Alpine salamanders at risk? The current status of an emerging fungal pathogen.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the 'salamander plague' ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

Microplastic pollution calls for urgent investigations in stygobiont habitats: A case study from Classical karst.

Microplastic pollution in karst systems is still poorly studied, despite the presence of protected species and habitats, and important water reserves. Vulnerable key species hosted in these habitats could consume or assimilate microplastics, which can irreversibly damage management efforts, and thus ecosystems functionality. This can be particularly true for subterranean water habitats where microplastic pollution effects on wildlife management programs are not considered. The aim of this study is to provide a case study from the Classical Karst Region, which hosts peculiar habitats and key species protected at European level, such as the olm Proteus anguinus. As this area has been deeply exploited and modified over time, and is adjacent to highways, roads and railways, which could contribute to pollution within the karst system, threatening the ecosystems, it provides a perfect model system. In this study we collected and investigated water and sediment samples from aquatic environments of surface and subterranean habitats hosting several subterranean environment-adapted organisms. Examined particles were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light. Furthermore, spectroscopic analyses were carried out in order to identify microplastics typology. Microplastics were found in all examined habitats. In water, microplastics concentration ranged from 37 to 86 items/L, in sediments from 776 to 2064 items/kg. Fibre-shape was the main present, followed by fragments and beads, suggesting multiple sources of pollution, especially textile products. Most of the particles were fluorescent under UV light and were mainly transparent, while not-fluorescent ones were especially black, blue or brown. Samples contained especially polyesters and copolymers. These results highlight intense MP pollution in karst areas, with significant impacts on water quality, and potential effects on subterranean environment-dwelling species. We stress the importance of monitoring pollution in these critical environments for biodiversity and habitat conservation: monitoring in karst areas must become a priority for habitat and species protection, and water resources management, improving analyses on a larger number of aquatic surface and subterranean habitats.

Decline and Extinction of the Italian Agile Frog Rana latastei from Core Areas of Its Range.

Detecting the trends of species and populations is fundamental to identifying taxa with high conservation priority. Unfortunately, long-term monitoring programs are challenging and often lacking. The Italian agile frog Rana latastei is endemic to Northern Italy and adjacent countries, is considered vulnerable by the IUCN, and is protected at the European level. However, quantitative estimates of its decline are extremely scarce. In this study, we document the trends in abundance and distribution of Rana latastei within Monza Park, which currently represents the area closer to the type locality of the species and holds unique genetic features. Wetlands within the park were monitored from 2000 to 2023; counts of egg clutches were taken as a measure of reproductive output and the abundance of breeding females. In 2000, the species occurred over a significant proportion of the park. Total abundance showed strong yearly variation but remained rather constant from 2000 to 2019. However, Rana latastei disappeared from the park around 2021 and was never detected in 2022-2023. The decline is probably related to the joint effect of multiple factors, including the conversion of breeding sites for farming, inappropriate water management, invasive alien species, and severe drought. The local extinction of Rana latastei occurred despite legal protection, highlighting the need for more effective and stringent tools for the conservation of European biodiversity.

Larval development and poor trophic resource availability: Local adaptations and plasticity in a widespread amphibian species.

Theory predicts that, in organisms with complex life cycles, if the earlier-stage limiting factor induces weak later-stage phenotypes, the development of the later-stage trait should evolve to reduce carry-over effects. Local adaptations could thus favour decoupling of later stages. However, decoupling is not always possible. In this study, we used a widespread amphibian, the European fire salamander (Salamandra salamandra), to assess the role of local adaptations to environmental stressful conditions experienced at the larval stage. We exposed 150 larvae from different altitudes to two conditions: rich food and poor food condition. Conditions in early life stages can affect an individual's traits, either as a direct effect or mediated through outcomes in successive life stages. To distinguish between effects of rearing conditions and local adaptation, we searched for a causal model. The causal model detected effects of both food treatment and population origin (altitude) on all life stages. Larvae reared under rich food condition metamorphosed earlier, had higher growth rates and reached smaller size at metamorphosis. Significant differences occurred between larvae of different origin: low-altitude individuals performed poorly under the poor food treatment. Moreover, larvae from higher altitudes were slower with rich food and faster with poor food compared to those from lower altitudes. Our results underline that environmental conditions and local adaptation can interplay in determining the plasticity of larval stages, still adaptations can maximize the growth efficiency of early stages in oligotrophic environments, leading to divergent pathways across populations and environmental conditions.

Condition- and context-dependent variation of sexual dimorphism across lizard populations at different spatial scales.

The evolution of sexual dimorphism (SD) is driven by intricate interplays between sexual and natural selection. When it comes to SD variation within populations, however, environmental factors play a major role. Sexually selected traits are expected to be strongly dependent on individual body condition, which is influenced by the local environment that individuals experience. As a consequence, the degree of SD may also depend on resource availability. Here, we investigated the potential drivers of SD expression at two sexually dimorphic morphometric traits, body size (snout vent length) and head shape (head geometric morphometrics), in the Italian wall lizard (Podarcis siculus). We assessed the existence of condition- and context-dependent SD across ten islands of the Aeolian archipelago (southern Italy), at within- and among-population scales. We observed strong geographical variation of SD among islands, and tested three potential SD predictors related to resource availability (individual body condition, ecosystem productivity, temperature). Body condition and ecosystem productivity were the main drivers of body size SD variation, and body condition was also the main driver for head shape SD. Our results highlight that the expression of SD in the Italian wall lizard is both condition- and context-dependent. These results are congruent at within- and among-populations scales highlighting that spatial multi-scale analysis represents a useful approach to understand patterns of SD expression.

Factors determining the dorsal coloration pattern of aposematic salamanders.

Aposematic bright colors have a key role for animal defense and can be expressed through metabolic production or by acquiring pigments from diet. Aposematic coloration can be related to both local adaptations and availability of trophic resources. The European fire salamander (Salamandra salamandra) shows significant color variability and occurs across a broad range of habitats. Here we combined field observations with common rearing experiments to disentangle the role of environmental conditions and local adaptations in determining aposematic coloration of salamander populations. We assessed color variation and measured habitat features and food availability in adults from 25 populations. Furthermore, we reared newborn larvae from 10 populations under different food availability and analyzed color of metamorphs. To assess color pattern, we measured the percentage of yellow covering the body, and the Hue, Saturation and Value of yellow coloration. Adult showed strong variation of color pattern; variation was strongly related to the individual's size, to habitat productivity and to food availability. Under common garden conditions, differences between populations were not anymore evident, and coloration was only affected by resource availability during larval development. Our results suggest that environmental conditions and food availability are more important than local adaptations in determining differences in aposematic color pattern.

Assessing Population Trends of Species with Imperfect Detection: Double Count Analyses and Simulations Confirm Reliable Estimates in Brown Frogs.

Most animal species are detected imperfectly and overlooking individuals can result in a biased inference of the abundance patterns and underlying processes. Several techniques can incorporate the imperfect detection process for a more accurate estimation of abundance, but most of them require repeated surveys, i.e., more sampling effort compared to single counts. In this study, we used the dependent double-observer approach to estimate the detection probability of the egg clutches of two brown frog species, Rana dalmatina and R. latastei. We then simulated the data of a declining population at different levels of detection probability in order to assess under which conditions the double counts provided better estimates of population trends compared to naïve egg counts, given the detectability of frog clutches. Both species showed a very high detection probability, with average values of 93% for Rana dalmatina and 97% for R. latastei. Simulations showed that not considering imperfect detection reduces the power of detecting population trends if detection probability is low. However, at high detection probability (>80%), ignoring the imperfect detection does not bias the estimates of population trends. This suggests that, for species laying large and easily identifiable egg clutches, a single count can provide useful estimates if surveys are correctly timed.

A New Disease Caused by an Unidentified Etiological Agent Affects European Salamanders.

New pathologies are causing dramatic declines and extinctions of multiple amphibian species. In 2013, in one fire salamander population of Northern Italy, we found individuals with undescribed cysts at the throat level, a malady whose existence has not previously been reported in amphibians. With the aim of describing this novel disease, we performed repeated field surveys to assess the frequency of affected salamanders from 2014 to 2020, and integrated morphological, histological, and molecular analyses to identify the pathogen. The novel disease affected up to 22% of salamanders of the study population and started spreading to nearby populations. Cysts are formed by mucus surrounding protist-like cells about 30 µm long, characterized by numerous cilia/undulipodia. Morphological and genetic analyses did not yield a clear match with described organisms. The existence of this pathogen calls for the implementation of biosecurity protocols and more studies on the dynamics of transmission and the impact on wild populations.

N-mixture models reliably estimate the abundance of small vertebrates.

Accurate measures of species abundance are essential to identify conservation strategies. N-mixture models are increasingly used to estimate abundance on the basis of species counts. In this study we tested whether abundance estimates obtained using N-mixture models provide consistent results with more traditional approaches requiring capture (capture-mark recapture and removal sampling). We focused on endemic, threatened species of amphibians and reptiles in Italy, for which accurate abundance data are needed for conservation assessments: the Lanza's Alpine salamander Salamandra lanzai, the Ambrosi's cave salamander Hydromantes ambrosii and the Aeolian wall lizard Podarcis raffonei. In visual counts, detection probability was variable among species, ranging between 0.14 (Alpine salamanders) and 0.60 (cave salamanders). For all the species, abundance estimates obtained using N-mixture models showed limited differences with the ones obtained through capture-mark-recapture or removal sampling. The match was particularly accurate for cave salamanders in sites with limited abundance and for lizards, nevertheless non-incorporating heterogeneity of detection probability increased bias. N-mixture models provide reliable abundance estimates that are comparable with the ones of more traditional approaches, and offer additional advantages such as a smaller sampling effort and no need of manipulating individuals, which in turn reduces the risk of harming animals and spreading diseases.

Field-recorded data on the diet of six species of European Hydromantes cave salamanders.

The availability of data on the feeding habits of species of conservation value may be of great importance to develop analyses for both scientific and management purposes. Stomach flushing is a harmless technique that allowed us to collect extensive data on the feeding habits of six Hydromantes species. Here, we present two datasets originating from a three-year study performed in multiple seasons (spring and autumn) on 19 different populations of cave salamanders. The first dataset contains data of the stomach content of 1,250 salamanders, where 6,010 items were recognized; the second one reports the size of the intact prey items found in the stomachs. These datasets integrate considerably data already available on the diet of the European plethodontid salamanders, being also of potential use for large scale meta-analyses on amphibian diet.