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.

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.

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.

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.

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.

Loss of genetic diversity and increased embryonic mortality in non-native lizard populations.

Many populations are small and isolated with limited genetic variation and high risk of mating with close relatives. Inbreeding depression is suspected to contribute to extinction of wild populations, but the historical and demographic factors that contribute to reduced population viability are often difficult to tease apart. Replicated introduction events in non-native species can offer insights into this problem because they allow us to study how genetic variation and inbreeding depression are affected by demographic events (e.g. bottlenecks), genetic admixture and the extent and duration of isolation. Using detailed knowledge about the introduction history of 21 non-native populations of the wall lizard Podarcis muralis in England, we show greater loss of genetic diversity (estimated from microsatellite loci) in older populations and in populations from native regions of high diversity. Loss of genetic diversity was accompanied by higher embryonic mortality in non-native populations, suggesting that introduced populations are sufficiently inbred to jeopardize long-term viability. However, there was no statistical correlation between population-level genetic diversity and average embryonic mortality. Similarly, at the individual level, there was no correlation between female heterozygosity and clutch size, infertility or hatching success, or between embryo heterozygosity and mortality. We discuss these results in the context of human-mediated introductions and how the history of introductions can play a fundamental role in influencing individual and population fitness in non-native species.

Sexual selection drives asymmetric introgression in wall lizards.

Hybridisation is increasingly recognised as an important cause of diversification and adaptation. Here, we show how divergence in male secondary sexual characters between two lineages of the common wall lizard (Podarcis muralis) gives rise to strong asymmetries in male competitive ability and mating success, resulting in asymmetric hybridisation upon secondary contact. Combined with no negative effects of hybridisation on survival or reproductive characters in F1-hybrids, these results suggest that introgression should be asymmetric, resulting in the displacement of sexual characters of the sub-dominant lineage. This prediction was confirmed in two types of secondary contact, across a natural contact zone and in two introduced populations. Our study illustrates how divergence in sexually selected traits via male competition can determine the direction and extent of introgression, contributing to geographic patterns of genetic and phenotypic diversity.

Sexual dimorphism in digit length ratios in two lizard species.

Sexual dimorphism in digit length ratios has been reported for humans, a few other mammals, and two bird species. This dimorphism is thought to arise via an interaction between the prenatal exposure of the embryo to sex hormones and the Hox genes, which are highly conserved among vertebrates and control the development of both the appendices, including fingers and toes, and the urogenital system. In this study, we report on sexual dimorphism in 2D:3D, 2D:4D, and 3D:4D contralateral ratios of the forelimbs in two species of oviparous lizards, the common wall lizard (Podarcis muralis) and the tree skink (Mabuya planifrons), as measured on museum specimens. We found that male P. muralis had a larger 2D:4D ratio on both sides and larger 2D:3D ratio on the left side than females, whereas in M. planifrons, males had lower 2D:3D ratios than females on the left side. The two species show opposite patterns of sexual dimorphism in body size, males being larger than females in P. muralis, and the reverse in M. planifrons, suggesting that interspecific variation of sex differences in digit ratios could be associated with sex-specific growth trajectories. There was a limited evidence for directional asymmetry in digit ratios. Therefore, our findings provide the first evidence that digit ratios are sexually dimorphic in any reptile species and are consistent with the idea that the genetic link between limb development and the urogenital system had been established with the evolution of the earliest terrestrial tetrapods. Importantly, many lizard species with genetic sex determination, including the ones we studied, are oviparous and may represent valuable animal models for experimental tests of the association between prenatal exposure to androgens or estrogens and digit ratios.