into three groups. Our results highlight that, while there are probably only two Hyla species in Israel, one population of one of the species might qualify as a separate evolutionarily significant unit. Our findings elucidate the taxonomic status of Hyla frogs in Israel and provide the basis for determining appropriate management and conservation priorities.

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Population genetic analysis of the recently rediscovered Hula painted frog (Latonia nigriventer) reveals high genetic diversity and low inbreeding.

After its recent rediscovery, the Hula painted frog (Latonia nigriventer) has remained one of the world's rarest and least understood amphibian species. Together with its apparently low dispersal capability and highly disturbed niche, the low abundance of this living fossil calls for urgent conservation measures. We used 18 newly developed microsatellite loci and four different models to calculate the effective population size (Ne) of a total of 125 Hula painted frog individuals sampled at a single location. We compare the Ne estimates to the estimates of potentially reproducing adults in this population (Nad) determined through a capture-recapture study on 118 adult Hula painted frogs captured at the same site. Surprisingly, our data suggests that, despite Nad estimates of only ~234-244 and Ne estimates of ~16.6-35.8, the species appears to maintain a very high genetic diversity (HO = 0.771) and low inbreeding coefficient (FIS = -0.018). This puzzling outcome could perhaps be explained by the hypotheses of either genetic rescue from one or more unknown Hula painted frog populations nearby or by recent admixture of genetically divergent subpopulations. Independent of which scenario is correct, the original locations of these populations still remain to be determined.

Living quarters of a living fossil-Uncovering the current distribution pattern of the rediscovered Hula painted frog (Latonia nigriventer) using environmental DNA.

One of the greatest challenges of effective conservation measures is the correct identification of sites where rare and elusive organisms reside. The recently rediscovered Hula painted frog (Latonia nigriventer) has not been seen for many decades and was therefore categorized extinct. Since its rediscovery in 2011, individuals from the critically endangered species have been found, with great effort, only in four restricted sites. We applied the environmental DNA (eDNA) approach to search for new populations of the Hula painted frog in suitable aquatic habitats. We further used the eDNA data to classify the landscape factors associated with the species distribution and to predict its suitable habitats. We sampled 52 aquatic sites in the Hula Valley during the spring of 2015 and 2016 and amplified the samples with a species-specific qPCR assay. DNA of the Hula painted frog was detected in 22 of the sites, all of which clustered within three main areas. A boosting classification model showed that soil type, vegetation cover and the current and former habitats are all key predictors of the frog's current distribution. Intriguingly, the habitat suitability models reveal a high affinity of the species to its long-lost habitat of the historical wetlands. Our findings encourage a series of informed searches for new populations of this threatened frog and provide guidance for future conservation management programmes. In the era of global conservation crisis of amphibians, developing the eDNA approach, a reliable detection method for many critically endangered and elusive amphibians, is of particular importance.

Skin microbiota differs drastically between co-occurring frogs and newts.

Diverse microbial assemblages inhabit amphibian skin and are known to differ among species; however, few studies have analysed these differences in systems that minimize confounding factors, such as season, location or host ecology. We used high-throughput amplicon sequencing to compare cutaneous microbiotas among two ranid frogs (Rana dalmatina, R. temporaria) and four salamandrid newts (Ichthyosaura alpestris, Lissotriton helveticus, L. vulgaris, Triturus cristatus) breeding simultaneously in two ponds near Braunschweig, Germany. We found that bacterial communities differed strongly and consistently between these two distinct amphibian clades. While frogs and newts had similar cutaneous bacterial richness, their bacterial composition strongly differed. Average Jaccard distances between frogs and newts were over 0.5, while between species within these groups distances were only 0.387 and 0.407 for frogs and newts, respectively. At the operational taxonomic unit (OTU) level, 31 taxa exhibited significantly different relative abundances between frogs and newts. This finding suggests that chemical or physical characteristics of these amphibians' mucosal environments provide highly selective conditions for bacterial colonizers. Multi-omics analyses of hosts and their microbiota as well as directed efforts to understand chemical differences in the mucosal environments (e.g. pH), and the specificities of host-produced compounds against potential colonizers will help to better understand this intriguing pattern.

Amphibian skin microbiota exhibits temporal variation in community structure but stability of predicted Bd-inhibitory function.

Host-associated microbiomes are increasingly recognized to contribute to host disease resistance; the temporal dynamics of their community structure and function, however, are poorly understood. We investigated the cutaneous bacterial communities of three newt species, Ichthyosaura alpestris, Lissotriton vulgaris and Triturus cristatus, at approximately weekly intervals for 3 months using 16S ribosomal RNA amplicon sequencing. We hypothesized cutaneous microbiota would vary across time, and that such variation would be linked to changes in predicted fungal-inhibitory function. We observed significant temporal variation within the aquatic phase, and also between aquatic and terrestrial phase newts. By keeping T. cristatus in mesocosms, we demonstrated that structural changes occurred similarly across individuals, highlighting the non-stochastic nature of the bacterial community succession. Temporal changes were mainly associated with fluctuations in relative abundance rather than full turnover of bacterial operational taxonomic units (OTUs). Newt skin microbe fluctuations were not correlated with that of pond microbiota; however, a portion of community variation was explained by environmental temperature. Using a database of amphibian skin bacteria that inhibit the pathogen Batrachochytrium dendrobatidis (Bd), we found that the proportion of reads associated with 'potentially' Bd-inhibitory OTUs did not vary temporally for two of three newt species, suggesting that protective function may be maintained despite temporal variation in community structure.