European Cave Salamander Research Articles

Four years monitoring of the endangered European plethodontid salamanders

The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021–2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.

Inferring on Speleomantes Foraging Behavior from Gut Contents Examination.

We here provide the first comprehensive analysis and discussion on prey consumed by the European cave salamanders of the genus Speleomantes. Our study stems from the need to shed light on the still unknown foraging behavior adopted by Speleomantes cave salamanders. Starting from the published datasets on gut contents from all Speleomantes species (including hybrids), we here discuss additional information (i.e., species ecology, lower taxonomic level), which were systematically omitted from those data sets. We analyzed a data set consisting of 17,630 records from 49 categories of consumed prey recognized from gut contents of 2060 adults and juveniles Speleomantes. Flying prey accounted for more than 58% of the prey items, while elongated crawling prey accounted for no more than 16% of the diet within a single population. Among the total recognized prey items, only three can be surely ascribed to the group of strictly-cave species (i.e., troglobites), meaning that European cave salamanders mostly forage in surface environment, and therefore represent one of the major drivers of allochthonous organic matter in subterranean environments. Some of the consumed prey seemed to be aquatic, allowing us to hypothesize whether Speleomantes are able to catch prey from a shallow body water. Furthermore, European cave salamanders possess the ability to prey upon taxa characterized by particular anti-predator defenses, while morphological constraints seem to be the most important limit to prey consumption. For each specific case, we provide insights and propose hypotheses concerning the foraging behavior that need to be tested to properly understand the foraging behavior of this cryptic salamanders.

Different patterns of introgression in a three species hybrid zone among European cave salamanders.

Hybrid zones occur where genetically distinct populations meet, mate and produce offspring with mixed ancestry. In Plethodontid salamanders, introgressive hybridization is a common phenomenon, where hybrids backcross with parental populations leading to the spread of new alleles into the parental genomes. Whereas many hybrid zones have been reported in American Plethodontid salamanders, only a single hybrid zone has been documented in European plethodontids so far, which is located at the Apuan Alps in the Italian Peninsula. Here, we describe a previously unreported hybrid zone in the Northern Apennines involving all the three Plethodontid salamander species inhabiting the Italian Peninsula. We found 21 new Speleomantes sites of occurrence, from a hitherto unexplored area located at the boundaries between three Speleomantes species ranges. Using mitochondrial (Cytb and ND2 genes) and nuclear markers (two diagnostic SNPs at the NCX1 gene), we revealed a three-way contact zone where all the three mainland species hybridize: S. strinatii, S. ambrosii and S. italicus. We observed a strong mitonuclear discordance, with mitochondrial markers showing a conspicuous geographic pattern, while diagnostic nuclear SNPs coexisted in both the same populations and individuals, providing evidence of hybridization in many possible combinations. The introgression is asymmetric, with S. italicus mitogenome usually associated with S. a. ambrosii and, to a lesser extent, to S. strinatii nuclear alleles. This finding confirms that Plethodontid are a group of choice to investigate hybridization mechanisms and suggests that behavioural, genetic and ecological components may concur in determining the direction and extent of introgression.

European Plethodontid Salamanders on the Forest Floor: Testing for Age-Class Segregation and Habitat Selection

Spatial distribution of animals is affected by environmental and social factors, acting both at inter- and intraspecific levels, and generating patterns of segregation or aggregation. Several studies investigated age-class segregation of the European Cave Salamander Speleomantes strinatii, in underground environments, showing a clear spatial segregation. We investigated the spatial distribution of S. strinatii on the forest floor, on 111 plots surveyed three times/season for two consecutive seasons, in northern Italy during autumn 2017 and spring 2018. We analyzed count data to model co-abundance of adults and juveniles, using a conditional two-species N-mixture model, incorporating environmental covariates. In contrast with what was observed in underground environments, we recorded no spatial segregation between juvenile and adult of S. strinatii on the forest floor. Instead, we found that adults and juveniles showed different responses to environmental features.

Interspecific and interpopulation variation in individual diet specialization: Do environmental factors have a role?

Individual diet specialization (IS) has important community- and population-level implications and its ecological drivers are actively investigated. Here, to test the hypothesis that local environmental conditions may influence IS in wild populations, we analyzed the stomach contents of 395 individuals from eight populations of five allopatric species of European cave salamanders (genus Hydromantes). We assessed whether their degree of individual diet specialization (1) scaled positively with the respective niche widths, in agreement with Van Valen's niche variation hypothesis (NVH), and (2) could be predicted by satellite-derived climatic and vegetation characteristics of the sites where the populations live. Consistent with the NVH, the degree of individual diet specialization increased with the populations' total niche width. Furthermore, two variables describing local nonarboreal vegetation cover and habitat heterogeneity successfully predicted the variation in individual specialization across the eight populations. Climatic factors had a generally low predictive power, with individual specialization in low- and high-elevation populations showing contrasting patterns of co-variation with air temperature in the warmest quarter of the year. However, independently from elevation, specialization peaked under conditions of high nonarboreal vegetation cover and high precipitation regimes. We discussed the results against two mutually nonexclusive scenarios hypothesizing different mechanisms linking environmental factors to salamanders' trophic strategy at an individual and population level. We concluded that satellite-derived climatic and vegetation variables to date generally adopted to model Grinnellian niches might also be useful in predicting spatial variations in dietary habits of populations, that is, their Eltonian niches.

Same Diet, Different Strategies: Variability of Individual Feeding Habits across Three Populations of Ambrosi’s Cave Salamander (Hydromantes ambrosii)

European cave salamanders of the genus Hydromantes are a group of eight species endemic to Italy and south-eastern France. Knowledge on the trophic niche of European Hydromantes is poor, and the few available studies only partially investigate their feeding habits. We performed an in-depth study on the trophic niche of the Ambrosi’s cave salamander (H. ambrosii), assessing the potential divergences among three different populations. All the populations had a similar diet composition, showing a wider trophic niche in fall compared to spring. In only one population, “true specialists” were present; however, in all three populations, generalist individuals always represented the larger proportion. Interspecific and intraspecific competition did not play an important role in determining individual dietary specialisation in H. ambrosii; contrarily, the characteristics of the surrounding environment seemed to be an important factor. The best body conditions were observed in the population located in the site where the non-arboreal vegetation cover was the highest. Besides providing new information on the trophic niche of H. ambrosii, we here showed that studies encompassing both intrinsic and extrinsic factors at the population level are needed to fully understand the trophic dynamics occurring among European cave salamanders.

On the stability of the dorsal pattern of European cave salamanders (genus Hydromantes)

Photographic identification is an emerging method for recognising wild animals. This harmless methodology allows researchers to identify “naturally marked” individuals and therefore study their specific ecology and behaviour. However, before incurring potential data loss, it is recommended to test the methodology on the target species and evaluate the pros and cons. We assessed the reliability of photographic identification in adult Hydromantes salamanders from three species. Specifically, we assessed whether the dorsal pattern of adult salamanders changed over time, thus evaluating its potential use as a reliable marking methodology. We used capture-mark-recapture and controlled conditions (i.e. individuals kept in fauna boxes) to evaluate potential changes in the dorsal pattern of Hydromantes through time. We did not observe any change in the dorsal pattern in the three species during the study period. Photographic identification might be a useful marking technique for these endangered species. However, these animals are usually found in environments generally lacking light and thus, researchers must be careful in setting up proper light conditions to produce suitable pictures for individual identification of Hydromantes.

Vitellogenin in the European cave salamander, Proteus anguinus: Its characterization and dynamics in a captive female as a basis for non-destructive sex identification.

Vitellogenin (Vtg) is a precursor protein of egg yolk proteins in oviparous and ovoviviparous vertebrates. Except in a case of exposure to estrogenic endocrine disruptors, Vtg is a female-specific protein and could be used as a molecular marker for sex identification. This would be especially useful in the case of the endangered European cave salamander Proteus anguinus in which sexes are indistinguishable according to external morphology, which hinders the establishment of a successful captive breeding program. Here we describe the identification, partial characterization, and purification of Vtg from P. anguinus. Vtg was identified in the plasma of a vitellogenic proteus female with visible oocytes. The identification of this protein was accomplished by mass spectrometry analysis. Two-dimensional gel electrophoresis revealed proteus Vtg as a mix of 190 kDa isoforms with isoelectric points in the pH range 5.3-6.0. Vtg was purified from proteus blood by gel filtration followed by anion-exchange chromatography. Using specific staining of SDS-PAGE gels, the Vtg was found to be phosphorylated and lipidated. Unlike the case in some other aquatic vertebrates, in P. anguinus, Vtg was not present in detectable amounts in cutaneous mucus. Degradation of oocytes in the captive vitellogenic female was accompanied by simultaneous decrease of Vtg concentration. Over a period of 10 months, the concentration of Vtg dropped from maximal to sub-detectable. Our results show that Vtg is a promising molecular marker for sex identification and ovary maturation in P. anguinus, which could contribute to the development of a viable program for captive reproduction of this unique species.

Composition of the cutaneous bacterial community of a cave amphibian, Proteus anguinus.

The European cave salamander Proteus anguinus is a charismatic amphibian endemic to the concealed and inaccessible subterranean waters of the Dinaric Karst. Despite its exceptional conservation importance not much is known about its ecology and interactions with the groundwater microbiome. The cutaneous microbiota of amphibians is an important driver of metabolic capabilities and immunity, and thus a key factor in their wellbeing and survival. We used high-throughput 16S rRNA gene sequencing based on seven variable regions to examine the bacteriome of the skin of five distinct evolutionary lineages of P. anguinus and in their groundwater environment. The skin bacteriomes turned out to be strongly filtered subsamples of the environmental microbial community. The resident microbiota of the analyzed individuals was dominated by five bacterial taxa. Despite an indicated functional redundancy, the cutaneous bacteriome of P. anguinus presumably provides protection against invading microbes by occupying the niche, and thus could serve as an indicator of health status. Besides conservation implications for P. anguinus, our results provide a baseline for future studies on other endangered neotenic salamanders.

What shapes the trophic niche of European plethodontid salamanders?

The trophic niche is a life trait that identifies the consumer’s position in a local food web. Several factors, such as ontogeny, competitive ability and resource availability contribute in shaping species trophic niches. To date, information on the diet of European Hydromantes salamanders are only available for a limited number of species, no dietary studies have involved more than one species of the genus at a time, and there are limited evidences on how multiple factors interact in determining diet variation. In this study we examined the diet of multiple populations of six out of the eight European cave salamanders, providing the first data on the diet for five of them. In addition, we assessed whether these closely related generalist species show similar diet and, for each species, we tested whether season, age class or sex influence the number and the type of prey consumed. Stomach condition (empty/full) and the number of prey consumed were strongly related to seasonality and to the activity level of individuals. Empty stomachs were more frequent in autumn, in individuals far from cave entrance and in juveniles. Diet composition was significantly different among species. Hydromantes imperialis and H. supramontis were the most generalist species; H. flavus and H. sarrabusensis fed mostly on Hymenoptera and Coleoptera Staphylinidae, while H. genei and H. ambrosii mostly consumed Arachnida and Endopterygota larvae. Furthermore, we detected seasonal shifts of diet in the majority of the species examined. Conversely, within each species, we did not find diet differences between females, males and juveniles. Although being assumed to have very similar dietary habits, here Hydromantes species were shown to be characterized by a high divergence in diet composition and in the stomach condition of individuals.

The first ecological study on the oldest allochthonous population of European cave salamanders (Hydromantes sp.)

The introduction of allochthonous species often represents a serious problem for ecosystems and native species. Usually, these cases involve common widespread species that show high adaptability and strong competitiveness against local species. Within amphibians, the introduction of allochthonous species mainly deals with anurans, while cases involving caudata are few and poorly studied. We report the first assessment of an introduced population of European plethodontid salamanders. This population is located in the French Pyrenees and represents the oldest allochthonous Hydromantes population. We reconstructed the history of its introduction and collected data on the ecology and feeding habits of this population. Our results show that this population is stable and reproductive, showing strong similarities to Italian mainland species of Hydromantes. This study provided the base for further studies focused on this allochthonous population of European cave salamanders.

First microsatellite data on Proteus anguinus reveal weak genetic structure between the caves of Postojna and Planina

Abstract The European cave salamander, Proteus anguinus, or proteus, is the largest obligate cave animal in the world. It is an endangered and charismatic species of high conservation importance for subterranean waters. Conservation genetic studies are hampered by the extreme size and repetitiveness of its nuclear genome. The aim of the study was to develop and characterize the first microsatellite markers for proteus, and test their informativeness at the level of individuals, populations and between populations in the Postojna and Planina caves in Slovenia. Twenty‐three novel polymorphic microsatellite markers were amplified in 201 individuals from both caves using three multiplex reactions. The number of alleles per locus varied from three to nine. The loci are largely unlinked and conform to Hardy–Weinberg genotype frequencies. Genetic equilibrium and an FST value of 0.0024 suggest a nearly panmictic population in both caves separated by some 10 km of subterranean river course, while Bayesian clustering detected weak genetic structure. The microsatellites described fill the gap of urgently needed nuclear markers in Proteus that can be applied in genetic mark–recapture studies, population monitoring and identification of management units to assist conservation efforts.

Editorial 2012

Editorial 2012

The free‐radical damage theory: Accumulating evidence against a simple link of oxidative stress to ageing and lifespan

Recent work on a small European cave salamander (Proteus anguinus) has revealed that it has exceptional longevity, yet it appears to have unexceptional defences against oxidative damage. This paper comes at the end of a string of other studies that are calling into question the free-radical damage theory of ageing. This theory rose to prominence in the 1990s as the dominant theory for why we age and die. Despite substantial correlative evidence to support it, studies in the last five years have raised doubts over its importance. In particular, these include studies of mice with the major antioxidant genes knocked out (both singly and in combination), which show the expected elevation in oxidative damage but no impact on lifespan. Combined, these findings raise fundamental questions over whether the free-radical damage theory remains useful for understanding the ageing process, and variation in lifespan and life histories.

Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus

The European cave salamander Proteus anguinus Laurenti 1768 is one of the best-known subterranean animals, yet its evolutionary history and systematic relationships remain enigmatic. This is the first comprehensive study on molecular evolution within the taxon, using an mtDNA segment containing the control region (CR) and adjacent sequences. Two to seven tandem repeats of 24–32 bp were found in the intergenic spacer region (VNTR1), and three, four or six repeats, 59–77 bp each, in the 3′ end of the CR (VNTR2). Different molecular mechanisms account for VNTR2 formation in different lineages of Proteus. The overall CR variation was lower than that of the spacer region, the 3′ end of the cytb gene, or the tRNA genes. Individual genes and the concatenated non-repetitive sequences produced similar, well resolved maximum likelihood, Bayesian inference and parsimony trees. The numbers of repeat elements as well as the genealogy of the VNTR2 repeat units were mostly inconsistent with the groupings of the non-repetitive sequences. Different degrees of repeat array homogenization were detected in all major groups. Orthology was established for the first and the second VNTR2 elements of some populations. These two copies may therefore be used for analyses at the population level. The pattern of CR sequence variation points to strong genetic isolation of hydrographically separated populations. Genetic separation of the major groups of populations is incongruent with the current division into subspecies.

A non-lethal method to sample gastrointestinal parasites from terrestrial salamanders

The usual method to obtain gastrointestinal parasites from amphibians is dissecting sacrificed animals (e.g. Pritchard and Kruse, 1982; Goater and Goater, 2001 and references therein). However, the recent worldwide decline of amphibian populations (e.g. Daszak et al., 2003; Stuart et al., 2004) and the raising conservation concern about endangered and rare species strongly demand the finding of alternative and less disruptive methods that may reduce the overcollection of amphibians from their natural habitats. This is especially the case of endangered species that are in need of protection, such as European cave salamanders belonging to the genus Speleomantes Dubois, 1984 (Amphibia, Urodela, Plethodontidae). These fully terrestrial salamanders, found in SW France and Italy including Sardinia (Lanza et al., 1995), are totally protected by the European Union 92/43 directive for the conservation of biodiversity, known as “Habitat and Species”. Little is known about gastrointestinal parasites of Speleomantes (Ricci, 1988; Ben Slimane and Durette-Desset, 1995) and, recently, a new species of Distoichometra (Cestoda, Nematotaeniidae) was described from preserved specimens of S. strinatii studied for the first time by Pastorino (1974) (Buriola et al., in press). Thus, the examination