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Copulatory organs are a key trait in reproductive compatibility and sexual isolation. The role of male genitalia in boosting mating success is well known and is often the outcome of behavioural and biological constraints, although no clear and common interpretation about their evolution appears broadly applicable. In snakes, hemipenial morphology has often been described under the perspective of sexual selection, taking into consideration both behavioural and morphological traits of both sexes. We investigated hemipenial morphology and ornamentation in the two subspecies of Hierophis viridiflavus, a male-male combating colubrid, and compared it to the sister species H. gemonensis, to assess intraspecific variation in size of genitalia and ornamentation richness. The male intromitted organ of this species is unilobed and bulbous, with rich ornamentations consisting of basal spines and apical calyculations. We detected no statistically significant difference in hemipenial size, basal spine count, and spine length between the two subspecies, suggesting that no copulatory barrier is present between the two clades. Although hemipenial morphology and anatomy do not seem suitable to assess intraspecific variability as shown in this study, they are often highly variable at the family or genus level, suggesting that the evolution of male genitalia is linked to phylogenetic relationships and that hemipenial divergence might be correlated to lineage splitting despite not being necessarily the main cause. Future studies should be aimed at investigating hemipenial morphology and anatomy across species under the perspective of comparative phylogeny and reproductive behaviours to address thoroughly the constraints of hemipenial evolution and development.

We here report the unexpected presence of the Italian wall lizard (Podarcis siculus campestris) on Gorgona Island, in the Parco Nazionale Arcipelago Toscano (Tuscan Archipelago, Tyrrhenian Sea, Tuscany, Central Italy). Field observations were carried out in 2020 confirming its presence on the island, where it had never been reported before. We recorded 37 GPS points of the species in three major areas of Gorgona (with 50 lizard records) and about 180 visual counts regarding all age classes (newborns, juveniles and adults). The species was found in the urban area (site of state prison) and in two grassy and bushed areas, around and along olive tree plantations. Seven individuals were captured and their tails were used to assess the sequence variation of the mitochondrial CYB gene. Biometrical parameters were also evaluated for six of these individuals. We detected three distinct CYB haplotypes that were compared to Podarcis siculus CYB sequences available in public databases. They resulted identical or phylogenetically closest to those found in mainland Tuscany. One haplotype, found in three specimens, was identical to one previously detected at Orti Bottagone (WWF Oasis in Piombino), while the other two haplotypes were most similar to haplotypes reported in the Giannella peninsula and Pisa, respectively.

How a native gecko manages to coexist with an alien rodent in the Mediterranean since thousands of years? What kind of eco-ethological adaptations or evolutionary adjustments enables this gecko to persist? The present study explores the interaction between the endemic European Leaf-toed gecko (Euleptes europaea) and the alien Black rat (Rattus rattus). In the last 30 years, we compared 26 populations inhabiting “rat” and “rat-free” islands and islets in Tunisia, Sardinia, Corsica and Southern France. Geckos’ populations can persist despite the occurrence of rats. In the presence of rats: 1) geckos’ average body size tends to decrease towards medium-sized individuals; 2) geckos shift their spatial behaviour avoiding to forage “in the open”; 3) geckos’ body condition is not affected by the presence of rats. Moreover, shortly after rats’ eradication, geckos’ population structure seems to change and larger sized geckos prevail while the spatial behaviour is much more conservative. The mechanisms driving the interactions between the two species still need to be explained. Rats could represent a stressor for geckos, compete for space, be pest vectors and even predators. Coexistence of natives and aliens requires adaptive plasticity and evolutionary adjustments. In contexts where the risk of reinvasion is high, eradication programs need to be carefully evaluated, since the arrival of “new rats” on an island could have much more damaging effects on the insular biota than those caused by the eradicated population.

Disentangling the effects of single releasers in animal communication is a demanding task because a releaser often consists of a combination of different key stimuli. Territorial communication in reptiles usually depends on visual, chemical, and acoustic stimuli, but the role of each of them depends on phylogeny. Lacertids are modern lizards that rely mainly on chemical cues for their communication, but they also use aggressive displays based on visual recognition. We experimentally tested the visual stimuli that release an aggressive response in the males of a typical lacertid, the common wall lizard (Podarcis muralis), testing the effects of silicone models and mirrored images in captivity. The response to models and control (a blank sheet) was not significantly different and these stimuli did not release any aggressive behaviour. On the contrary, the reflected image in a mirror caused overt aggression (i.e., bites against it) in 63% of tested individuals. The results clearly demonstrate the role of visual stimuli in territorial communication, but only as a combined effect of shape and motion, differently from other lizard families for which shape is enough to stimulate aggressive responses. Mirrors can be useful tools to investigate aggression related to physiological and morphological aspects in lacertid lizards.

Invasive species are a major threat to biodiversity and alien herpetofauna already caused conservation problems in Italy and in the world. Pelophylax kurtmuelleri (Gayda, 1940) is a water frog native to Southern Balkans, that was introduced in NW Italy (Liguria) in 1941, from where it has spread to Piedmont, Lombardy and recently to Emilia Romagna. Surprisingly, during its expansion, the Balkan frog seemed not to be able to cross the Po River (with a single exception). So, we investigated the reasons for such directional limitation of range expansion, focusing on the potential role of the Po River. Combining ecological niche and connectivity models, we adopted a two-step process in order to assess: (i) if the habitat suitability differs between the two sides of the Po Plain; (ii) if the Po River could act as a barrier in terms of dispersal. Results showed that the northern side of the plain seems less suitable than the southern one, even if many suitable patches occur along the main left tributaries of the Po River and in the hilly part of the plain. The Po River behaves like a barrier for the Balkan frog, but the only known point on the north bank of the river indicates that it may be able to only slow the dispersal and not to completely stop it. So, the most probable interpretation for the absence of the Balkan frog from the Northern Po Plain, is the combination of habitat suitability and connectivity issues: the former makes less probable that new viable populations can establish in the North, the latter decreases the northwards flux of the frogs. Given these findings, the Balkan frog seems able to spread in the whole Northern Italy and the colonization of the northern Po plain could happen shortly.

Photo-identification is used for individual recognition of several animal species. It gives the possibility to take photos of large species from a distance or to avoid invasive marking techniques in small animals. For amphibians, the use of non-invasive marking methods is even more relevant in the light of their global decline. Here we use the photo-identification data from a population of Triturus carnifex to validate the photo-identification software I3S Pattern. This recently developed utility has never been applied to amphibians. The software proved to be efficient and accurate for individual recognition for this species. Contrarily to the previous releases of the I3S family, I3S Pattern is particularly suitable for amphibians characterized by a complex individual pattern of large blotches or irregular spots, which are not readily identified by eye.

Ecological immunology attempts to explain the variability of immune response among individuals by invoking costs and trade-offs, which may optimize the immune defence against pathogens. In ectotherms body temperature is correlated to that of the surrounding environment, so that their entire physiology, including immune functions, is influenced by the environmental temperature. We used in vitro phytohaemoagglutinin (PHA) stimulation in order to assess the effects of temperature on cell mediated adaptive response in male and female Common wall lizards (Podarcis muralis). Cell cultures were prepared from blood samples, inoculated with PHA and incubated at 22°C, 25°C, 32°C, and 38°C for three days. PHA stimulation caused proliferation of T-lymphocytes, but the effect depended on the incubation temperature. Lymphocyte proliferation was significantly impaired at both 22°C and 38°C compared to 32°C, which represented the highest levels of activation. Furthermore, lymphocyte activation was more variable in males while females were less immune suppressed than males at low temperatures. Differences between sexes suggest a possible influence of steroid hormones.

The Montpellier snake Malpolon monspessulanus is a wide-ranging species that inhabits Western and Eastern Europe, North Africa and Middle East. Four clades have been recognised as two species, M. insignitus and M. monspessulanus, each with two subspecies. Clades have been substantially identified on the basis of molecular data, pholidosis and colouration, while morphometric traits have been ignored. We compared head shape of 54 specimens belonging to three out of the four clades (M. insignitus insignitus, M. i. fuscus, and M. monspessulanus monspessulanus) by means of geometric morphometrics. We found a significant differentiation: the supraocular and frontal area showed the largest amount of variation, being respectively much thinner in M. i. insignitus, a bit less thin in M. i. fuscus and definitely wider in M. m. monspessulanus. Our findings are fully in agreement with the genetic studies and phylogeny explains more than 20% of the observed variation, supporting the taxonomic distinction inside the genus Malpolon. The functional and/or adaptive meaning of the observed differences is not clear, but it seems unlikely that it may be related to diet. Combining morphological data with phylogeography and environmental features, we formulated an explanatory hypothesis that allowed a precise and testable prediction.

For their rapid and alarming decline, the Italian populations of the Italian spadefoot toad (Pelobates fuscus insubricus) are of high conservation importance. In this study we examined habitat use by the spadefoot toads in north-west Italy, where one of the largest remaining populations lives. We used compositional analysis and logistic regression models to elaborate land-use and soil composition in order to determine the habitat preferences of the spadefoot toads. We found that the two main variables predicting the occurrence of spadefoot toads in the study area were the percentages of sandy-loam soil texture and the presence of Entisols. Our results showed that spadefoot toads preferred soils that keep soft and malleable structure: Entisols with sand texture, where sand represents the major component, and mature soils, with a high degree of pedogenesis and a relatively high natural fertility and humidity. Conversely, Pelobates fuscus avoids Inceptisols, probably too hard to be dug by the species. This study demonstrates that, at least in the Italian range, the choice of areas in which to reintroduce P. fuscus, or where re-create favourable habitats for it, must take into account the soil types, which, in intensely cultivated areas, seems to be decisive for the possibility of survival of the species in the medium and long time.

Female common wall lizards (Podarcis muralis) typically reproduce annually and lay more than one clutch per season. In this study, we tested whether females store sperm between clutches and between years by manipulating mating opportunities of females through appropriate experiments. Our results revealed that females are definitely unable to store sperm for medium or long-term, suggesting they necessary need to repeatedly mate with males to fertilize their eggs. Finally, by comparing our results to other similar multi-clutched species, we conclude that sperm storage probably does not constitute a selective advantage for species with a promiscuous reproductive system based on multiple mating in populations with high densities.

Photographic identification is a promising marking technique alternative to the toe-clipping, but is time consuming, particularly when a large number of individuals is involved. For this reason several authors had frequently preferred the toe-clipping. In this study we analysed the black spot pattern of ventral scales of wall lizards (Podarcis muralis) and we showed that photographic identification is an effective method for recognizing individuals, and the error of this technique is much less than that of the toe-clipping arising from natural toe loss. Moreover, the numerically encoding of the black spot pattern may radically reduce the time needed to compare the pictures of large samples of individuals, solving one of the more important obstacle against the use of photographic identification.

Vipera berus (Linnaeus, 1758) is the terrestrial snake showing the widest distribution in the world, occuring from Great Britain, France and Northern Italy to the Sakhalin Island and North Korea (Nilson, 1980; Saint Girons, 1980; Nilson et al., 1994; Nilson & Andrén, 1997a). However, adders do not occur uniformly over their distribution area, but are scattered in several isolated populations (Nilson & Andrén, 1997a). Frequently, ecological traits of borderline and isolated populations differ from those living in the core area of the distribution range of the species, and might be subjected to higher risks of stochastic extinction and higher differentiation rates (Mayr, 1970). For example, meadow vipers (Vipera ursinii) show a highly fragmented distribution, many of isolated groups being different subspecies (Nilson & Andrén, 1997b, 2001).