Martina Muraro

Genetic non‐invasive sampling (gNIS) methods, which enable DNA extraction from naturally shed materials, offer a powerful, low‐impact tool for studying elusive species, minimising disturbance and ethical concerns. However, such approaches require careful assessment of sample quality and data accuracy to ensure reliable genetic results. The hazel dormouse ( Muscardinus avellanarius ) is an arboreal rodent that plays a crucial role in ecosystems. Despite conservation concerns arising from its low population densities, which are closely linked to habitat quality, it remains poorly understood due to monitoring challenges. This pilot study, conducted in the high Agri Valley (Basilicata, Italy), evaluates a non‐invasive genetic sampling protocol for the hazel dormouse using hair‐tubes. We investigated the genotyping efficiency, the reliability and informativeness of selected microsatellite markers and the impact of hair sample quantity and trap placement duration on genotyping success and error rates. We used 126 hair tube traps at 14 sampling sites, obtaining reliable genotypes for 14 individuals out of 32 hair samples collected, with a genotyping efficiency significantly affected by both the number of hairs collected and the collecting time interval. The microsatellite marker panel showed a high number of alleles and a low probability of identity, proving to be informative for population monitoring. The results underscore the potential of this non‐invasive approach for monitoring elusive and conservation‐priority species while highlighting the need to minimise field collection time and optimise trap design to improve DNA quality and genotyping reliability.

The common practice of using subspecies as conservation targets raises the question of whether efforts are focused on preserving conspicuous components of the species’ phenotypic variability rather than evolutionarily significant units. To address this question, in this study we performed a comprehensive morphological and genetic assessment on all the subspecies of wall lizard described for the Aeolian Archipelago (Italy) to determine whether they represent distinct evolutionary lineages and/or discrete phenotypic partitions. Further, we applied a monophyly test to 70 subspecies belonging to seven wall lizard species occurring in Italy, based on our results and on previous phylogeographic studies. We found that none of the Aeolian subspecies represents a distinct evolutionary lineage, despite some morphological differentiation of island populations across the archipelago, suggesting a very recent origin of island populations and of the observed phenotype variation. Across seven wall lizard species, tests revealed that lizard subspecies rarely (< 9% of cases) match evolutionary units. This study demonstrates that intraspecific taxonomy of wall lizards is a poor predictor of phylogeographic partitions and evolutionary units, and therefore of limited use (if not dangerous) for defining conservation and management units. A better approach would be relying on the integration of genomic and phenotypic data to assess the evolutionary significance and conservation value of phenotypic and genetic units within species.

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.

Detecting the trends of species and populations is fundamental to identify 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 the Monza Park, which currently represents the area closer to the type locality of the species. Wetlands within the park were monitored from 2000 to 2023; counts of egg clutches were taken as a measure of reproductive output and of 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 2022-23. 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.

Hybridization is a widespread phenomenon in animals, and hybrid heterosis/breakdown could be key processes determining the evolutionary dynamics of hybrids. Indeed, hybrids are not consistently disadvantaged compared to the parental lineages, as was historically assumed. Multiple processes could lead to performance differences between parental lineages and their hybrids. Despite many studies evaluated the performance of hybrids, a quantitative synthesis is required to assess the general pattern. Here we used meta-analytic and meta-regression approaches to quantify the fitness differences between parental lineages and their hybrids, and to identify possible processes that could lead to these differences. Specifically, we tested biological and methodological parameters that could determine differences in performance between hybrids and parental lineages. Hybrid performance was extremely variable across studies, being often significantly higher or lower compared to the mean performance of their parents. Nevertheless, the averaged hybrid performance was similar to the fitness of parental lineages, with differences across studies related to how performance was assessed. Genetic divergence between parental lineages, and the approach used to identify hybrids were the parameters most strongly related to variation in hybrid performance. Performance was lower for hybrids between distantly related lineages. Furthermore, study settings and the use of imprecise approaches for hybrid identification (e.g. morphology-based) can bias assessments of performance. Studies performed on wild populations and using genetic approaches for hybrid identification detected more often a decreased hybrid performance, compared to laboratory studies. We highlight the importance of appropriate settings for a realistic understanding of the evolutionary impacts of hybridization.

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.

Amphibians are an exemplary case of the current biodiversity crisis, being among the vertebrates suffering the fastest decline. Population dynamics of amphibians can result from processes acting at different scales. Both the local characteristics of breeding wetlands and the features of the surrounding landscape can strongly affect the temporal dynamics of amphibian populations. European newts are particularly threatened by land‐use change and invasive alien species. While it is known that newts are declining across Europe, few studies have performed broad‐scale assessments of their decline, either because abundance dynamics are more complex to analyse than presence/absence data or because they require a high sampling effort and long‐term monitoring. In this study, we show that long‐term distribution data can be combined with demographic models to quantify the decline in abundance of newt species at the regional scale, and to assess the importance of multiple factors in determining abundance dynamics. We performed multiple surveys between 1996 and 2020 and used N‐mixture models in a Bayesian framework. We then calculated abundance changes between the first and the last sampling season, which were performed with an average timespan of 13 years across all wetlands. Both Italian crested newts and smooth newts showed large declines, with an average estimated abundance loss between the first and last sampling season of 57% and 63%, respectively. Local characteristics of the wetlands were the main determinants of abundance dynamics: the abundance of both species showed a positive relationship with the area and the permanence of the wetland and a negative relationship with the presence of fish. Additionally, the abundance of Italian crested newts was negatively related to the presence of invasive crayfish. No relationship was detected between abundance and terrestrial habitat availability or connectivity measures. Despite uncertainties in the absolute values of estimated abundance, the striking regional‐scale decline of newts is evident. Among the major determinants of population dynamics, fish and crayfish presence increased their prevalence in the study area, while other factors remained more stable. Management actions aimed at eradicating or controlling invasive fish and crayfish might halt abundance loss and even revert this declining trend. The application of N‐mixture models to long‐term data from representative sites permits the analysis of temporal trends of species at the regional scale even when data come from complex monitoring schemes. We found large declines in abundance of two newt species, suggesting that European newts may be more threatened than previously thought.

Aim: Understanding which factors determine the variation in population size across space and time is crucial to plan sound conservation interventions. Amphibians are often characterized by large demographic changes, therefore a better understanding of factors driving these changes could help mitigate their global crisis. We investigated drivers of abundance dynamics of two similar frog species to understand why they met different fates in the same study area.Location: Northern Italy.Methods: In seven different years between 2004 and 2020, we performed repeated counts of egg masses of two similar frog species, the agile frog (Rana dalmatina) and the Italian agile frog (Rana latastei), in 31 wetlands, and used Bayesian models to estimate the relationships between frog abundance and candidate drivers acting at the scale of (i) site: wetland surface, shading percentage and the presence of the invasive red swamp crayfish (Procambarus clarkii); (ii) landscape: forest cover around wetlands; (iii) climate: yearly precipitation and (iv) spatially structured population (SSP): clutch incidence of the focal species and crayfish incidence in the surrounding landscape.Results: The two species showed sharp differences in population size and trends: R. dalmatina was abundant and showed a stable trend throughout the entire study period; R. latastei showed low abundance in the first years and then almost disappeared. The abundance of R. dalmatina was positively related to forest cover, shading, wetland area and precipitation, while negatively related to the occurrence of invasive crayfish at both local and SSP level. R. latastei abundance increased with wetland area and precipitation, while models were not able to detect relationships with other factors.Main conclusions: The high sensitivity to drought and demographic stochasticity could have contributed to the quasi‐extinction of R. latastei in the study area, highlighting that similar species can meet different fates even under the same environmental conditions.