Major advancements in ecology and biodiversity conservation have been made thanks to methods for marking and individually tracking animals. Marking animals is both widely used and controversial due to the potential consequences for animal welfare, which are often incompletely evaluated prior to implementation. Two outstanding knowledge gaps concerning the welfare consequences of individual marking are their short-term behavioural impacts and the relative impacts from marking versus the handling of animals while carrying out procedures. We addressed these knowledge gaps through an experimental study of alpine newts (Ichthyosaura alpestris) in which we varied handling and marking procedures. Examining individual responses to handling, toe clipping and visible implant elastomer (VIE) injection over 21 days showed that handling and marking elicited increased newt activity and hesitancy to feed compared to animals that did not get handled or marked. These effects were apparent even when animals were handled only (not marked), and marking did not further increase the magnitude of responses. Increases in newt activity and feeding hesitancy were transient; they were not observed in the weeks following handling and marking. While previous studies emphasise the welfare impacts of marking procedures themselves, these findings highlight that handling alone can elicit behavioural changes with possible costs to welfare. Yet, the transient nature of behavioural responses suggests that immediate costs of handling may be subsequently compensated for in the short term.

Edge effects change biodiversity patterns and ecological processes, particularly in tropical forests. To understand the synergistic impact of multiple edges, this study examines how edge influence (EI) is associated with life-history traits (snout-vent length and body temperature), diversity and microhabitat of amphibians as well as habitat characteristics in a tropical forest in Ecuador. We used EI, a metric that calculates cumulative effects across all nearby edges, in combination with five environmental variables that are part of the amphibians’ microhabitat (temperature, humidity, slope, canopy cover and leaf litter depth) to understand how their biodiversity patterns are impacted. Our results show that most amphibian species tend to be habitat specialists, and many had an affinity for forest edges and warmer habitats. We do not find significant correlations between EI and amphibian life-history traits and diversity. Our findings corroborate previous results that many amphibian species tend to be positively associated with habitat fragmentation and show that this association is likely driven by thermal regulation.

Acanthocephalans constitute a small taxonomic group related to rotifers and specialized in a parasitic lifestyle. Anurans act as paratenic and definitive hosts and infections always occur trophically. Our objective is to describe and summarize the richness of acanthocephalans in Neotropical anurans. We conducted a literature review in the main research databases, compiling data published until August 2021. We identified 66 articles with records of acanthocephalan-anuran association, 53.03% were carried out in Brazil. We detected 108 species of anurans from 11 families parasitized by acanthocephalans. With the exception of Bufonidae, Hylidae and Leptodactylidae, which are relatively well-studied families, interaction with acanthocephalans remains largely unexplored for most anuran species. We found six families of acanthocephalans: Centrorhynchidae, Echinorhynchidae, Oligacanthorhynchidae, Cavisomidae, Neoechinorhynchidae and Plagiorhynchidae. Centrorhynchidae and Echinorhynchidae presented the largest number of taxa associated with anurans. The largest number of records corresponded to acanthocephalans in the larval stage (cystacanths), for which anurans act as paratenic hosts. We observed a lack of specific taxonomic resolution in the identifications of most reports, because a large part of the records in the larval stage make morphological identification difficult. Brazil, Mexico, Paraguay, Argentina, Ecuador and Peru are the countries with the most records, while Costa Rica, Venezuela, Colombia, Chile and Uruguay exhibited the lowest publication numbers, resulting in gaps in the distribution of acanthocephalans. We expanded the known number of anuran species parasitized by acanthocephalans, compared to the last published review. Overall, we aim to contribute to the understanding of diversity within this intriguing but understudied group.

Cosmocercoides Wilkie, 1930 are gastrointestinal parasites commonly found in amphibians and reptiles, with 4 species reported from the Neotropical region. In the present study, a new species of Cosmocercoides, namely Cosmocercoides amapari n. sp. is described using integrated approaches such as light and scanning microscopy, and DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. The specimens were collected from the large intestine of 3 species of hylid frogs in Amapá, Brazil. The new species can be distinguished from its congeners by morphological traits, including the pattern of caudal papillae, absence of the gubernaculum, 2 poorly sclerotized spicules, presence of lateral alae and somatic papillae along the body. In addition, our molecular analyses and phylogenetic reconstructions strongly support the status of the new taxon, which clustered poorly with a large clade of Cosmocerca spp. Cosmocercoides amapari n. sp. is the 29th species of the genus, the 5th from the Neotropical region, the third reported in Brazil, the second described from the Amazon region and the first belonging to the Neotropical region with molecular data.

In the present study, we aimed to describe the composition of endoparasites associated with anurans from an altitudinal rainforest enclave in northeastern Brazil. Additionally, we tested if microhabitat use influences endoparasite abundance and richness, as well as the hypothesis that larger frogs tend to be more parasitized. We sampled 306 individuals from 25 anuran species that were necropsied and analysed using a stereomicroscope. The total endoparasite prevalence was 79.08%, with a parasitic community consisting of 46 taxa. Overall, we found the common pattern described for Neotropical amphibians, which is the predominance of generalist and direct-cycle parasites. Twenty new host records and two possible new parasite species were found, highlighting the importance of this type of inventory. We also observed that microhabitat use was associated with a significant difference in parasite richness between groups, in which arboreal and terrestrial species, and aquatic and arboreal species contributed to these differences. Moreover, larger frogs tended to be more parasitized regarding only an interspecific view. Our results suggest that parasite richness is directly related to infection cycle and how the host exploits its habitat.

The intracellular localization of Ca2+, Ca2+-ATPase, Calmodulin, and Calbindin D-28KD have been studied in testes of the toad Leptodactylus chaquensis, using ultracytochemical and immunohistochemical techniques. The Ca2+ presences in the nucleus and into the mitochondria of the germ cells, together with the activity of Ca2+-ATPase detected in the nuclear envelope and mitochondrial crests, suggest the participation of this transporter in the storage of Ca2+. In Sertoli cells, Ca2+ deposits were also found in vesicles and lamellar bodies. Calmodulin and Calbindin D-28KD were revealed in the cytoplasm of both cell types. At the spermatozoon level, the cation deposits were located in the subacrosomal space and in the acrosomal vesicle. Ca2+-ATPase activity was observed in the acrosomal and plasma membranes of the gamete that suggests the existence of a transport system responsible for maintaining low cytoplasmic Ca2+ levels. The activity of Ca2+-ATPase and the location of Ca2+ deposits in gamete tail would be related to flagellar movement. The colocalization of Ca2+ deposits and their binding proteins in efferent duct cells would probably be associated with secretory activity. Considering that intracellular Ca2+ is present in different gonadal cells, this work would provide a better understanding of the cation importance in the testicular functions of this species.

We investigated trophic resource partitioning in seven syntopic anurans from low- and mid-elevation stream habitats of a tropical riparian ecosystem by utilising stomach content analysis (SCA) and stable isotope analysis (SIA). Our SCA data revealed dietary similarities, narrow trophic niche breadth, and low dietary niche overlap in Ansonia muelleri, Limnonectes magnus, Occidozyga laevis, Megophrys stejnegeri, Pulchrana grandocula, Sanguirana mearnsi, and Staurois natator which could be attributed to these anurans’ selection of available local prey items. We confirmed ant-specialisation (myrmecophagy) of the Mindanao island endemic bufonid A. muelleri based on our temporal SCA dietary data. Our SIA estimates of assimilation of potential prey sources confirmed that L. magnus, P. grandocula, and O. laevis are generalist predators, opportunistically feeding on locally abundant insect prey items. This study on trophic resource partitioning in syntopic anurans provides the first picture of trophic interactions, i.e., predation and competition in stream communities in tropical riparian zones of a watershed ecosystem in northeast Mindanao of the southern Philippines.

Paramos are high-elevation tropical Andean ecosystems above the tree line that display variable temperature and frequent freezing spells. Because a significant anuran community lives in this environment, physiological protection against freezing must characterise individuals in this community. Antifreeze protection has been studied in amphibians from other communities, and it is likely that Paramo anurans rely on the same underlying molecules that convey such protection to Nearctic species. However, given the pervasive presence of freezing spells in the Paramos year-round, the processes of activating protection mechanisms may differ from that of seasonal counterparts. Accordingly, this study investigated cryoprotection strategies in high-elevation tropical frogs, using as a model the terrestrial and nocturnal genus Pristimantis, specifically P. bogotensis, P. elegans and P. nervicus from Paramos, and the warm ecosystem counterparts P. insignitus, P. megalops and P. sanctaemartae. We focused on freeze tolerance and its relationship with glucose accumulation and ice formation. Under field conditions, the highest elevation P. nervicus exhibited higher glucose concentration at dawn compared to noon (1.7 ± 0.6 mmol/L versus 3.5 ± 1.32 mmol/L). Under experimental thermal freeze exposure for 2 hours between −2 and −4 ºC, the glucose concentration of the three Paramo species increased but physiological diversity was evident (P. nervicus 126%; P. bogotensis 100%; and P. elegans 55%). During this test, body ice formation was assessed calorimetrically. The species with the highest body ice formation was P. bogotensis (17% ± 5.37; maximum value: 63%; n = 8), followed by P. nervicus (5% ± 3.27; maximum value: 11%; n = 5) and P. elegans (0.34% ± 0.09; maximum value: 1%; n = 4). The study shows physiological diversity both within a genus and across the amphibian community around the freezing contour. Overall, Paramo species differ in freezing physiology from their low-elevation counterparts. Thus, climate shifts increasing freezing spells may affect the structure of communities in this zone.

Natural and anthropogenic stressors, including parasites and pesticides, may induce oxidative stress in animals. Measuring oxidative stress responses in sentinel species that are particularly responsive to environmental perturbations not only provides insight into host physiology but is also a useful readout of ecosystem health. Newly metamorphosed northern leopard frogs (Lithobates pipiens), a sentinel species, were collected from agricultural and non-agricultural wetlands exposed to varying concentrations of the herbicide atrazine. Significant effects of certain parasites' abundance and their interaction with atrazine exposure on frog oxidative stress were identified. Specifically, increased protein levels were detected in frogs infected with echinostome metacercariae. In addition, the nematode Oswaldocruzia sp. was significantly associated with increased thiol concentration and catalase activity. Significant parasite × atrazine interactions were observed for atrazine exposure and the abundance of Oswaldocruzia sp. on thiol, as thiol concentrations increased with parasite abundance at low atrazine localities and decreased in high atrazine wetlands. In addition, a significant interaction between the abundances of Oswaldocruzia sp. and gorgoderid trematodes on thiol concentrations was observed. These findings demonstrate that studies of oxidative stress on animals in natural ecosystems should account for the confounding effects of parasitism, particularly for amphibians in agricultural landscapes.

Degradation and habitat loss of natural grasslands in Southern Brazil has a negative impact on native organisms, potentially including the composition of anuran helminth communities. Here, we characterized the richness, abundance, taxonomic composition, prevalence and intensity of helminth infection in four anuran species. Host anurans were collected in 34 ponds (19 in native grasslands with livestock and 15 in agricultural cultivation) from the highland grasslands in the Brazilian states of Santa Catarina and Paraná. Our results showed a significant difference between native grasslands with livestock and agricultural cultivation regarding the structure of helminth communities for the hosts Aplastodiscus perviridis and Pseudis cardosoi. We also found a greater prevalence and intensity of infection in anurans in areas of agricultural cultivation than in native grasslands with livestock. We found that the environmental descriptors (local and landscape) seem to explain most of the differences in anuran parasitism recorded between native grasslands with livestock and agricultural areas. Thus, we emphasized that the loss of grassy habitat due to conversion to agricultural cultivation can alter helminth communities in anurans, with further work needed to understand the mechanisms involved.

Darwin's frogs Rhinoderma darwinii and Rhinoderma rufum are the only known species of amphibians in which males brood their offspring in their vocal sacs. We propose these frogs as flagship species for the conservation of the Austral temperate forests of Chile and Argentina. This recommendation forms part of the vision of the Binational Conservation Strategy for Darwin's Frogs, which was launched in 2018. The strategy is a conservation initiative led by the IUCN SSC Amphibian Specialist Group, which in 2017 convened 30 governmental, non-profit and private organizations from Chile, Argentina and elsewhere. Darwin's frogs are iconic examples of the global amphibian conservation crisis: R. rufum is categorized as Critically Endangered (Possibly Extinct) on the IUCN Red List, and R. darwinii as Endangered. Here we articulate the conservation planning process that led to the development of the conservation strategy for these species and present its main findings and recommendations. Using an evidence-based approach, the Binational Conservation Strategy for Darwin's Frogs contains a comprehensive status review of Rhinoderma spp., including critical threat analyses, and proposes 39 prioritized conservation actions. Its goal is that by 2028, key information gaps on Rhinoderma spp. will be filled, the main threats to these species will be reduced, and financial, legal and societal support will have been achieved. The strategy is a multi-disciplinary, transnational endeavour aimed at ensuring the long-term viability of these unique frogs and their particular habitat.

Amphibians on African mountains are threatened by habitat loss and fragmentation, pollution, disease and climate change. In particular, there have been recent reports of declines of montane endemic frogs in Cameroon. Mount Bamboutos, although home to numerous species of endemic amphibians, has no official protection and its amphibian populations have so far not been studied quantitatively. We surveyed frog assemblages on this mountain along a gradient of forest modification over a 2-year period. Through visual encounter surveys stratified across forest and farmland, we found that threatened montane amphibian species are closely associated with forested areas, particularly the Critically Endangered Leptodactylodon axillaris and Endangered Leptodactylodon perreti, Astylosternus ranoides and Cardioglossa oreas. Using the updated inventory of amphibians, which includes species with broader ranges across Africa, we found 69% of amphibian species on Mount Bamboutos to be threatened. We did not record several species present in historical records, which suggests they may have disappeared from this mountain, including Cardioglossa pulchra, Phrynobatrachus steindachneri, Phrynobatrachus werneri, Sclerophrys villiersi, Werneria bambutensis and Wolterstorffina mirei. The pattern of change detected in the amphibian community is consistent with declines on other mountains in the country, with a loss of Phrynobatrachus, Werneria and Cardioglossa spp., but persistence of Astylosternus, Arthroleptis and Leptodacty-lodon. The observed relationships of land-use patterns and amphibian diversity suggest that ongoing land-use changes could extirpate the remaining montane endemic frog species, particularly L. axillaris and L. perreti. Preserving a network of connected forest patches is therefore critical to save the endemic amphibians of Mount Bamboutos.

Habitat fragmentation is a major cause of biodiversity loss in agricultural landscapes. Studying habitat connectivity in fragmented landscapes is therefore pivotal for better understanding the factors that shape faunal communities in anthropogenic landscapes. Amphibians have limited dispersal abilities, strong site fidelity and often perform seasonal movements to reach relatively distant breeding habitats. This calls for a better knowledge of which landscape features might promote dispersal, especially in crops. We applied graph-theoretic network analyses to a set of 35 waterbodies embedded in 10 rice fields in a savanna–rain forest ecotone, Tocantins, Brazil, to assess the importance of landscape features (forest patches, waterbodies) for anuran functional connectivity within the entire network. We used taxonomic, functional and phylogenetic diversity as proxies of anuran functional connectivity (i.e. dispersal ability intrinsic to the taxa), based on previous associations uncovered between species diversity metrics and landscape connectivity. We found that, assuming individuals belonging to each of the 14 amphibian species recorded are unable to disperse over 800 m, forest and waterbody area and abundance are the most important predictors of waterbody importance for connectivity. Hence, pond network connectivity for amphibians in rice crops depends on (1) abundant and large forest patches in the area surrounding waterbodies, and (2) a network of abundant waterbodies.

Digenetic trematodes of the genus Clinostomum are cosmopolitan parasites infecting fishes, amphibians, reptiles, and snails as intermediate hosts. Despite the broad geographical distribution of this genus, debate about the number of species and how they vary in host use has persisted. To better understand patterns of infection among host species and across life stages, we used large-scale field surveys and molecular tools to examine five species of amphibians and seven species of fishes from 125 California ponds. Among the 12,360 examined hosts, infection was rare, with an overall prevalence of 1.7% in amphibians and 9.2% in fishes. Molecular evidence indicated that both groups were infected with Clinostomum marginatum. Using generalized linear mixed effects models, host species identity and host life stage had a strong influence on infection status, such that Lepomis cyanellus (green sunfish) (49.3%) and Taricha granulosa (rough skinned newt) (9.2%) supported the highest overall prevalence values, whereas adult amphibians tended to have a higher prevalence of infection relative to juveniles (13.3% and 2.5%, respectively). Experimentally, we tested the susceptibility of two amphibian hosts (Pseudacris regilla [Pacific chorus frog] and Anaxyrus boreas [western toad]) to varying levels of cercariae exposure and measured metacercariae growth over time. Pseudacris regilla was 1.3× more susceptible to infection, while infection success increased with cercariae exposure dose for both species. On average, metacarcariae size increased by 650% over 20 days. Our study highlights the importance of integrating field surveys, genetic tools, and experimental approaches to better understand the ecology of host–parasite interactions.

The Acheulian site of Gesher Benot Ya’aqov (GBY) and the Mousterian site of Nahal Mahanayeem Outlet (NMO) are open-air sites situated on the bank of the Upper Jordan River at its southern estuary in the Hula Valley, Israel. Both sites were deposited on the shore of a paleo-Lake Hula, a shallow body of water that persisted throughout a considerable part of the Pleistocene as a fresh-water lake. Most of the amphibian and squamate taxa recovered are aquatic species related to the natural biota of the Hula Valley, alongside some terrestrial species. Twelve amphibian and squamate taxa were recovered at each site. Most of the species recovered from the archaeological contexts do not differ from extant Hula Valley amphibians and squamates, with the exception of a varanid (Varanus sp.) recovered at GBY and the possible presence of the eastern fourlined ratsnake (cf. Elaphe sauromates). The snake’s presence could indicate slightly cooler conditions during human occupation at NMO. A noteworthy continuity in species presence is observed throughout the many archaeological horizons as well as in comparison to the current Hula basin fauna. This suggests a surprising similarity in environmental conditions over a significant portion of the Quaternary in this region.

Open palates with large interpterygoid vacuities are a diagnostic characteristic of temnospondyl amphibians, the most species-rich group of early tetrapods. Aside from their functional roles, several other aspects of such vacuities, such as their variation and spatial relationships relative to the orbits, have received only scarce attention. The present work examines patterns of shape and size changes in the orbits and vacuities of temnospondyls using a time-calibrated phylogeny of 69 temnospondyl taxa and 13 additional early tetrapod ‘outgroups' (colosteids, an embolomere, ‘microsaurs' and nectrideans). Orbit and vacuity outlines are quantified in a comparative framework using standard eigenshape analyses. In addition, we employ a series of ratios of linear measurements of both orbits and vacuities, and subject them to a phylogenetic principal component analysis in order to evaluate their proportional changes relative to the skull and to one another. Finally, we examine rates of evolutionary change and their associated shifts for shape and size for both structures, and assess the strength and significance of the correlations between these two variables using phylogenetic generalised least squares analyses. Although orbits and vacuities have fairly simple outlines, they both reveal complex models of proportional change across the temnospondyl phylogeny. These changes exhibit strong phylogenetic signal, that is, trait covariance among taxa is predicted by tree topology. We discuss the hypothesis that, early in tetrapod evolution, the functional role of the vacuities was related to the accommodation of the anterior jaw muscles. Only later in evolution did such vacuities serve to accommodate the eye muscles only.