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.

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.

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.

Status of the largest extant population of the critically endangered Aeolian lizard Podarcis raffonei (Capo Grosso, Vulcano island).

The Aeolian wall lizard Podarcis raffonei is an island endemic that survives only on three tiny islets, and on the Capo Grosso peninsula of the Vulcano island, thus is among the European vertebrates with the smallest range and one of the most threatened by extinction. This species is declining due to competition and hybridization with the non-native lizard Podarcis siculus, but a regular monitoring program is lacking. Here we assessed the size and status of the Capo Grosso population of P. raffonei on Vulcano. In September 2015 we captured 30 individuals showing the typical brown phenotype of P. raffonei, while one single male showed a green phenotype, apparently intermediate between P. raffonei and the non-native Podarcis siculus. In May 2017, only 47% of 131 individuals showed the typical brown phenotype (P. raffonei-like) and 53% showed the green phenotype (P. siculus-like). Based on N-mixture models and removal sampling the estimated size of the Capo Grosso population was of 800-1300 individuals in 2017, being similar to 2015; available data suggest that the total range of the species could be as small as 2 ha. The frequency of individuals with the typical P. raffonei phenotype dramatically dropped between two samplings with a parallel increase of individuals displaying the green phenotype. Observation on outdoor captive-bred individuals demonstrates plasticity for colouration in P. raffonei individuals from Capo Grosso, with several individuals showing the typical brown pattern in September 2017 and a green pattern in March 2021. Non-exclusive hypotheses, including hybridization with P. siculus and plasticity in colour pattern of P. raffonei, are discussed to explain the phenotypic shifts of the P. raffonei population of Capo Grosso. While genomic evidence is required to reach conclusions and investigate eventual hybridization, it is urgent to undertake a programme for the monitoring and management of this lizard.

Manual Sampling and Video Observations: An Integrated Approach to Studying Flower-Visiting Arthropods in High-Mountain Environments.

Despite the rising interest in biotic interactions in mountain ecosystems, little is known about high-altitude flower-visiting arthropods. In particular, since the research in these environment can be limited or undermined by harsh conditions and logistical difficulties, it is mandatory to develop effective approaches that maximize possibilities to gather high-quality data. Here we compared two different methods, manual sampling and video observations, to investigate the interactions between the high-mountain arthropod community and flowers of Androsace brevis (Primulaceae), a vulnerable endemic alpine species with a short flowering period occurring in early season. We manually sampled flower-visiting arthropods according to the timed-observations method and recorded their activity on video. We assessed differences and effectiveness of the two approaches to estimate flower-visiting arthropod diversity and to identify potential taxa involved in A. brevis pollination. Both methods proved to be effective and comparable in describing the diversity of flower visitors at a high taxonomic level. However, with manual sampling we were able to obtain a fine taxonomic resolution for sampled arthropods and to evaluate which taxa actually carry A. brevis pollen, while video observations were less invasive and allowed us to assess arthropod behavior and to spot rare taxa. By combining the data obtained with these two approaches we could accurately identify flower-visiting arthropods, characterize their behavior, and hypothesize a role of Hymenoptera Apoidea and Diptera Brachycera in A. brevis pollination. Therefore, we propose integrating the two approaches as a powerful instrument to unravel interactions between flowering plants and associated fauna that can provide crucial information for the conservation of vulnerable environments such as high-mountain ecosystems.

Rapid adaptation to invasive predators overwhelms natural gradients of intraspecific variation.

Invasive predators can exert strong selection on native populations. If selection is strong enough, populations could lose the phenotypic variation caused by adaptation to heterogeneous environments. We compare frog tadpoles prior to and 14 years following invasion by crayfish. Prior to the invasion, populations differed in their intrinsic developmental rate, with tadpoles from cold areas reaching metamorphosis sooner than those from warm areas. Following the invasion, tadpoles from invaded populations develop faster than those from non-invaded populations. This ontogenetic shift overwhelmed the intraspecific variation between populations in a few generations, to the point where invaded populations develop at a similar rate regardless of climate. Rapid development can have costs, as fast-developing froglets have a smaller body size and poorer jumping performance, but compensatory growth counteracts some costs of development acceleration. Strong selection by invasive species can disrupt local adaptations by dampening intraspecific phenotypic variation, with complex consequences on lifetime fitness.

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.