Salamandrina Perspicillata Research Articles

Safe and genotyped. A non-invasive method for extraction of amphibian DNA from water baths and its application on Northern spectacled salamanders, Salamandrina perspicillata (Savi 1821)

The global decline of amphibian populations, with 40.7% of species classified as threatened, calls for innovative and ethical approaches in conservation genetics. Molecular biology advancements have introduced environmental DNA (eDNA) analysis, primarily focusing on aquatic environments. However, the present study explores a novel non-invasive protocol using water samples to extract DNA from terrestrial and semi-terrestrial amphibians, specifically the endangered Italian endemic salamander, Salamandrina perspicillata (Savi, 1821). Unlike traditional invasive methods involving tissue sampling, this protocol immerses animals briefly, eliminating the need for digit or tail amputations or manipulation for buccal swabs. The study validated the protocol through DNA extraction, amplification, and sequencing, yielding results comparable to traditional methods. The non-invasive nature of the protocol aligns with the 3Rs principles (Replace, Reduce, Refine) and offers a streamlined, stress-minimizing alternative for studying protected and endangered species. Future experiments should also explore further refinements, including reduced soaking times and additional applications, such as skin microbiota analysis. This protocol represents a significant step towards ethical and effective research practices in amphibian conservation genetics, encouraging a paradigm shift in wildlife research ethics. Continued innovation in non-invasive methodologies is essential for comprehensive understanding and robust conservation strategies amid the ongoing biodiversity crisis.

Mutual Avoidance in the Spectacled Salamander and Centipede: A Discrepancy between Exploratory Field and Laboratory Data.

Interactions between amphibians and arthropods encompass a wide range of ecological relationships, predominantly characterized by predator-prey dynamics, with adult amphibians as the predators. In some instances, the roles are reversed. This study focuses on the potential predator-prey relationship between the spectacled salamander (Salamandrina perspicillata) and the centipede Scolopendra cingulata in Central Italy. Building upon previous research on chemical cue perception in amphibians, we investigated potential olfactory cue-mediated avoidance behaviours exhibited by S. perspicillata towards the potential predator S. cingulata through field observations and manipulative experiments. In a natural site, we estimated the degree of negative co-occurrence between the study species under shelters and found an avoidance pattern between S. perspicillata and S. cingulata in refuges. However, when the study species were forced to choose between sharing or not sharing a given shelter, through a manipulative experiment, the avoidance pattern was not confirmed. Potential determinants contributing to the avoidance pattern observed in nature are discussed. Our exploratory results represent a good example of how what often appears to be a strong observation-based pattern in natural settings needs to be carefully scrutinized. Hypotheses testing through experiments in controlled environments remains a valuable approach to exclude potentially misleading processes.

Foraging success is differently affected by local climate in two syntopic forest-dwelling salamanders

Amphibians are small ectothermic vertebrates with high permeable skins that are highly constrained by environmental conditions, such as humidity and temperature. Therefore, terrestrial salamanders regulate their surface activity, including foraging, to minimize evaporative water loss. The influence of local micro-climatic factors on the foraging success of European forest dwelling salamanders has rarely been analysed in detail. We examined the role of local climatic conditions on the foraging success, measured by the total number of prey items, of two syntopic forest-dwelling salamanders: Strinati’s cave salamander, Speleomantes strinatii (family Plethodontidae), and Northern spectacled salamander, Salamandrina perspicillata (family Salamandridae). We evaluated the effects of rainfall, temperature and body size on the foraging success of these two species, in eight sampling sites in the Northern Apennines of Italy, for a total of 11 salamander populations. Our findings show that different climatic variables affected the two species differently. Salamandrina perspicillata foraged more intensively during periods of prolonged rainfall with higher temperatures. On the contrary, S. strinatii foraging success was negatively influenced by rising temperature, while rainfall seemed to have no significant effect. Interestingly, body size increased success in S. perspicillata but not in S. strinatii. Overall, our findings show that, when foraging in the same environment, these two species are conditioned by different climatic factors possibly due to their differences in overall body size and their ability to resist dehydration.

Is the Northern Spectacled Salamander Salamandrina perspicillata aposematic? A preliminary test with clay models

Aposematism is a visual communication system in which bright and contrasted coloured prey warn predators about their unprofitability. The Northern Spectacled Salamander Salamandrina perspicillata, a small terrestrial salamander endemic to Italy, displays a uniform dark dorsal colouration and a contrasted ventral side in which a bright red colour is displayed by coiling the tail over the body. In amphibians, this behaviour, known as “Unkenreflex”, is usually considered to be aposematic. In this study, we used realistic plasticine replicas to test this aposematic hypothesis in the Northern Spectacled Salamander. Of the 199 clay models placed in a natural habitat, 165 (83%) were recovered and 39 (24%) showed some sign of predation. The head of the models was more attacked than expected by chance (P = 0.042), suggesting that potential predators were perceiving models as real prey. However, there were no differences in the proportion of dorsal (18/83 = 22%), and ventral (21/82 = 26%) models attacked by predators. Therefore, contrary to expectations our experiment did not support the aposematic hypothesis. However, predation experiments with clay models have limitations and our results should be considered as preliminary, deserving further research to better understand the Northern Spectacled salamander prey-predator system.

An updated check-list of Italian amphibians and reptiles

This paper represents an update of the list of Italian amphibians and reptiles published 15 years ago by Razzetti et al. (2006) and of the checklist published in 1993 by the late Benedetto Lanza. At present, the Italian herpetofauna includes 101 species (41 amphibians and 60 reptiles) and an amphibian taxon of hybrid origin. Seven species and one subspecies are allochthonous and became naturalized within the last century. Since the last published list, a new species has been described (Vipera walser), five taxa have been raised to species rank (Salamandrina perspicillata, Speleomantes sarrabusensis, Zootoca carniolica, Malpolon insignitus and Natrix helvetica) while three taxa have been downgraded to subspecies. All the relevant taxonomic changes based upon new research have been discussed, including tentative revisions and controversial taxa. Nine species reported or listed dubitatively in Lanza’s 1993 list are excluded here.

First observation of partial neoteny in <em>Salamandrina perspicillata</em> (Savi, 1821)

Different forms of neoteny were recorded in some Italian amphibian species: full (absolute or temporary) and partial. However, this physiological phenomenon was never observed in the Italian endemic Salamandrina perspicillata. The authors report the first record of partial neoteny in an individual of this species, a leucistic larva observed in a drinking trough in the Valle del Treja Regional Park (Latium - Central Italy).

Skewed sex ratio in a forest salamander: artefact of the different capture probabilities between sexes or actual ecological trait?

Sex ratio is an essential demographic parameter and distortions from a balanced sex ratio may have contrasting effects on the population dynamics. However, observation of distorted sex ratio using counts or captures may reflect an actual ecological trait of the studied population but may also be an artefact due to different capture probabilities of males and females. We compared results obtained from Counts and Capture-Marking-Recapture (CMR) on both sexes in a population of a forest dwelling salamander, Salamandrina perspicillata, and we investigated if males and females had different capture probabilities. We surveyed available literature to compare information on sex ratio from other populations of S. perspicillata. The sex ratio from our counts was 0.65 and was significantly male-biased as reported in other studies. The estimated sex ratio from CMR data was 0.57. Although males showed higher recapture rates than females in every capture session, these differences were not statistically significant. Therefore, the skewed sex ratio towards males is not only an artefact due to different capture probabilities between males and females but reflects an actual demographic trait, although the magnitude of the skeweness was overestimated by counts.

Conservation of salamanders in managed forests: Methods and costs of monitoring abundance and habitat selection

Ecological traits affect species’ responses to human impacts. Amphibians are declining worldwide and one of the major causes of such decline is habitat loss. Forestry practices have a primary role in determining habitat loss and fragmentation for amphibians. Thus, researchers should provide forest practitioners with essential information in order to address proper forest management plans. Here, we studied ecological requirements in the terrestrial phase of salamanders and we tested the feasibility of repeated counts to infer habitat determinants of salamanders’ abundance in order to guide forest management plans. We employed the N-mixture models for the analysis of repeated count data of an Italian endemic salamander (Salamandrina perspicillata) on seventy-seven 100 m2 plots, within a central-Italian forest. Modelling salamanders abundance as a function of site specific habitat features allowed us to give precise guidelines for forest management. Harvesting should be conducted preferably on south facing slopes, since salamanders’ occurrence and abundance are higher on northern slopes. Forest operations should be avoided or reduced within a buffer of some 150m from reproductive sites. Since salamanders use tree bases as shelters, patches of forest with larger trees and higher canopy cover should be retained, ensuring the availability of moist shelters. The amount of the cost for the whole monitoring, from plot installation to data analyses was 4872 Euros (about 5558 USD). Given the ease of application and inexpensiveness of this sampling protocol, we encourage its employment in similar situations, in order to gather useful information, which are essential to couple forestry practices with species conservation strategies.

Photographic identification method (PIM) using natural body marks: A simple tool to make a long story short

Individual marking allows researchers to collect important data using Mark-recapture method. Photographic Identification Method (PIM) has obtained favorable opinions, being effective and less intrusive with respect to traditional marking procedures. Here, we developed and tested the efficacy of a software-aided PIM based on different characteristics, which act as natural marks. Target species were the northern spectacled salamander (Salamandrina perspicillata, Amphibia: Urodela) and the stag beetle (Lucanus cervus, Coleoptera: Lucanidae). We considered as natural mark the ventral pattern of S. perspicillata and, for the first time in L. cervus, the presence/number of small denticles on male mandibles. We selected 4 independent characteristics, each one with several states, for each species. We asked participants, with different degree of familiarity with the species, to identify recaptured individuals through manual PIM. To assess the advantages of the software, we compared the time spent by experts of the species to identify an individual through manual PIM and software-aided PIM. We further compared our semi-automated method with a fully automated one. The software efficiency was calculated by increasing the number of characteristics used to filter individuals from the database (from 1 to 4). The chosen characteristics proved their reliability for individual identification and can be used as natural marks. The proposed software-aided PIM shorten the identification time, does not led to mismatching errors and proved its efficacy regardless of the taxa/characteristic used. Thus, this tool can be consider a more practical alternative to conventional marking techniques, in particular for sensitive and threatened species that need quick and effective monitoring programs carried out with non-invasive sampling methods.

Olim palus , where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the Circeo National Park (Central Italy)

The Circeo National Park lies in a territory that was deeply shaped by human activity, and represents one of the few remaining patches of plain wetland habitat in Central Italy. In this study distribution and few demographic information of the amphibians in the Park were provided. Seven species and 25 bibliographic and 84 original breeding sites were recorded, and population size estimations were carried out for a population of these three species: Pelophylax sinkl esculentus , Bufo balearicus and Rana dalmatina . For the studied populations of pool frog and green toad the operational sex ratio and the demographic effective population size was also estimated. For Rana dalmatina , which is strictly associated to forest environment, a positive and significant correlation between the number of egg clutches and maximum depth of the swamps was found. The State plain forest is the most important habitat for amphibians’ conservation in the park. The occurrence of dangerous alien species was investigated and they are evaluated as the major threat for amphibians in the park, especially the crayfish Procambarus clarkii in the State plain forest. Index of Calling Survey were performed for anurans and the medians did not differ among species. The potential distribution of amphibians in the Park was evaluated by building a species distribution model. Finally, the absence of three species reported in literature in the 60’s of the last century ( Bombina pachypus, Salamandrina perspicillata, Rana italica ) is also discussed.

Habitat trees and salamanders: Conservation and management implications in temperate forests

Among vertebrates the concept of “habitat tree” in temperate forests, involving tree size and microhabitat occurrence, has been investigated mainly for birds and mammals. However, trees are also used by many amphibian species or sometimes by whole amphibian families that have evolved adaptations for living on trees. While there is a self-evident link between arboreal amphibians, which occur mainly in tropical forests, and trees, the relationship between trees and forest ground-dwelling amphibians is less widely studied and more difficult to understand. As a consequence, the effect of forestry practices on amphibian communities are commonly considered at the landscape scale, identifying habitat alteration and loss as major threats. We provide the first evidence that the combination of forestry practices and species conservation strategies may act at a smaller scale than those traditionally adopted: the single-tree scale. Our study focused on the Italian endemic spectacled salamander (Salamandrina perspicillata). In a hectare forest stand in central Italy we marked nearly 400 trees individually and for each we measured the DBH, the number of buttresses in the stump (BUT) and the number of cavities between soil and stump (HOL). Salamanders were sampled and individually marked at each tree in eleven sampling occasions, during 2013 and 2014. We discovered that the concept of “habitat tree” may also hold for salamanders, and that DBH and HOL are the best predictors of tree suitability for salamanders. Moreover, using the number of captures for the same individuals on the same tree, occurring in different years, we were able to distinguish between trees that act as permanent or temporary shelters for salamanders. Permanent shelters were not only used by the same individuals in different years, but also hosted a larger number of salamanders, and were characterized by a larger number of HOL. Our findings may be considered of importance for drawing up forest management plans and achieving conservation objectives. During forest harvest operations, for the purpose of salamander conservation we suggest retaining trees with a larger DBH (>30cm) and a high number of HOL. Finally, since BUT is highly correlated with HOL but is less sensitive to variations during time, we suggest this parameter be used as a proxy of HOL.

Environmental factors associated with amphibian breeding in streams and springs: effects of habitat and fish occurrence

Streams are among the most threatened aquatic habitats for amphibians. Amphibians often demonstrate a complex community structure and investigations are mainly performed on pond-dwelling species, whereas data regarding stream-dwelling species is still scarce. The aim of this study was to evaluate the relevance of stream features and the effects of the occurrence of predatory fish on the community structure of amphibians. To assess the occurrence of breeding among amphibians (in Northern Apennine streams), sampling was performed on 57 stream and spring sites between early March and late May 2014. The research process recorded the presence of four breeding species (Salamandra salamandra, Salamandrina perspicillata, Bufo bufo, and Rana italica) and discovered that the community structures of amphibians were significantly connected to different habitat features. The investigation showed that fish presence is the primary habitat feature that affects the communities of amphibians: B. bufo was the only amphibian species not affected by fish presence, while the other species avoided sites with fish. Sun exposure, depth of water and slope inclination played important roles in affecting amphibian breeding. Our study confirms that fish presence in small streams can be detrimental for different amphibian species.

Development of microsatellites for the genus Salamandrina: A tool to discriminate between northern and southern spectacled salamanders (Salamandrina perspicillata and Salamandrina terdigitata) and their hybrids

Ten microsatellite loci were developed for the northern spectacled salamander, Salamandrina perspicillata, and cross-amplification was obtained for the southern spectacled salamander, Salamandrina terdigitata. These two species are the sole representative of the genus Salamandrina, a threatened amphibian endemic to the Italian peninsula. The two salamanders show a parapatric distribution with a contact zone in Central Italy. All but one locus were polymorphic and six microsatellites were found diagnostic for species discrimination. We genotyped a total of 169 individuals. Allelic diversity was 5.1 ± 1.5 SE in S. perspicillata and 1.9 ± 0.31 SE in S. terdigitata. Mean observed and expected heterozygosity were 0.51 ± 0.06 SE and 0.61 ± 0.07 SE, respectively in the northern spectacled salamander and 0.35 ± 0.15 SE and 0.35 ± 0.09 SE, respectively in the southern spectacled salamander. These new markers will provide an excellent tool to assess population structure and genetic diversity in Salamandrina and will be used to investigate natural hybridization events between northern and southern spectacled salamanders.

Generalisation within specialization: inter-individual diet variation in the only specialized salamander in the world.

Specialization is typically inferred at population and species level but in the last decade many authors highlighted this trait at the individual level, finding that generalist populations can be composed by both generalist and specialist individual. Despite hundreds of reported cases of individual specialization there is a complete lack of information on inter-individual diet variation in specialist species. We studied the diet of the Italian endemic Spectacled Salamander (Salamandrina perspicillata), in a temperate forest ecosystem, to disclose the realised trophic niche, prey selection strategy in function of phenotypic variation and inter-individual diet variation. Our results showed that Salamandrina is highly specialized on Collembola and the more specialized individuals are the better performing ones. Analyses of inter-individual diet variation showed that a subset of animals exhibited a broader trophic niche, adopting different foraging strategies. Our findings reflects the optimal foraging theory both at population and individual level, since animals in better physiological conditions are able to exploit the most profitable prey, suggesting that the two coexisting strategies are not equivalent. At last this species, feeding on decomposers of litter detritus, could play a key role determining litter retention rate, nutrient cycle and carbon sequestration.

She gets many and she chooses the best: polygynandry inSalamandrina perspicillata(Amphibia: Salamandridae)

Polyandry is a widespread mating strategy, found in a broad number of taxa. Among amphibians, polyandry, and multiple paternity as its direct consequence, is quite common in salamanders, especially within Ambystomatidae and Plethodontidae. In the suborder Salamadroidea the existence of two different types of spermatheca allows several kinds of polyandry strategies to appear. We used multilocus microsatellite genotyping to investigate the presence of polyandry and its effects on the paternity in a previously unstudied species with a terrestrial habit, Salamandrina perspicillata. We collected gravid females in their natural habitat and analysed the paternity of the offspring by using the software COLONY and GERUD. We found that all the analysed clutches had been fertilized by 2–4 males and that in every clutch one male had sired most of the offspring. Our results confirmed that polyandry is an important component of the mating system of this species, suggesting that females are able to recognize the sperm of the male that will provide a genetic benefit for their offspring. We found evidence of female cryptic choice based on males' genetic dissimilarity: (1) males who sire most of the offspring of a given female tend to be genetically different from their sexual partner; (2) a same male, when mated with two females, sired a proportion of the offspring inversely correlated with his genetic similarity to the female; (3) genetic dissimilarity between mating partners is positively correlated with offspring heterozygosity. According to the genetic compatibility model, we hypothesized that in the observed non resource-based mating system the indirect benefit for the offspring should reflect interactions between paternal and maternal genomes rather than the inheritance of the so-called ‘good genes’. This study suggests a polygynandrous mating system for the study species and provides the first report in a salamandrid species in natural condition that reproductive success of males is correlated with genetic dissimilarity between mates. Moreover, we found evidence of an offspring benefit (higher heterozygosity) derived from the most genetically dissimilar father.

APHIS: A new software for photo-matching in ecological studies

Unique body characteristics are increasingly used for individual recognition to avoid the effort and the potential negative effects of capture–mark–recapture technique. As a consequence there is a growing demand for computer procedures to assist users in photo-recognition of an individual. We present a new software for photo-matching developed to minimize the pre-processing time and maximize the speed of the matching procedure. In APHIS photos can be processed in batches of hundreds and users can select between two alternative matching procedures, one interactive, built as an extension of existing and freely available software, and one automatic. We assessed its performance in terms of individual recognition and time efficiency and illustrate its use with real capture-photo–recapture studies on a reptile and an amphibian species, the Balearic Lizard Podiarcis lilfordi and the Northern spectacled salamander Salamandrina perspicillata, with contrasting skin patterns.

Contrasted influences of moon phases on the reproduction and movement patterns of four amphibian species inhabiting different habitats in central Italy

Many studies have provided evidence that prey adjust their behaviour to adaptively balance the fitness effects of reproduction and predation risk. Nocturnal terrestrial animals should deal with a range of environmental conditions during the reproductive season at the breeding sites, including a variable amount of natural ambient light. High degrees of illumination are expected to minimize those behaviours that might increase the animal detection by predators. Therefore, under habitat variable brightness conditions and in different ecosystems, the above mentioned behaviours are expected to depend on the variation in predation risk. Although moon effects on amphibian biology have been recognized, the direction of this influence is rather controversial with evidences of both increased and depressed activity under full moon. We tested in four nocturnal amphibian species (Hyla intermedia, Rana dalmatina, Rana italica, Salamandrina perspicillata) the effects of different (i) light conditions and (ii) habitats (open land vs. dense forest) on the reproductive phenology. Our results showed that the effects of the lunar cycle on the study species are associated with the change in luminosity, and there is no evidence of an endogenous rhythm controlled by biological clocks. The habitat type conditioned the amphibian reproductive strategy in relation to moon phases. Open habitat breeders (e.g., ponds with no canopy cover) strongly avoided conditions with high brightness, whereas forest habitat breeders were apparently unaffected by the different moon phases. Indeed, for all the studied species no effects of the moon phase itself on the considered metrics were found. Rather, the considered amphibian species seem to be conditioned mainly by moonlight irrespective of the moon phase. The two anurans spawning in open habitat apparently adjust their oviposition timing by balancing the fitness effects of the risk to be detected by predators and the reproduction.

What goes in does not come out: different non-lethal dietary methods give contradictory interpretation of prey selectivity in amphibians

To date the most commonly used non-lethal methods in amphibian dietary studies are stomach flushing and faecal analysis. In this study, we compared the outcome of these two methods in interpreting prey selectivity when the available prey community in the environment is known. Stomach flushed contents and faeces were obtained from the same 27 individuals of the spectacled salamander (Salamandrina perspicillata) from a site in Central Italy. The interpretation of the population prey selection strategy varied in relation to the method used. Stomach content analysis suggested that salamanders were highly specialized on springtails, while faecal contents indicated a generalist trophic strategy. Prey selectivity indexes were also highly divergent: the analysis of stomach contents indicated a significant positive selection upon springtails, while exactly the opposite conclusion was obtained when faecal contents were analyzed. The results confirm that in amphibians, stomach analysis provides more reliable dietary data in comparison to faecal analysis. This is related to the fact that soft-bodied prey items tend to be more fully digested, disappearing in faeces while highly chitinized and less digestible prey taxa tend to increase their relative abundances in faeces.

Different season, different strategies: Feeding ecology of two syntopic forest-dwelling salamanders

Trophic niche may be the most important ecological dimension for some vertebrate groups and in particular for terrestrial amphibians, that are important predators of soil invertebrates. In general, resource partitioning occurs between syntopic species with similar ecological niches, and coexistence patterns seem to be regulated by temporal resource variability. However most of the generalization on foraging strategies of terrestrial salamanders are extrapolated from studies on New World temperate species, thus we investigated the seasonal effect of resource variation in an European forest ecosystem, in which two ecologically similar but phylogenetically distinct salamander species are found. The diet of adult and juvenile cave salamanders (Speleomantes strinati), and of adult spectacled salamander (Salamandrina perspicillata) was obtained by stomach flushing, and results showed large seasonal changes both in prey availability and in salamander realised trophic niche. Values of trophic diversity were similar and niche overlaps were large among all salamander groups in spring, during high prey availability. Conversely in autumn, when a two-fold reduction in prey biomass was observed, there was a clear niche partitioning as the smaller S. perspicillata shifted from a generalist to a specialized trophic strategy. Juvenile Speleomantes strinatii, that largely overlapped in size with S. perspicillata, did not show any change in diet, suggesting that the feeding strategies were species-specific and not size-mediated. The observed patterns of variation in feeding ecology indicate that similar predators may react differently to changing prey availability to enhance niche partitioning. We also observed an increased energy intake during autumn for S perspicillata and S. strinatii juveniles, possibly related to differences in microhabitat use and activity patterns.

Aspects of olfactory mediated orientation and communication in Salamandrina perspicillata (Amphibia Caudata): an experimental approach

Chemical communication is known to be of great importance in animals, particularly in the dynamic processes involving social behaviours and spatial orientation. Yet in many animal species exhibiting social behaviours, the mechanisms at the base of interindividual recognition and spatial orientation are virtually unknown. We addressed this issue in a series of laboratory experiments using Salamandrina perspicillata, an Italian endemic salamander, by means of Y-maze test. Individuals did not discriminate between the soil from the site of capture and other substrates. Salamanders were able to recognise their own chemical cue by selecting the arm housing the paper soaked in their body secretions, so demonstrating their capacity for following a trail of their own chemical cues. No interindividual (either homosexual or heterosexual) attraction was observed. Therefore, substrate-borne chemical cues produced by conspecifics of S. perspicillata seem to have no sociosexual significance for individuals from their own populations. Our results show a clear influence of chemical cues on individual behaviour in S. perspicillata. Although our data reveal such a behavioural trait, no evidence of gregariousness was found in the tested population.