Species-Specific Spatial Patterns of Variation in Sexual Dimorphism by Two Lizards Settled in the Same Geographic Context.

The evolution of sexual dimorphism (SD) results from intricate interactions between sexual and natural selections. Sexually selected traits are expected to depend on individual condition, while natural selected traits should not be. Islands offer an ideal context to test how these drivers interact with one another, as the size is a reliable proxy for resource availability. Here, we analysed SD in body size (snout-vent length) and head shape (assessed by geometric morphometric) in two species of lizards (Podarcis muralis and P. siculus) inhabiting the Tuscan archipelago (Central Italy). We found a strong SD variation among islands in both species. Furthermore, in P. muralis emerged some significant correlations between SD and island size, supporting the occurrence of possible effects of individual condition on SD. By contrast, SD in P. siculus followed opposite trajectories than in P. muralis, suggesting that in this species, natural selection could play a major role as a driver of SD. Our findings show that natural and sexual selection can interact in complex ways, and the responses are species-specific. Therefore, spatial patterns of variation in SD may strongly differ among species, even when they settle in the same geographic contest.

Condition- and context-dependent variation of sexual dimorphism across lizard populations at different spatial scales.

The evolution of sexual dimorphism (SD) is driven by intricate interplays between sexual and natural selection. When it comes to SD variation within populations, however, environmental factors play a major role. Sexually selected traits are expected to be strongly dependent on individual body condition, which is influenced by the local environment that individuals experience. As a consequence, the degree of SD may also depend on resource availability. Here, we investigated the potential drivers of SD expression at two sexually dimorphic morphometric traits, body size (snout vent length) and head shape (head geometric morphometrics), in the Italian wall lizard (Podarcis siculus). We assessed the existence of condition- and context-dependent SD across ten islands of the Aeolian archipelago (southern Italy), at within- and among-population scales. We observed strong geographical variation of SD among islands, and tested three potential SD predictors related to resource availability (individual body condition, ecosystem productivity, temperature). Body condition and ecosystem productivity were the main drivers of body size SD variation, and body condition was also the main driver for head shape SD. Our results highlight that the expression of SD in the Italian wall lizard is both condition- and context-dependent. These results are congruent at within- and among-populations scales highlighting that spatial multi-scale analysis represents a useful approach to understand patterns of SD expression.

Haemosporidian infections in wild populations of Podarcis muralis from the Italian Peninsula.

Parasites can significantly influence the ecology, behaviour and physiology of their hosts sometimes with remarkable effects on their survivorship. However, endemic parasites or those not associated with obvious clinical disease have been partly neglected in the past decades comparatively to the most pathogenic ones. Apicomplexa are an important example of blood parasites that have been broadly investigated, although it can be difficult to determine the effects of infections at the population level, especially in widespread species. Such is the case of the common wall lizard (Podarcis muralis). We investigated 61 populations across Italy between 2008 and 2017 and recorded snout­vent length, latitude, date of collection and took blood samples for parasite count. We modelled parasite prevalence and load in a Bayesian framework. Parasites were present in all populations but 1 and in 13 of them all individuals were parasitized. We recorded almost identical responses for probability of infection and parasite load in both sexes, directly proportional to body size and inversely proportional to latitude, with a peak in cooler months. Therefore, haemosporidians can be very common in P. muralis, although their presence can vary significantly. Moreover, such a high prevalence makes it necessary to investigate to what extent haemosporidians affect hosts' survivorship, taking into consideration abiotic and biotic factors such as temperature, hormone levels and immune response.

Population Genomics of Wall Lizards Reflects the Dynamic History of the Mediterranean Basin.

The Mediterranean Basin has experienced extensive change in geology and climate over the past six million years. Yet, the relative importance of key geological events for the distribution and genetic structure of the Mediterranean fauna remains poorly understood. Here, we use population genomic and phylogenomic analyses to establish the evolutionary history and genetic structure of common wall lizards (Podarcis muralis). This species is particularly informative because, in contrast to other Mediterranean lizards, it is widespread across the Iberian, Italian, and Balkan Peninsulas, and in extra-Mediterranean regions. We found strong support for six major lineages within P. muralis, which were largely discordant with the phylogenetic relationship of mitochondrial DNA. The most recent common ancestor of extant P. muralis was likely distributed in the Italian Peninsula, and experienced an "Out-of-Italy" expansion following the Messinian salinity crisis (∼5 Mya), resulting in the differentiation into the extant lineages on the Iberian, Italian, and Balkan Peninsulas. Introgression analysis revealed that both inter- and intraspecific gene flows have been pervasive throughout the evolutionary history of P. muralis. For example, the Southern Italy lineage has a hybrid origin, formed through admixture between the Central Italy lineage and an ancient lineage that was the sister to all other P. muralis. More recent genetic differentiation is associated with the onset of the Quaternary glaciations, which influenced population dynamics and genetic diversity of contemporary lineages. These results demonstrate the pervasive role of Mediterranean geology and climate for the evolutionary history and population genetic structure of extant species.

Climate Shapes the Geographic Distribution and Introgressive Spread of Color Ornamentation in Common Wall Lizards.

AbstractClimate can exert an effect on the strength of sexual selection, but empirical evidence is limited. Here, we tested whether climate predicts the geographic distribution and introgressive spread of sexually selected male color ornamentation across 114 populations of the common wall lizard, Podarcis muralis. Coloration was highly structured across the landscape and did not reflect genetic differentiation. Instead, color ornamentation was consistently exaggerated in hot and dry environments, suggesting that climate-driven selection maintains geographic variation in spite of gene flow. Introgression of color ornamentation into a distantly related lineage appears to be ongoing and was particularly pronounced in warm climates with wet winters and dry summers. Combined, these results suggest that sexual ornamentation is consistently favored in climates that allow a prolonged reproductive season and high and reliable opportunities for lizard activity. This pattern corroborates theoretical predictions that such climatic conditions reduce the temporal clustering of receptive females and increase male-male competition, resulting in strong sexual selection. In summary, we provide compelling evidence for the importance of climate for the evolution of color ornamentation, and we demonstrate that geographic variation in the strength of sexual selection influences introgression of this phenotype.

Patterns of variations in dorsal colouration of the Italian wall lizard Podarcis siculus.

Research on animal colouration has grown exponentially in the last decade thanks to multidisciplinary approaches. Most studies are focused on trade-offs between communication and mimicry, which represent the two main constraints and drivers of the evolution of body colourations. Reptiles are excellent model species for investigating this field of study and lizards in particular show great variability of body colourations and their functions. We studied the lizard Podarcis siculus, analysing the variations of dorsal colour of three populations and obtained clear patterns of seasonal and ontogenetical variation of dorsal colour. According to baseline colour, males were greener and brighter than females, although no difference in saturation was recorded. According to seasonal variations, analyses showed that both sexes significantly vary in colour over the year: males reached higher peaks of hue and saturation later than females during spring, while females showed higher peaks of brightness and reached earlier similarly to hue and saturation. Ontogenetic variations were recorded only in males, which become greener, less bright and saturated with growing size. Therefore, our results suggest the occurrence of two opposing strategies in colour expression between sexes: males' dorsal colouration plays a major role in communication, while females are more crypsis-oriented.

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.

Close encounters of the three morphs: Does color affect aggression in a polymorphic lizard?

Color polymorphism is genetically controlled, and the process generating and maintaining morphs can affect speciation/extinction rates. Color badges are useful signals in intraspecific communication because they convey information about alternative strategies and can potentially decrease unnecessary conflicts among different color morphs. Competition and aggressive interactions among color morphs can contribute to polymorphism maintenance. This could lead to an uneven spatial distribution of morphs in a population because the local frequency of each morph establishes the intensity of the competition and the fitness of each male. We used a polymorphic lizard, Podarcis muralis, to assess if aggression varies among morphs under two contrasting hypotheses: a heteromorphic versus homomorphic aggression. We used laboratory mirror tests after lizard color manipulation, and we verified the consistency of results with an analysis of the spatial distribution of morphs in a wild population. Both the experiments confirmed that aggression is more common during homomorphic than heteromorphic contests. The adoption of alternative behavioral strategies that minimize risks and costs could facilitate the stable coexistence of the phenotypes and reduce competition. A bias in aggression would advantage rarer morph, which would suffer less harassment by common morphs obtaining a fitness advantage. This process would be negatively-frequency-dependent and would stabilize polymorphism, possibly contributing to sympatric speciation.

Morph-specific seasonal variation of aggressive behaviour in a polymorphic lizard species.

The persistence of colour polymorphism (CP) within a given population is generally associated with the coexistence of alternative reproductive strategies, each one involving specific trade-offs among behavioural, morphological, physiological, and other life histories. Common wall lizard (Podarcis muralis), is a medium-sized diurnal lizard, showing CP in three main colours (yellow, white, and red) on throat and belly, and a morph-specific pattern for both immunocompetence and seasonal variation of T levels. Yellow males show low stamina with high plasma T levels at the beginning of the season, while white males show high stamina with a higher plasma T levels at the end of the season. We hypothesised the presence of two strategies: a risky one, characterised by high aggressiveness played by yellow-morph, and a conservative one by white morph with low aggressiveness. Thus, we tested the aggressive response to conspecifics of yellow and white morphs using a mirror inserted into their cage, mimicking an intrusion of a stranger in their territories, throughout the breeding season (from April to July, 117 trials). We considered three types of aggressive response, with different levels of aggressiveness: (i) bite against the image reflected in the mirror, (ii) seconds spent by the individuals into the half mirrored cage, and (iii) number of times the lizard entered the half mirrored cage. We also considered the number of tongue flicking as explorative behaviour variable. All lizards were tested after a period of acclimatisation to the captivity conditions. Results demonstrate that yellow males showed a higher aggressive response in the early season and a decrease aggressive response towards the end, whereas white males showed an opposite pattern.

Statistical methodology for the evaluation of leukocyte data in wild reptile populations: A case study with the common wall lizard (Podarcis muralis).

The leukocyte profile has the potential to be a reliable method to measure health conditions and stress in wild animals, but limitations occur because current knowledge on reference intervals is largely incomplete, especially because data come from studies on captive animals involving few individuals from single populations. Here we propose a general framework for achieving reliable leukocyte reference intervals, encompassing a set of internal and external factors, potentially affecting the leukogram. To do so, we present a systematic survey of the hematology of the common wall lizard, Podarcis muralis, involving 794 lizards from 54 populations over the whole geographic range of the species in Italy. Reference intervals for white blood cell (WBC) and leukocyte differential count were obtained by using linear mixed models in a Bayesian framework. The application of the procedure clearly showed that both internal (sex and size) and external (latitude and season) factors are a source of variation of leukocyte profile. Furthermore, the leukogram of common wall lizard has a strong variability among populations, which accounts for more than 50% of the whole variation. Consequently, some common assumptions used in studies on captive individuals are no longer supported in wild populations, namely, i) any group of individuals is a representative sample, ii) any population is representative of all others, iii) geographic clines do not occur over the species range, and iv) seasonal variation has limited effects. We encourage researchers aimed at the definition of leukocyte reference intervals for wild populations of reptiles to involve a large number of populations over a wide geographic range in ad hoc statistical models to disentangle local and geographic effects on leukocyte profile variation.

Spatial variation in gene flow across a hybrid zone reveals causes of reproductive isolation and asymmetric introgression in wall lizards.

Hybrid zones provide insights into the evolution of reproductive isolation. Sexual selection can contribute to the evolution of reproductive barriers, but it remains poorly understood how sexual traits impact gene flow in secondary contact. Here, we show that a recently evolved suite of sexual traits that function in male-male competition mediates gene flow between two lineages of wall lizards (Podarcis muralis). Gene flow was relatively low and asymmetric in the presence of exaggerated male morphology and coloration compared to when the lineages share the ancestral phenotype. Putative barrier loci were enriched in genomic regions that were highly differentiated between the two lineages and showed low concordance between the transects. The exception was a consistently low genetic exchange around ATXN1, a gene that modulates social behavior. We suggest that this gene may contribute to the male mate preferences that are known to cause lineage-assortative mating in this species. Although female choice modulates the degree of reproductive isolation in a variety of taxa, wall lizards demonstrate that both male-male competition and male mate choice can contribute to the extent of gene flow between lineages.

Seasonal Variations in Femoral Gland Secretions Reveals some Unexpected Correlations Between Protein and Lipid Components in a Lacertid Lizard.

Animals modulate intraspecific signal shape and intensity, notably during reproductive periods. Signal variability typically follows a seasonal scheme, traceable through the expression of visual, acoustic, chemical and behavioral patterns. The chemical channel is particularly important in lizards, as demonstrated by well-developed epidermal glands in the cloacal region that secrete lipids and proteins recognized by conspecifics. In males, the seasonal pattern of gland activity is underpinned by variation of circulating androgens. Changes in the composition of lipid secretions convey information about the signaler's quality (e.g., size, immunity). Presumably, individual identity is associated with a protein signature present in the femoral secretions, but this has been poorly investigated. For the first time, we assessed the seasonal variability of the protein signal in relation to plasma testosterone level (T), glandular activity and the concentration of provitamin D3 in the lipid fraction. We sampled 174 male common wall lizards (Podarcis muralis) over the entire activity season. An elevation of T was observed one to two months before the secretion peak of lipids during the mating season; such expected delay between hormonal fluctuation and maximal physiological response fits well with the assumption that provitamin D3 indicates individual quality. One-dimensional electrophoretic analysis of proteins showed that gel bands were preserved over the season with an invariant region; a result in agreement with the hypothesis that proteins are stable identity signals. However, the relative intensity of bands varied markedly, synchronously with that of lipid secretion pattern. These variations of protein secretion suggest additional roles of proteins, an issue that requires further studies.

Morph-specific protein patterns in the femoral gland secretions of a colour polymorphic lizard.

Colour polymorphism occurs when two or more genetically-based colour morphs permanently coexist within an interbreeding population. Colouration is usually associated to other life-history traits (ecological, physiological, behavioural, reproductive …) of the bearer, thus being the phenotypic marker of such set of genetic features. This visual badge may be used to inform conspecifics and to drive those decision making processes which may contribute maintaining colour polymorphism under sexual selection context. The importance of such information suggests that other communication modalities should be recruited to ensure its transfer in case visual cues were insufficient. Here, for the first time, we investigated the potential role of proteins from femoral gland secretions in signalling colour morph in a polymorphic lizard. As proteins are thought to convey identity-related information, they represent the ideal cues to build up the chemical modality used to badge colour morphs. We found strong evidence for the occurrence of morph-specific protein profiles in the three main colour-morphs of the common wall lizard, which showed both qualitative and quantitative differences in protein expression. As lizards are able to detect proteins by tongue-flicking and vomeronasal organ, this result support the hypothesis that colour polymorphic lizards may use a multimodal signal to inform about colour-morph.

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.

Morph-specific assortative mating in common wall lizard females.

Color polymorphism often is associated with alternative reproductive strategies and may reflect different adaptive optima that coexist within populations. The equilibrium among morph frequencies is maintained by the occurrence of opposite selective pressures (disruptive vs. stabilizing), which promote polymorphism while preserving gene flow. Sexual selection may contribute on both sides, particularly when morphs do not mate randomly. Reptiles offer a good model, notably lizards. Nevertheless, previous studies on mate choice in polymorphic lizards have generated contrasting results, with some studies suggesting that female morphs might tune their preference depending on environmental/social conditions such as crowding. We experimentally manipulated the number of individuals a female common wall lizard Podarcis muralis perceives around her, to test if females of different morphs (white or yellow) tune their choice for white and yellow males in order to maximize the probability that hatchlings follow the strategy best adapted to the population density. Results showed that crowding experienced by females did not affect mate choice, arguing against a flexible choice strategy by females. However, white females significantly associated with white males, whereas yellow females did not significantly associate with yellow males. Thus, sexual selection could contribute to the maintenance of color polymorphism in this species by a mix of assortative and non-assortative mating strategies, which could maintain the equilibrium between gene divergence and gene flow among morphs.

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.

Inter- and intra-population variability of the protein content of femoral gland secretions from a lacertid lizard.

Femoral glands of male lizards produce waxy secretions that are involved in inter- and intraspecific chemical communication. The main components of these secretions are proteins and lipids, the latter having been extensively studied and already associated to male quality. On the opposite, the composition and role of proteins are nearly unknown, the only available information coming from few studies on iguanids. These studies got the conclusion that proteins might have a communicative function, notably they could signal individual identity. A generalization of these findings requires the extension of protein analysis to other lizard families, and the primary detection of some patterns of individual variability. Using the common wall lizard Podarcis muralis as a model species, the protein fraction of the femoral pore secretions was investigated to provide the first characterization of this component in a lacertid lizard and to explore its source of variability, as a first step to support the hypothesized communicative role. Samples of proteins from femoral secretions were collected from 6 Italian populations and subjected to 1-dimensional electrophoresis. The binary vector of the band presence/absence was used to define the individual profiles. Protein fraction is found to have a structured pattern, with both an individual and a population component. Although the former supports the potential communicative role of proteins, the latter offers a double interpretation, phylogenetic or environmental, even though the phylogenetic effect seems more likely given the climatic resemblance of the considered sites. Further studies are necessary to shed light on both these issues.

Seasonal variations of plasma testosterone among colour-morph common wall lizards (Podarcis muralis).

Sexual steroids influence reproductive behaviours and promote secondary sexual traits. In male lizards, increasing levels of testosterone (T) bolster conspicuous colouration, stimulate territoriality, and trigger antagonistic interactions among rivals. Moreover, in colour polymorphic species, reproductive strategy, aggressiveness and T levels can differ between morphs. Therefore, T level is considered as an important mechanism that regulates the expression of colour polymorphism and sexual behaviours of males. But in the polymorphic territorial wall lizard (Podarcis muralis), a lack of relationship between morphs and aggressiveness challenges the notion that T plays such a role. To examine this issue, we compared adult T levels among three colour morphs (white, yellow and red) through repeated sampling during the mating season. High T levels were observed at the onset of the mating season followed by a significant decrease, a pattern documented in other lizard species. Mean T levels did not differ among morphs. However, yellow males maintained significantly higher T levels over time and displayed a stronger subsequent decline. Overall, in this species, seasonal T patterns differ among morphs, not mean values. Previous studies revealed that T suppresses the immune response; suggesting that a strong initial investment promoted by high T levels may trade-off against immunity (maintenance). Further experimental investigations are required to clarify the relationship between T and reproductive effort in polymorphic species that exhibit complex temporal pattern of T levels.

Morphological variation in salamanders and their potential response to climate change.

Despite the recognition that some species might quickly adapt to new conditions under climate change, demonstrating and predicting such a fundamental response is challenging. Morphological variations in response to climate may be caused by evolutionary changes or phenotypic plasticity, or both, but teasing apart these processes is difficult. Here, we built on the number of thoracic vertebrae (NTV) in ectothermic vertebrates, a known genetically based feature, to establish a link with body size and evaluate how climate change might affect the future morphological response of this group of species. First, we show that in old-world salamanders, NTV variation is strongly related to changes in body size. Secondly, using 22 salamander species as a case study, we found support for relationships between the spatial variation in selected bioclimatic variables and NTV for most of species. For 44% of species, precipitation and aridity were the predominant drivers of geographical variation of the NTV. Temperature features were dominant for 31% of species, while for 19% temperature and precipitation played a comparable role. This two-step analysis demonstrates that ectothermic vertebrates may evolve in response to climate change by modifying the number of thoracic vertebrae. These findings allow to develop scenarios for potential morphological evolution under future climate change and to identify areas and species in which the most marked evolutionary responses are expected. Resistance to climate change estimated from species distribution models was positively related to present-day species morphological response, suggesting that the ability of morphological evolution may play a role for species' persistence under climate change. The possibility that present-day capacity for local adaptation might help the resistance response to climate change can be integrated into analyses of the impact of global changes and should also be considered when planning management actions favouring species persistence.

On the identity of Chironius flavolineatus (Serpentes: Colubridae).

The snake genus Chironius Fitzinger, 1826 is endemic to the Neotropical region, occurring from Honduras to Uruguay and northeastern Argentina. Some species of the genus have taxonomic and/or nomenclatural problems, such as C. flavolineatus which lacks agreement in the literature about its authorship and type locality. Some researchers have been suggesting Jan (1863) as the author of the species since he first described C. flavolineatus based on two specimens. However, other researchers report that Jan's description is so incomplete that it is not possible to ascertain what snake he had in mind and therefore suggest Boettger (1885) as the author, since he was the first to provide a detailed description of the species. In the present study one of the syntypes of C. flavolineatus, supposedly destroyed in Second World War, was found. Thus, the taxonomic identity of C. flavolineatus was redefined, its lectotype was designated and the authorship of the taxa is attributed to Jan (1863).