Green algae usually assigned to the genus Oophila are known to colonize egg capsules of amphibian egg masses across the Nearctic and Palearctic regions. We study the phylogenetic relationships of these algae using a phylotranscriptomic data set of 76 protein-coding single-copy nuclear genes. Our data set includes novel RNAseq data for six amphibian-associated and five free-living green algae, and draft genomes of two of the latter. Within the Oophila clade (nested within Moewusinia), we find samples from two European frogs (Rana dalmatina and R. temporaria) closely related to those of the North American frog R. aurora (Oophila subclade III). An isolate from the North American R. sylvatica (subclade IV) appears to be sister to the Japanese isolate from the salamander Hynobius nigrescens (subclade J1), and subclade I algae from Ambystoma maculatum are sister to all other lineages in the Oophila clade. Two free-living algae (Chlamydomonas nasuta and Cd. pseudogloeogama) are nested within the Oophila clade, and a strain of the type species of Chlorococcum (Cc. infusionum) is related to this assemblage. Our phylotranscriptomic tree suggests that recognition of different species within the Oophila clade (\"clade B\" of earlier studies) is warranted, and calls for a comprehensive taxonomic revision of Moewusinia.

The chytrid Batrachochytrium dendrobatidis (Bd) is a widespread fungus causing amphibian declines across the globe. Although data on Bd occurrence in Eastern Europe are scarce, a recent species distribution model (SDM) for Bd reported that western and north-western parts of Ukraine are highly suitable to the pathogen. We verified the SDM-predicted range of Bd in Ukraine by sampling amphibians across the country and screening for Bd using qPCR. A total of 446 amphibian samples (tissue and skin swabs) from 11 species were collected from 36 localities. We obtained qPCR-positive results for 33 samples including waterfrogs (Pelophylax esculentus complex) and fire- and yellow-bellied toads (Bombina spp.) from 8 localities. We found that Bd-positive localities had significantly higher predicted Bd habitat suitability than sites that were pathogen-free. Amplification and sequencing of the internal transcribed spacer (ITS) region of samples with the highest Bd load revealed matches with ITS haplotypes of the globally distributed BdGPL strain, and a single case of the BdASIA-2/BdBRAZIL haplotype. We found that Bd was non-randomly distributed across Ukraine, with infections present in the western and north-central forested peripheries of the country with a relatively cool, moist climate. On the other hand, our results suggest that Bd is absent or present in low abundance in the more continental central, southern and eastern regions of Ukraine, corroborating the model-predicted distribution of chytrid fungus. These areas could potentially serve as climatic refugia for Bd-susceptible amphibian hosts.

There is an increased interest in the evolution and development of newts from the genus Triturus because: (1) morphological differentiation among the nine constituent species largely corresponds to different ecological preferences, (2) hybridization between different species pairs has various evolutionary outcomes in terms of life history traits and morphology, and (3) the genus expresses a balanced lethal system that causes arrested growth and death of half of the embryos. These features provide natural experimental settings for molecular, morphological, and life-history studies. Therefore, we produce a staging table for the Balkan crested newt (T. ivanbureschi). We provide detailed descriptions of 34 embryonic stages based on easily observable and interpretable external morphological characters, to ensure reproducibility. Compared with previous staging tables for Triturus, we include a vastly increased sample size and provide high-resolution photographs in lateral, ventral, and dorsal view, complemented by videos of specific developmental periods, and accompanied by detailed explanations on how to delineate the specific stages. Our staging table will serve as a baseline in comparative studies on Triturus newts: an emerging model system in evolutionary and developmental studies.

Acanthocephalans, in their adult stage, are obligatory parasites of many types of vertebrates, including anuran amphibians. Their complex life cycle always involves an arthropod intermediate host but may include non-obligatory strategies that could improve transmission success, such as paratenic infections. In paratenic hosts, these parasites are normally found loose in the body cavity or encysted in internal organs. Here, we present the first report of acanthocephalans found encysted under the skin of an amphibian (i.e., external to its body cavity). The specimen, a clay robber frog [Haddadus binotatus (Spix, 1824)], had been collected in an Atlantic Forest area in southeastern Brazil. Upon examination of the frog, we recovered two specimens of acanthocephalan (Order Echinorhynchida) encysted under the skin of its venter. Considering the host\'s relatively small size and its thin ventral musculature, we believe that the acanthocephalans may have accidentally trespassed the muscular tissue while attempting to encyst in the frog\'s internal body wall.

Nonvisual opsins are transmembrane proteins expressed in the eyes and other tissues of many animals. When paired with a light-sensitive chromophore, nonvisual opsins form photopigments involved in various nonvisual, light-detection functions including circadian rhythm regulation, light-seeking behaviors, and seasonal responses. Here, we investigate the molecular evolution of nonvisual opsin genes in anuran amphibians (frogs and toads). We test several evolutionary hypotheses including the predicted loss of nonvisual opsins due to nocturnal ancestry and potential functional differences in nonvisual opsins resulting from environmental light variation across diverse anuran ecologies. Using whole-eye transcriptomes of 81 species, combined with genomes, multitissue transcriptomes, and independently annotated genes from an additional 21 species, we identify which nonvisual opsins are present in anuran genomes and those that are also expressed in the eyes, compare selective constraint among genes, and test for potential adaptive evolution by comparing selection between discrete ecological classes. At the genomic level, we recovered all 18 ancestral vertebrate nonvisual opsins, indicating that anurans demonstrate the lowest documented amount of opsin gene loss among ancestrally nocturnal tetrapods. We consistently found expression of 14 nonvisual opsins in anuran eyes and detected positive selection in a subset of these genes. We also found shifts in selective constraint acting on nonvisual opsins in frogs with differing activity periods, habitats, distributions, life histories, and pupil shapes, which may reflect functional adaptation. Although many nonvisual opsins remain poorly understood, these findings provide insight into the diversity and evolution of these genes across anurans, filling an important gap in our understanding of vertebrate opsins and setting the stage for future research on their functional evolution across taxa.

The amphibian taxon includes three orders that present different morphological characteristics: Anura, Caudata, and Apoda. Their skin has a crucial role: it acts as an immune organ constituting a physical, chemical, immunological, and microbiological barrier to pathogen insult and conducts essential physiological processes. Amphibians have developed specialized features to protect the vulnerable skin barrier, including a glandular network beneath the skin surface that can produce antimicrobial and toxic substances, thus contributing to the defense against pathogens and predators. This study aims to characterize Langerhans cells in the skin of Lithobates catesbeianus (order: Anura; Shaw, 1802), Amphiuma means (order: Caudata; Garden, 1821), and Typhlonectes natans (order: Apoda; Fischer, 1880) with the following antibodies: Langerin/CD207 (c-type lectin), Major Histocompatibility Complex (MHC)II, and Toll-like receptor (TLR)2 (expressed by different types of DCs). Our results showed Langerhans cells positive for Langerin CD/207 in the epidermis of the three species; moreover, some antigen-presenting cells (APCs) in the connective tissue expressed TLR2 and MHCII. The distribution of the Langerhans cells is very similar in the three amphibians examined, despite their different habitats. A greater knowledge of the amphibian immune system could be useful to better understand the phylogeny of vertebrates and to safeguard amphibians from population declines. Furthermore, the similarities between amphibians\' and human skin concerning immunological features may be useful in both biology and translational medicine.

The species richness in the Neotropics has been linked to environmental heterogeneity and a complex geological history. We evaluated which biogeographic processes were associated with the diversification of Monkey tree frogs, an endemic clade from the Neotropics. We tested two competing hypotheses: the diversification of Phyllomedusinae occurred either in a \"south-north\" or a \"north-south\" direction in the Neotropics. We also hypothesized that marine introgressions and Andean uplift had a crucial role in promoting their diversification. We used 13 molecular markers in a Bayesian analysis to infer phylogenetic relationships among 57 species of Phyllomedusinae and to estimate their divergence times. We estimated ancestral ranges based on 12 biogeographic units considering the landscape modifications of the Neotropical region. We found that the Phyllomedusinae hypothetical ancestor range was probably widespread throughout South America, from Western Amazon to Southern Atlantic Forest, at 29.5 Mya. The Phyllomedusines\' ancestor must have initially diverged through vicariance, generally followed by jump-dispersals and sympatric speciation. Dispersal among areas occurred mostly from Western Amazonia towards Northern Andes and the South American diagonal of dry landscapes, a divergent pattern from both \"south-north\" and \"north-south\" diversification hypotheses. Our results revealed a complex diversification process of Monkey tree frogs, occurring simultaneously with the orogeny of Northern Andes and the South American marine introgressions in the last 30 million years.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

Tonic immobility is an antipredator defence in which the prey animal remains motionless after physical contact with the predator, pretending to be dead. This behaviour has been observed among a variety of taxa but has received only little attention in amphibian larvae. During our field studies with fire salamander larvae, we observed that larvae from different habitats display tonic immobility after handling. In our study site, we find larvae in pond and stream habitats, that differ in several aspects such as their stress response and their risk-taking behaviour, likely caused by the very different habitat conditions. We measured the time that the tonic immobility behaviour was displayed but found no difference between larvae from the two habitat types. Likewise, we also found no correlation between the size of the larvae and the duration of displaying the behaviour. In conclusion, we found that fire salamander larvae show tonic immobility, but found no evidence that the different habitat conditions influence the tonic immobility behaviour.

Advertisement calls in frogs have evolved to be species-specific signals of recognition and are therefore considered an essential component of integrative taxonomic approaches to identify species and delineate their distribution range. The species rich genus Microhyla is a particularly challenging group for species identification, discovery and conservation management due to the small size, conserved morphology and wide distribution of its members, necessitating the need for a thorough description of their vocalization. In this study, we provide quantitative description of the vocal behaviour of Microhyla nilphamariensis, a widely distributed south Asian species, from Delhi, India, based on call recordings of 18 individuals and assessment of 21 call properties. Based on the properties measured acrossed 360 calls, we find that a typical advertisement call of M. nilphamariensis lasts for 393.5 ±  57.5 ms, has 17 pulses on average and produce pulses at rate of 39 pulses/s. The overall call dominant frequency was found to be 2.8 KHz and the call spectrum consisted of two dominant frequency peaks centered at 1.6 KHz and 3.6 KHz, ranging between 1.5-4.1 KHz. Apart from its typical advertisement call, our study also reveals the presence of three \'rare\' call types, previously unreported in this species. We describe variability in call properties and discuss their relation to body size and temperature. We found that overall dominant frequency 1 (spectral property) was found to be correlated with body size, while first pulse period (temporal property) was found to be correlated with temperature. Further, we compare the vocal repertoire of M. nilphamariensis with that of the congener Microhyla ornata from the western coast of India and Sri Lanka and also compare the call properties of these two populations of M. ornata to investigate intra-specific call variation. We find statistically significant differentiation in their acoustic repertoire in both cases. Based on 18 call properties (out of 20), individuals of each locality clearly segregate on PCA factor plane forming separate groups. Discriminant function analysis (DFA) using PCA factors shows 100% classification success with individuals of each locality getting classified to a discrete group. This confirms significant acoustic differentiation between these species as well as between geographically distant conspecifics. The data generated in this study will be useful for comparative bioacoustic analysis of Microhyla species and can be utilized to monitor populations and devise conservation management plan for threatened species in this group.