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Morphological analysis of a polymelia case in Rhinella dorbignyi (Anura: Bufonidae). This note describes the morphology of a supernumerary left hind limb in an adult female of Rhinella dorbignyi found in Atalaya (Magdalena, Buenos Aires). The case was studied through radiographs of the specimen in life, and once dead, the morphological approach included dissection to observe the appendicular muscles and diaphanization with double staining to observe the skeleton. The notable features of the supernumerary limb are: (1) absence of finger I, (2) shorter length of all portions of the limb and less robustness than in a normal hind limb, (3) anomalous junction with the pelvic girdle by means of a well marked femoral head with limb-type features associated to bipedal gait, (4) femur without muscles, and (5) tibia and fibula completely fused in midline. In short, the supernumerary limb is a reduced duplication of the left hind limb. [ABSTRACT FROM AUTHOR]

Simple Summary: At present, there are two hypotheses about the biogeographic origin of Kurixalus on the East Asian islands. We reconstructed the ancestral distribution of Kurixalus, based on complete sampling and accurate selection of biogeographical analysis models. The results showed that Kurixalus on the East Asian islands have originated from the Asian mainland through two long-distance colonization events (jump dispersal). In addition, the analyses of the tempo of diversification revealed that the diversification rate of Kurixalus showed a slight decreasing trend. The relevant results will help us to comprehensively and accurately understand the geographical origin of Kurixalus and improve our understanding of the origin history of the flora and fauna of Taiwan Island. The ancestral area of Kurixalus on the East Asian islands is under dispute, and two hypotheses exist, namely that distribution occurred only on the Asian mainland (scenario of dispersal) and that wide distribution occurred on both the Asian mainland and the East Asian islands (scenario of vicariance). In this study, we conducted biogeographic analyses and estimated the lineage divergence times based on the most complete sampling of species, to achieve a more comprehensive understanding on the origin of Kurixalus on the East Asian islands. Our results revealed that the process of jump dispersal (founder-event speciation) is the crucial process, resulting in the distribution of Kurixalus on the East Asian islands, and supported the model of the Asian mainland origin: that Kurixalus on the East Asian islands originated from the Asian mainland through two long-distance colonization events (jump dispersal), via the model of vicariance of a widespread ancestor on both the Asian mainland and the East Asian islands. Our results indicated that choices of historical biogeography models can have large impacts on biogeographic inference, and the procedure of model selection is very important in biogeographic analysis. The diversification rate of Kurixaus has slightly decreased over time, although the constant-rate model cannot be rejected. [ABSTRACT FROM AUTHOR]

Simple Summary: Recurrent successful breeding of the critically endangered Vanishing Rainfrog (Craugastor evanesco) in captivity has been challenging. Therefore, the objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this species in the near future. The Gonadotropin-Releasing Hormone agonist, at a concentration of 4 micrograms per gram of body weight, stimulated the production of high-quantity and quality spermatozoa. This study also described the morphology of C. evanesco spermatozoa for the first time. These results contributed new and valuable knowledge on the reproductive biology of C. evanesco and for the development of other assisted reproductive technologies that might be applied to this and other species' conservation programs. The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9—GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane. [ABSTRACT FROM AUTHOR]

Boana caiapo is a hylid treefrog with occurrence in the states of Goiás, Mato Grosso, Pará, and Tocantins. We provide a new record of the occurrence of B. caiapo in the state of Mato Grosso, Brazilian Cerrado (Neotropical Savana). The record extends its known distribution approximately 330 km Northwest of the type locality (Aragarças municipality, Goiás) and 263 km Northwest of the nearest known locality (Cocalinho municipality, Mato Grosso state). All localities belong to the Tocantins-Araguaia River basin, reinforcing the restricted distribution of this species. Studies related to new records of species are important to direct conservation proposals. [ABSTRACT FROM AUTHOR]

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Evaluation of changes in the leukogram of adult marsh frogs Pelophylax ridibundus inhabiting the polluted sedimentation lake of the Brikel Thermal Power Station (TPS), southern Bulgaria, as biomarkers for ecological stress is presented. For a comparison, data for frogs inhabiting a less disrupted habitat (reference site, RS) are presented. Leukopenia in the blood of individuals of P. ridibundus inhabiting the sedimentation lake of the Brikel TPS was detected but not in frogs from the reference site. Furthermore, the differential leukocyte count of the affected frogs was characterised by neutrophilia, eosinophilia, monocytosis, basopenia and lymphopenia. The values of the neutrophil-lymphocyte (N/L) ratio of the frogs from the lake of the Brikel TPS were higher than that of the ones inhabiting the RS. These changes in the leukogram are indicators of the exhaustion of the immune system and of activation of the organism's defences in response to stress. The changes in the leukogram are interpreted as caused by the deteriorated water quality in the sedimentation lake as it contained toxicants (reactive water-soluble forms of nitrogen and phosphates) and industrial ash. The results from this study confirm the role of parameter changes in the leukogram of P. ridibundus as diagnostic biomarkers for ecological stress. [ABSTRACT FROM AUTHOR]

The majority of the amphibian larvae spend their early life stage in aquatic medium. In such system, they respond to a broad array of stimuli (tactile, chemical and visual cues) and exhibit suitable behavioural responses. The ability of detection of food and predator was studied in the tadpoles of the Indian skipper frog Euphlyctis cyanophlyctis in the laboratory setup by using a rectangular glass test tank with end compartments (stimulus zones) providing exclusively visual and and (or) chemical food (either boiled spinach or tadpoles of Duttaphrynus melanostictus) and predator (tadpoles of Hoplobatrachus tigerinus). A test tadpole E. cyanophlyctis was held at the centre of the test tank for 5 min (acclimation) to perceive visual and (or) chemical food or predator cues. Then it was released from the centre of the test tank and allowed to associate or stay away from the caged stimulus subjects (food or predator), which were placed at one end of the test tank either in a glass beaker (visual cues) or in a mesh cage wrapped with cheese cloth (chemical cues) for a period of 10 min. The test tadpoles failed to detect either food or predator through visual cues. They spent almost an equal amount of time in the zone housing food or predator in a glass beaker as compared to the opposite zone, which was kept empty. However, they detected both food and predator solely through chemical cues. In tests with chemical food cues (either boiled spinach or tadpoles of D. melanostictus), they spent the majority of their time (67.25 % of total time) near chemical food cues rather than empty or visual food cues. In contrast, in tests with chemical predator cues (tadpoles of H. tigerinus), they spent the majority of their time (68.89 % of total time) away from the chemical predator cues. Therefore, the findings show that E. cyanophlyctis tadpoles detect both food and predator through chemical sensory mechanism rather than visual ones. [ABSTRACT FROM AUTHOR]

Studying intraspecific geographic variation in communication signals represents a good approach to understand the evolution of species diversification in animals. The poison frog Andinobates bombetes (Dendrobatidae) exhibits intraspecific geographic variation in the spectral traits of its advertisement call. However, it is still unknown whether there is concomitant variation in temporal call traits, and whether call variation is somehow linked to differences in warning coloration (red and yellow morphs). We analyzed spectral and temporal traits of the advertisement calls recorded from 100 males belonging to twelve populations of A. bombetes: nine bearing the red coloration, and three bearing the yellow one. After controlling for eventual effects of temperature and body size of males in call traits, we found geographic variation in both spectral and temporal call traits, yet such variation was unrelated to differences in warning coloration between populations. Previous research indicated that geographic variation in the spectral properties of this species advertisement call can be explained by concomitant differences in the acoustic abiotic environment. Nonetheless, a positive relationship between geographic distance and call divergence also supports the role of genetic drift in the evolution of acoustic signaling of A. bombetes. [ABSTRACT FROM AUTHOR]

We describe the call of Barbourula busuangensis, a peculiar and elusive fully aquatic frog endemic to the Palawan archipelago of western Philippines. This species is so poorly known that most of its basic natural history characteristics (reproductive mode and seasonality, diet, age structure, lifespan) remain to be discovered. Contrary to the calls of other frog species adapted to life in fast-flowing streams, the call of B. busuangensis has a very low fundamental frequency around 250 Hz, with an average duration of 1.2 seconds. Additionally, its pulsed, amplitude modulated structure differs from the tonal calls of all known species in its most closely-related (sister) clade, the genus Bombina. [ABSTRACT FROM AUTHOR]

Anurans communicate using different modes, such as acoustic, visual, and chemical signaling, which can be displayed independently or in a multimodal context. While knowledge of anuran acoustic communication has advanced substantially in recent decades, few reports have shown that amphibians communicate using seismic signals. Here we describe the vocal repertoire of the Neotropical marsupial frog, Fritziana tonimi, and report for the first-time a vocal response to a non-vocally induced plant-borne vibration in an anuran. Their advertisement call was emitted in a long call series. Calls had durations of 21-744 ms, were presented in 1-9 notes, had dominant frequencies of 2.24-3.10 kHz, and covered up to seven frequency bands. A single-pulse call resembling a short whistle was frequently emitted in response to a human touching a bromeliad leaf. We hypothesize that if F. tonimi could sense leaf movement produced by a conspecific, this call may be for reproduction or aggressive purposes. Further, this short call could have advantages over the more common long call series in that it could inform conspecifics of the presence of another male in the bromeliad while spending less energy and possibly lowering the risk of attracting predators. Future research could investigate whether other frogs communicate via seismic signals. [ABSTRACT FROM AUTHOR]

Reproduction of the western Amazonian tree frog Nyctimantis rugiceps, a species breeding in bamboo internodes and tree holes, was studied in the field and laboratory. Once a male had attracted a female, the pair attached 206-553 (mean = 357.4) eggs to the phytotelme wall at water level. Subsequently, the male left the site, while the female regularly returned to the hatched larvae after 2-9 (mean = 5.1) days and deposited 211-878 (mean = 446.6) unfertilized nutritive eggs that the larvae bit open and consumed when feeding for the first time, and subsequently swallowed wholly. Time to metamorphosis depended on the number of tadpoles present, but the numbers that hatched and reached metamorphosis were low (2-15 larvae). A possible case of egg parasitism by a Ranitomeya variabilis larva was observed. Skin secretions of a dying frog were probably a toxin known to be harmful even to humans. Some morphological features of N. rugiceps are discussed, especially with respect to breeding in phytotelmes and oophagy. [ABSTRACT FROM AUTHOR]

Harlequin Toads (Bufonidae: Atelopus) are among the most diverse Neotropical amphibians, but 84% of the known species are threatened with extinction mainly due to the fungal disease chytridiomycosis. With no effective tools to mitigate chytridiomycosis in the wild being available, captive breeding is currently the only option to ensure the long-term survival of many Harlequin Toad species. However, the life history and ecology of most species is still largely unknown. Using spatially explicit capture-recapture data from 2005-2013, we studied breeding habitat use and reproductive phenology of the critically endangered Rancho Grande Harlequin Toad, Atelopus cruciger. This species is a seasonal breeder that reproduces exclusively during the dry season. Adult males and females, but also juveniles, migrate from the forest to the river at the beginning of the dry season and return to the forest as soon as the rainy season begins, although a few adult males may remain on the riverbank throughout the year. Despite these seasonal displacements, the toads return to the same locations on the riverbank every year. Both female and male adult toads exhibit a strong breeding site fidelity, occupying small areas (= 5 m2) along the river margins. Adults were mainly active on rocky substrate and vegetation during the day, with males occupying significantly more elevated spots closer to the stream than females. Male territories tend to overlap, but we observed no aggressive or territorial defence behaviour. We discuss the implications of habitat use and high site fidelity for in situ and ex situ conservation measures of this critically endangered toad. [ABSTRACT FROM AUTHOR]

The Ethiopian Highlands are considered a biodiversity hotspot, harboring a high number of endemic species. Some of the endemic species probably diversified in situ; this is, for example, the case of a monophyletic clade containing 12 known species of grass frogs of the genus Ptychadena. The different species occur at elevations ranging from 1,500 to above 3,400 m and constitute excellent models to study the process of diversification in the highlands as well as adaptations to high elevations. In this study, we sampled 294 specimens across the distribution of this clade and used complete mitogenomes and genome-wide SNP data to better understand how landscape features influenced the population structure and dispersal of these grass frogs across time and space. Using phylogenetic inference, population structure analyses, and biogeographic reconstructions, we found that the species complex probably first diversified on the south-east side of the Great Rift Valley. Later on, species dispersed to the north-west side, where more recent diversification occurred. We further demonstrate that Ptychadena species have dispersed across the Great Rift Valley at different times. Our analyses allowed for a more complete understanding of the contribution of geological events, biogeographic barriers and climatic changes as drivers of species diversification and adaptation in this important biogeographic region. [ABSTRACT FROM AUTHOR]

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin. [ABSTRACT FROM AUTHOR]

During the breeding season, hundreds of anurans agglomerate at spawning sites, where a diversity of social contexts arise. In this scenario, anurans may alter their vocal repertoire according to the immediate social context, to overcome intraspecific competition. To evaluate the acoustic response of Boana goiana to successive interactions with a conspecific competitor, the present study was based on comparing the repetition rates of advertisement and aggressive calls of B. goiana focal males exposure to repeated artificial advertisement calls in 10 playback sessions. We observed that B. goiana males altered their acoustic behaviour in response to the simulated arrival of a new competitor. Males emitted less advertisement calls and more aggressive calls with the arrival of the competitors. Temperature and body condition were not important predictors of continuity of males in the experiment or the call repetition rates in the pre-playback session. However, the body condition influenced the total delta advertisement calls (differences in the call rate emission between end and the beginning of the experiment), indicating that higher values of body conditions allow males to show smaller reductions in the emission of advertisement calls over time in the presence of a competitor. [ABSTRACT FROM AUTHOR]

Recent molecular analyses cast doubt on the subspecific relationships of Japanese brown frogs Rana tagoi tagoi, R. t. okiensis, and R. t. yakushimensis. Many samples of R. tagoi from three main islands, i.e., Honshu, Shikoku, and Kyushu tend to form a genetic unit with rich variation. On the other hand, R. t. okiensis, R. t. yakushimensis, and some insular samples are clearly isolated from R. t. tagoi and each other. Thus, we propose to treat each subspecies of R. tagoi as full species and describe part of the Goto Islands populations as Rana matsuoi sp. nov. The new species and R. tagoi show a complicated distributional pattern within the Goto Islands but the former is distinguishable from the latter by their smaller male body size, relatively longer limbs, more developed toe webs, nuptial pad formation in breeding male, and advertisement call with repetition of short notes. [ABSTRACT FROM AUTHOR]

The Miyako toad, Bufo gargarizans miyakonis, is a subspecies endemic to the Miyako Islands of the Ryukyu Archipelago. The distribution of B. g. miyakonis is curious since no toads are found on other islands in the remaining part of the Ryukyu Archipelago. In this study, we conducted phylogenetic estimation and population genetic analyses using sequence data of the mitochondrial control region to clarify the phylogenetic position of B. g. miyakonis in the B. gargarizans species group and its population history. Results of phylogenetic analyses suggested that B. g. miyakonis was closely related to eastern populations of B. gargarizans and that there is incomplete lineage sorting among these populations. The divergence time between B. g. miyakonis and its closest lineages was estimated to be 0.54–0.75 Ma, the Middle Pleistocene, which was a little younger than the estimated value provided by the previous study. It is probable that the migration of the ancestor of B. g. miyakonis was strongly affected by the Middle Pleistocene glacial cycle. Population genetics analyses suggested that this subspecies experienced a rapid population size expansion, and that there is moderate genetic differentiation of sub-populations. Although the B. g. miyakonis population has a relatively high-level genetic diversity, it is also unique, having a significant number of accumulated mutations following rapid demographic expansion. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the diet of Rhinella marina by comparing the availability of food in its environment with its actual diet, and verify whether there is a relationship between the size and volume of its prey and the size of the toad and the width of its mouth. A total of 43 toads (95.55%) presented food items in the stomach. In general, R. marina feeds on the most abundant items in the environment with a niche amplitude of B=0.055. Hymenoptera was the most representative prey numerically. On the other hand, Diplopoda was the most representative prey in volume and frequency. Considering the food importance index (Ix), Diplopoda (Ix=0.47) was the most important part of the diet of R. marina. The relationships between the biometric characteristics of R. marina and its prey are significant with a low coefficient of determination. This result demonstrates that larger individuals predate smaller and lower volume prey, such as ants, and on large prey such as Diplopoda. In general, the diet of R. marina is specialized and is composed of arthropods that occur in greater abundance in nature, but mainly of Diplopoda and Hymenoptera, without selectivity regarding the size of the prey. [ABSTRACT FROM AUTHOR]

Acoustic communication is vital to the survival and fitness of many vertebrates. While successful communication is reliant on acoustic signals reaching an intended receiver and maintaining content fidelity, propagating signals are attenuated by environmental factors such as habitat type. The acoustic adaptation hypothesis (AAH) predicts that the acoustic properties of signals are selected for optimal transmission in the habitats in which they propagate. To date, consistent evidence to support the theory is lacking, but studies have typically been primarily based on localized datasets with small sample sizes. Citizen science is emerging as a potential means to overcome the challenges associated with broad‐scale sampling, allowing us to assess the validity of the AAH in frogs on a continental scale. We analysed the male advertisement calls of 674 individual banjo frogs (Limnodynastes dorsalis, L. dumerilii, L. interioris and L. terraereginae) from across their range, pairing these citizen science data with remotely sensed measures of habitat complexity. While we detected considerable variation in acoustic structure within and among banjo frog species, this was not strongly correlated with habitat complexity. Thus, we found no support for the AAH. However, with our study spanning an area of over 1.7 million km2, we conducted the largest‐scale test of the AAH in frogs to date, contributing to the global understanding of acoustic signal evolution. [ABSTRACT FROM AUTHOR]

The study of the peculiarities of energy metabolism in populations of the marsh frog Pelophylax ridibundus from several reservoirs of the Kamchatka Peninsula with different temperature regimes revealed the temperature compensation of this parameter in tadpoles. The linear dependence of the tadpoles' metabolic rate on the average annual temperature of habitat was shown. It was found that stable inherited differences in the intensity of tadpoles' metabolism were formed in 30–20 years from the moment of the first introduction of the species into the reservoirs of Kamchatka. An assumption is made about the limit of adaptive diapason of the species for the development stage 39–40 of tadpoles of the marsh frog. [ABSTRACT FROM AUTHOR]

The Andean Cordilleras of Colombia, especially the Cordillera Occidental, are among the areas with the highest diversity of Pristimantis frogs in the world. Within the Cordillera Occidental, the Serranía de los Paraguas is famous for its high diversity of sympatric species of Pristimantis frogs. In this study, we investigated acoustic frequency segregation in five sympatric and syntopic species of the genus Pristimantis inhabiting this Serranía. It is important to point out that three of the five species' calls are described for the first time: Pristimantis brevifrons, P. ptochus, and P. silverstonei. Our results showed that acoustic frequency segregation occurs among these five Pristimantis species. Another notable result was the inverse relationship between dominant frequency and male body size, which was corroborated in all species except in P. alius. Finally, we suggested that for improving our knowledge on species coexistence in Pristimantis, future research should also focus on species history and character evolution, given that the evolutionary history is also fundamental for explaining species coexistence. [ABSTRACT FROM AUTHOR]

Input of different spectral types of photoreceptors in amphibian colour vision has been assessed by studying the behaviour of male toads Bufo bufo and B. gargarizans in a laboratory. This method is based on the males' innate reaction of approaching and clasping any visual object during the breeding season. A pair of colour stimuli made of paper or displayed on a LCD monitor has been selected to match the toad's RGB colour space so that they differ by excitation of only a single spectral class of retinal photoreceptors. With pairwise presentation, the males are found to prefer a more 'bluish', or a less 'reddish', or a less 'greenish' stimulus from each pair. A microspectrophotometry of isolated rod and cone photoreceptors and an observation of behavioural responses in toads have revealed a trichromatic mesopic visual system based on a positive input from blue-sensitive photoreceptors and negative inputs from red-sensitive cones and green-sensitive rods. It is supposed that the observed colour preference and involvement of different chromatic mechanisms determine social interactions of anurans during mate choice. [ABSTRACT FROM AUTHOR]

Among populations of the same species, the diet composition varies depending on the environment, given the variation in diversity and abundance of available prey. Since Thoropa miliaris is a habitat specialist in reproduction, we tested the hypothesis that this specialization is also verified in its diet. We carried out the work in 10 localities in Serra do Mar and Serra da Mantiqueira, south‐eastern Brazil, where we collected frogs and evaluated prey availability. The environment did not provide significant variation in the diet of T. miliaris, and Formicidae was the prey category selectively consumed in greater volume, frequency and abundance in both mountain ranges. Therefore, we can state that T. miliaris has a selective diet on ants. [ABSTRACT FROM AUTHOR]

Repetitive elements have been identified in several amphibian genomes using whole genome sequencing, but few studies have used cytogenetic mapping to visualize these elements in this vertebrate group. Here, we used fluorescence in situ hybridization and genomic data to map the U1 and U2 small nuclear RNAs and histone H3 in six species of African clawed frog (genus Xenopus), including, from subgenus Silurana, the diploid Xenopus tropicalis and its close allotetraploid relative X. calcaratus and, from subgenus Xenopus, the allotetraploid species X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. Results allowed us to qualitatively evaluate the relative roles of polyploidization and divergence in the evolution of repetitive elements because our focal species include allotetraploid species derived from two independent polyploidization events — one that is relatively young that gave rise to X. calcaratus and another that is older that gave rise to the other (older) allotetraploids. Our results demonstrated conserved loci number and position of signals in the species from subgenus Silurana; allotetraploid X. calcaratus has twice as many signals as diploid X. tropicalis. However, the content of repeats varied among the other allotetraploid species. We detected almost same number of signals in X. muelleri as in X. calcaratus and same number of signals in X. pygmaeus, X. allofraseri, X. laevis as in the diploid X. tropicalis. Overall, these results are consistent with the proposal that allopolyploidization duplicated these tandem repeats and that variation in their copy number was accumulated over time through reduction and expansion in a subset of the older allopolyploids. [ABSTRACT FROM AUTHOR]

A solid basis to address the conservation challenges of amphibians requires an increased knowledge on their natural history and biology. Recent data on reproductive modes in amphibians suggest that they are much more complex and variable than previously thought but understudied. However, detailed information on the reproductive history is especially important to fill the current knowledge gaps. Following recent taxonomic changes in Ranitomeya variabilis, information about captive-breeding management, image-based measurements of total length and surface area of the silhouette for individuals from embryonic to metamorphic development, and detailed larval staging for captive-bred specimens are provided from French Guiana. The development of R. variabilis from the stage eight (Gosner 1960) through metamorphosis took 79 to 91 days (n = 6) with a survival rate of 46%. The developmental stages largely matched those of the generalized staging system of Gosner (1960), with differences in the stages when labia and teeth differentiation and atrophy of the oral apparatus occurred. Compared with other studies the total length of R. variabilis tadpoles was greater at given stages than those of R. variabilis from a Peruvian population and those of the sister species, R. amazonica. Other studies concerning growth curves based on surface area data revealed that R. variabilis tadpoles at peak size were larger than those of R. amazonica, R. imitator, R. reticulata, R. sirensis, and R. vanzolini, but smaller than R. benedicta. Our results represent the first embryonic and larval staging for R. variabilis, and detailed information is provided on their initial life phases. These data may facilitate the identification of R. variabilis tadpoles in the wild, as well helping to clarify the biogeographical distribution and taxonomic arrangement of the species. In addition, knowledge is added to the captive-breeding methodology for the species. [ABSTRACT FROM AUTHOR]

A new species of the genus Amolops, Amolops ailao sp. nov., is described from central Yunnan, China. The new species belongs to the A. mantzorum species group. Phylogenetic analyses based on the combination of mitochondrial 16S rRNA, COI, and cytb genes revealed that the new species is the sister taxon to Amolops ottorum with strong support. Genetically, the new species differs from A. ottorum by 5.0% in cytb sequences. Morphologically, the new species can be distinguished from known congeners by the combination of the following characters: true dorsolateral folds absent, but dorsolateral folds formed by series of glands present; circummarginal groove on tip of first finger absent; body size small (males SVL 33.0–35.1 mm and female SVL 41.3 mm); HW/SVL 0.32‒0.35; UEW/SVL 0.08‒0.10; THL/SVL 0.52‒0.56; vomerine teeth absent; interorbital distance narrower than internarial distance; tympanum distinct, less than half eye diameter; supratympanic fold present, indistinct; a pair of large tubercles on sides of cloaca; tibiotarsal articulation reaching beyond anterior corner of eye; and vocal sac absent. The cladogenesis events within the A. mantzorum group rapidly occurred from Pliocene 4.23 Mya to Pleistocene 1.2 Mya, coinciding with the recent intensive uplift of the Qinghai-Tibetan Plateau since the Pliocene. Combining findings in this study with the most recent taxonomic progress, we consider that there are 20 known Amolops species in Yunnan, China, accounting for the highest proportion of amphibian diversity of Yunnan, and five of them belong to the A. mantzorum group. Among different subfauna and water systems in Yunnan, the species diversity of Amolops in northwestern Yunnan and Nu River Basin is highest. [ABSTRACT FROM AUTHOR]

Amphibians exhibit diverse parental care behaviours, which may be performed by the female, male or both parents. In the Neotropical family Leptodactylidae, frogs in the genus Leptodactylus exhibit different parenting behaviours. The repertoire of care behaviours includes egg/nest attendance, nest chamber sealing, tadpole feeding with trophic eggs, and tadpole attendance associated with complex behaviours, such as pumping behaviour and channel digging. Based on the available information, we found that 23.8% of Leptodactylus species are known to exhibit post-fertilization parental care. Future studies should focus on mechanisms involved in parent-offspring communication, including acoustic and chemical signals. Moreover, behaviours such as provisioning with trophic eggs are not well understood and deserves further investigation. Because of these complex parental care behaviours, tadpole schooling, and relative easy observation, frogs in the genus Leptodactylus represent excellent models for studies interested in parent-offspring communication and evolution of parental care. [ABSTRACT FROM AUTHOR]

The Karpathos water frog (Pelophylax cerigensis) is classified as Critically Endangered according to the IUCN Red List and its distribution is restricted on the island of Karpathos, South Aegean Sea. Utilizing a next generation sequencing approach, we obtained whole mitochondrial genomes of P. cerigensis to determine the species phylogenetic position within the genus Pelophylax and to clarify the phylogenetic relationship between the species' population on Karpathos Island and the population of P. cf. bedriagae from the neighboring Rhodes Island. High-throughput sequencing generated mean ± SD = 227 945 ± 18 306 reads averaged per sample. High quality reads were assembled resulting in the complete mitogenome of P. cerigensis of a total size of 17 922 bp. Mitogenome organization was similar to other Pelophylax species, comprising of 13 Protein Coding Genes (PCGs), 22 tRNAs, two rRNAs and one Control Region. Overall mean genetic distance for the 13 PCGs, within the Ranidae family, ranged from 8.01% (COIII) to 11.6% (ATP8), while ratios of synonymous and non-synonymous substitutions were <1 in all the 13 PCGs, indicating purifying selection. Maximum Likelihood and Bayesian Inference analyses based on the concatenated sequences of 13 PCGs and two rRNAs (12S and 16S), showed that individuals from Karpathos and Rhodes form a well-supported monophyletic group with low genetic distance between them (p-distance = 0.1%). Hence, the Rhodes populations seem to belong to P. cerigensis and not to P. bedriagae as formerly thought, highlighting the need for revision of P. cerigensis current conservation status as well as further examination of the P. bedriagae species group. [ABSTRACT FROM AUTHOR]

Geographic peculiarities of population structure and hemicloning reproduction of the water frog Pelophylax esculentus complex of 904 samples within southern East European Plain were analyzed. The proportion of populations of P. ridibundus is 0.667 against those 0.042 of P. lessonae. The RE-type populations are the most common among mixed ones (0.153). The colonies of the two parental species and P. kl. esculentus (REL-type), as well as those of P. lessonae with P. kl. esculentus (LE-type), have the frequencies 0.072 and 0.046, respectively. All-hybrid populations (E-type) are not numerous across the region (0.017). In the populations of Central, Northern and Western Ukraine, the P. lessonae genome is eliminated during gametogenesis within hybrids while in the Lower Dnieper and Lower Danube drainages, genome of P. ridibundus is eliminated. In the Eastern Ukraine populations, hybrids usually produce diploid gametes or haploid gametes with the P. ridibundus genome, less often with the chromosome set of P. lessonae only, and even more rarely a mixture of different types of gametes. The predominance of P. ridibundus in hybrid communities and the elimination of the P. lessonae genome in hybrids inevitably leads to the transformation of hybrid populations into "pure" R-type populations. This circumstance makes hybridization with P. ridibundus a factor in the shrinking of P. lessonae populations. That trend is especially expressed in the Forest steppe zone, where hybridization proceeds most intensively, the P. lessonae genome is eliminated in hybrids, and unstable populations with a numerical superiority of P. ridibundus predominate. [ABSTRACT FROM AUTHOR]

Aim/Background: The present study, examined the impact of excess Vitamin A on feeding and pre-metamorphic tadpoles of Microhyla nilphamariensis to analyse its dose-dependent and stage-specific lethal and teratogenic effects. Materials and Methods: Feeding and pre-metamorphic stage tadpoles were subjected to varying concentrations of commercially available Vitamin A palmitate for different time intervals. The control and treated tadpoles were fixed 5, 10- and 15-Days Post Treatment (dpt) in neutral buffered formalin for morphological analyses, morphometry and histology. Results: In both feeding and pre-metamorphic stages, mortality was high when exposed to higher concentrations and longer duration (48-96 hr). When exposed for 24 hr, the treated tadpoles showed significant differences in morphometry, and abnormalities in the eye and intestine of the feeding stages and eye and kidneys of pre-metamorphic stages. Significantly, most of the treated tadpoles died before attaining metamorphosis and those that survived had a delayed metamorphosis with abnormalities. Conclusion: Vitamin A is lethal to the tadpoles of Microhyla nilphamariensis at high concentrations and at lower concentrations it affects the thyroid hormone function and causes abnormalities of various kinds. [ABSTRACT FROM AUTHOR]

Rana coreana is a brown frog species native to the Korean Peninsula. We characterized the complete mitochondrial genome of the species. The mitochondrial genome sequence of R. coreana is 22,262 bp and comprises 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and two control regions (CRs). The CR duplication and gene organization were identical to those observed in Rana kunyuensis and Rana amurensis. A total of 13 protein-coding genes were used to examine the phylogenetic relationships between this species and the genus Rana. R. coreana living on the Korean Peninsula, formed a cluster with R. kunyuensis and R. amurensis, with R. coreana showing the closest phylogenetic affinity for R. kunyuensis. [ABSTRACT FROM AUTHOR]

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer's disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.

Abstract Background Sex ratios of animal populations are important factors of population demographics. In pond-breeding amphibians, the operational sex ratio (OSR) among the breeding population is usually male-biased. Also, in European green toads (Bufotes viridis), males usually outnumber females at breeding sites, while the sex ratio of the total adult population (ASR) is assumed to be balanced. It has been suggested that sex-specific breeding behavior causes male-predominance at the breeding sites. We used a dataset of 5 years of street patrols to test this hypothesis. For this we analyzed local sex ratios of green toads in terrestrial habitats and at two artificial breeding ponds. We expected temporal and/or spatial changes of local sex ratios which would indicate sex dependent differences in breeding behavior. Results Overall observed ASR among 2111 green toads, counted in the course of street patrols from 2016 to 2020, was slightly male-biased (ASR = 0.56, annual ASRs = 0.49–0.63). Based on the data of 1631 toads (920 males, 711 females) captured within a radius of 300 m around nine main breeding sites, temporal and spatial variations in local ASRs were evaluated. Resulting values were compared to the calculated OSR at two artificial breeding ponds in 2021 (645 adult: 553 males, 92 females). Estimates predict more equally distributed females and males prior to the main breeding season. During breeding season, males predominated at both breeding sites (B1: 0.83, B2: 0.89), whereas females are estimated to outnumber males in terrestrial habitats. Proportions of females highly significantly increased with advancing time of the year and increasing distance to the breeding sites. While males tended to accumulate in proximity to water bodies, females dispersed soon after breeding to more distant areas. Conclusions Observed sex ratios in the studied green toad population changed with time and sampling site, deviating from the population-wide sex ratio. Expanding sampling effort in amphibian conservation assessments in time and space, i.e., outside the main breeding season and away from the breeding sites, would be important to encompass such variations.

Abstract Reproductive biology is an important topic that is well explored in many vertebrates, but information about frogs’ reproductive mechanisms could be improved. Therefore, this review aims to provide organized and specific information on frog reproduction. First, we developed schemes that illustrate the general information regarding reproductive biological mechanisms in frogs in a specific way. Then, we described the physiological, histological, and morphological mechanisms of each organ of the reproductive system of male and female frogs. Finally, this manuscript may contribute to a broader understanding of anuran reproductive biology. Since, understanding frogs’ reproductive system permits one to make a comparison with reproduction with other anurans.

Hyperolid reed frogs are one of the few families of Anurans known to possess glands that emit volatile compounds used in chemical communication. Hyperolius cinnamomeoventris, a model species, possesses a gular gland on its vocal sac that emits chemicals, and sends visual and auditory signals during calling. Previous investigations have shown that the glandular compounds are typically macrocyclic lactones. However, in this work, we show that another major constituent of the male specific gland is (10R,1S,6R,7R,10R)-amorph-4-ene-10β-ol [(1R,4R,4aR,8aS)-4-isopropyl-1,6-dimethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-ol]. This compound was synthesized for the first time and has the opposite configuration to amorph-4-ene-10β-ol known from plants. A short synthesis using an organocatalytic approach through a tandem Mannich/intramolecular Diels–Alder reaction led to a mixture of cadinols, which was used for the assignment of the natural cadinol structures and their stereoisomers.

Abstract Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad (Bufotes viridis) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007–2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent ‘wasteland’. Ecological managing of such landscape could vastly increase urban biodiversity.

The geographic distribution of phenotypic variation among closely related populations is a valuable source of information about the evolutionary processes that generate and maintain biodiversity. Leapfrog distributions, in which phenotypically similar populations are disjunctly distributed and separated by one or more phenotypically distinct populations, represent geographic replicates for the existence of a phenotype, and are therefore especially informative. These geographic patterns have mostly been studied from phylogenetic perspectives to understand how common ancestry and divergent evolution drive their formation. Other processes, such as gene flow between populations, have not received as much attention. Here, we investigate the roles of divergence and gene flow between populations in the origin and maintenance of a leapfrog distribution in Phyllobates poison frogs. We found evidence for high levels of gene flow between neighbouring populations but not over long distances, indicating that gene flow between populations exhibiting the central phenotype may have a homogenizing effect that maintains their similarity, and that introgression between 'leapfroging' taxa has not played a prominent role as a driver of phenotypic diversity in Phyllobates. Although phylogenetic analyses suggest that the leapfrog distribution was formed through independent evolution of the peripheral (i.e. leapfrogging) populations, the elevated levels of gene flow between geographically close populations poise alternative scenarios, such as the history of phenotypic change becoming decoupled from genome-averaged patterns of divergence, which we cannot rule out. These results highlight the importance of incorporating gene flow between populations into the study of geographic variation in phenotypes, both as a driver of phenotypic diversity and as a confounding factor of phylogeographic inferences.

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.

Abstract Reed bamboo is a major ecological and economic resource for many animals, including humans. Nonetheless, the influence of this plant's evolutionary role on the morphology of animal species remains unexplored. Here, we investigated the significance of bamboo habitats as ecological opportunities in shaping the skull morphology of bush frogs (Raorchestes) from the Western Ghats, Peninsular India. We applied a three‐dimensional (3D) geometric morphometric approach to capture the skull shape of 55 species of bush frogs. We visualized the skull shape variables in phylomorphospace with principal component analysis and performed phylogenetic generalized least‐squares analysis to assess the impact of cranial size (evolutionary allometry) and habitat (bamboo or non‐bamboo) on cranial shape. We quantified the morphological disparity between bamboo and non‐bamboo bush frogs' skull shape, and employed RRphylo, a phylogenetic ridge regression method, to access the evolutionary rate and rate shifts of skull shape change. The phylomorphospace delineated bamboo and non‐bamboo bush frogs. While cranial shape exhibited a significant but smaller association with size, its association with habitat type was non‐significant. We detected, however, significant differences in skull shape between the two frog groups, with bamboo frogs showing higher morphological disparity and a remarkable shift in the evolutionary rate of skull shape diversification. These findings underscore the role of reed bamboo in the evolution of skull shape in the radiation of frogs, endemic to the Western Ghats. We demonstrate that the association between the members of two distinct endemic clades (bamboo reeds and bamboo frogs) is the outcome of a deep‐time ecological opportunity that dates back to the Miocene.

We describe three new species of landfrogs, genus Pristimantis, from near the summit of Mt. Kopinang, one of the several high points of the Wokomung Massif, a large horseshoe-shaped tepui (= mesa) in west-central Guyana. Pristimantis koki n. sp. is known from 1,067 to 1,525 m elevation. It is characterized by small-sized adults averaging 12.4 mm SVL (snout-vent length) in males and 18.4 mm in females; a pointed, depressed, elongated snout; lack of an obvious tympanum, vocal slit, or sac; and diagnostically black pigment prominently arranged around the anus fringed by light pigment. When handled, P. koki seems to emit volatile organic compounds and leaves a slightly numbing taste at the base of the human tongue. Pristimantis kopinangae n. sp. is known from three specimens collected at approx. 1,385 m elevation on the Wokomung Massif and two specimens from slightly higher in elevation on Mt. Ayanganna. About the size of most Pristimantis inhabiting the Guyana uplands and highlands (20-30 mm SVL), it is characterized by 2-3 light yellow inguinal flash-mark blotches, short broadly round snout, large eye with a blue iris, white skin of chin and areolate belly with dark brown vermiculations; and absence of a tympanum. Pristimantis kalamandeenae n. sp. is known from three specimens collected on the Wokomung Massif including an amplexing pair at approx. 1,550 m elevation. Similar in size to P. kopinangae, it is characterized by an acuminate snout, black iris, obvious tympanum, and uniform tan pigmentation dorsally after dark that becomes uniformly dark brown in daytime. Phylogenetic results show that P. koki and P. kopinangae are sister species and are members of a larger assemblage of related species endemic to the Pantepui Region within the P. unistrigatus species group. Pristimantis kalamandeenae is not closely related to these species, instead forming a clade with the P. lacrimosus species group. The three new species occur in sympatry with at least five other Pristimantis species on the Wokomung Massif, the greatest known Pristimantis species richness on a single tepui of the Guiana Shield. [ABSTRACT FROM AUTHOR]

Previous molecular analyses of the frog genus Stefania have shown that species boundaries in that group are often difficult to delineate when solely based on morphology. As a consequence, "taxonomically cryptic" species are not uncommon in the genus. Several highland Stefania species remain to be described, some potentially critically endangered due to their highly restricted geographic ranges. One case is the microendemic Stefania population from the summit of Murisipán-tepui, a poorly explored table-top mountain in the Los Testigos Massif, a small tepui mountain range located north to the much larger Chimantá Massif in southern Venezuela. That population, mistaken as S. satelles for two decades, was later reported as Stefania sp. 2 and belongs to the S. ginesi clade. The new species is phylogenetically distinct but phenotypically similar to S. satelles, a taxon restricted to its type-locality, i.e. the summit of Aprada-tepui in Venezuela. The new species is described based on morphology and cranial osteology. Molecular divergences with S. satelles are high (> 8%) in the barcoding fragment of 16S rRNA. Amended definitions for the two other described species in the S. ginesi clade (S. ginesi and S. satelles) are also provided. The new species should be listed as critically endangered according to IUCN criteria. [ABSTRACT FROM AUTHOR]

We describe a new species of terrestrial-breeding frog of the genus Pristimantis from the Peruvian Yungas ecoregion of central Peru, Junin Department. The description is based on the observation of morphological features, color patterns of fourteen specimens, bioacoustic traits of the advertisement calls, and a phylogenetic analysis using a sequence fragment of 16S rRNA. The new species is mainly characterized by having a rounded or truncate snout in dorsal view, presence of dorsolateral folds, tympanic membrane and annulus distinct, absence of flash marks on the hidden surfaces of flanks and hindlimbs, and a small size (maximum SVL 15.6 mm in males and 19.3 mm in females). We provide diagnostic traits to differentiate the new species from phylogenetically close relatives, as well as morphologically similar and sympatric species of Pristimantis. [ABSTRACT FROM AUTHOR]

Simple Summary: Roundworms of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent study revealed that only one species, Oswaldocruzia filiformis, parasitizes anurans, lizards and snakes in European Russia. In this work we studied nematodes of the genus Oswaldocruzia in the European green toads from the Middle Volga region using traditional morphological and molecular genetic methods. Descriptions, original drawings, and data on molecular phylogenetic analysis for studied nematodes were produced. The results of our study showed that green toads are parasitized by two Oswaldocruzia species, Oswaldocruzia ukrainae and O. filiformis. We found broad variability in morphology of the studied nematodes, which can lead to inaccurate identification of closely related species of Oswaldocruzia. The data obtained in our study should contribute to the knowledge of Oswaldocruzia nematodes and host-parasite associations in general. Nematodes of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent molecular analysis of Oswaldocruzia nematodes revealed that only Oswaldocruzia filiformis, which possesses high morphological variability, parasitizes amphibians and reptiles in European Russia. Here we present the study of Oswaldocruzia nematodes from the European green toad Bufotes viridis (Anura, Bufonidae) collected at different localities of the Middle Volga region in 2018–2022. We analyzed the morphological characteristics of the Oswaldocruzia spp. taxonomy together with novel molecular phylogenetic data. The data on phylogenetic analysis (based on partial CoxI mtDNA gene sequences) showed that Bufotes viridis is parasitized by two Oswaldocruzia species, the host-specific parasite Oswaldocruzia ukrainae and species generalist Oswaldocruzia filiformis. Broad morphological variability was revealed in O. ukrainae nematodes both from the same host specimen and from various toad individuals from different localities. Our results highlight the need for further biodiversity research of morphologically similar Oswaldocruzia species from amphibians and reptiles in the Western Palearctic using molecular genetic methods. [ABSTRACT FROM AUTHOR]