Small Island Effects on the Thermal Biology of the Endemic Mediterranean Lizard Podarcis gaigeae.

Ectotherms are vastly affected by climatic conditions as they rely on external sources of heat to regulate their body temperature, and changes in their habitat thermal quality could seriously affect their overall biology. To overcome the problems of a thermally unfavorable habitat, lizards need to either adjust their thermoregulatory behavior or respond to directional selection and shift their preferred body temperatures. To assess the impact of habitat thermal quality on the thermoregulatory profile, we studied multiple islet and 'mainland' populations of the Skyros wall lizard Podarcis gaigeae, an endemic lacertid to Skyros Archipelago, Greece. We evaluated the effectiveness of thermoregulation (E) using the three main thermal parameters: body (Tb), operative (Te), and preferred (Tpref) temperatures. We first hypothesized that the spatial homogeneity, the scarcity of thermal shelters, and the exposure to higher winds on islets would result in more demanding climate conditions. Second, we anticipated that islet lizards would achieve higher E in response to the lower thermal quality therein. As hypothesized, thermal parameters differed between populations but not in the expected manner. Skyros 'mainland' habitats reached higher temperatures, had more intense fluctuations, and were of lower thermal quality. As a result, lizards showed higher accuracy, precision, and effectiveness of thermoregulation. Noteworthy, we found that lizards from different populations have shifted their thermal profile and preferred body temperatures to cope with the particular conditions prevailing in their habitats. The latter supports the labile view on the evolution of thermoregulation.

Origin matters: Using a local reference genome improves measures in population genomics.

Genome sequencing enables answering fundamental questions about the genetic basis of adaptation, population structure and epigenetic mechanisms. Yet, we usually need a suitable reference genome for mapping population-level resequencing data. In some model systems, multiple reference genomes are available, giving the challenging task of determining which reference genome best suits the data. Here, we compared the use of two different reference genomes for the three-spined stickleback (Gasterosteus aculeatus), one novel genome derived from a European gynogenetic individual and the published reference genome of a North American individual. Specifically, we investigated the impact of using a local reference versus one generated from a distinct lineage on several common population genomics analyses. Through mapping genome resequencing data of 60 sticklebacks from across Europe and North America, we demonstrate that genetic distance among samples and the reference genomes impacts downstream analyses. Using a local reference genome increased mapping efficiency and genotyping accuracy, effectively retaining more and better data. Despite comparable distributions of the metrics generated across the genome using SNP data (i.e. π, Tajima's D and FST ), window-based statistics using different references resulted in different outlier genes and enriched gene functions. A marker-based analysis of DNA methylation distributions had a comparably high overlap in outlier genes and functions, yet with distinct differences depending on the reference genome. Overall, our results highlight how using a local reference genome decreases reference bias to increase confidence in downstream analyses of the data. Such results have significant implications in all reference-genome-based population genomic analyses.

Tail regeneration alters the digestive performance of lizards.

Tissue regeneration is a fundamental evolutionary adaptation, which is well known in lizards that can regenerate their entire tail. However, numerous parameters of this process remain poorly understood. Lizard tail serves many functions. Thus, tail autotomy comes with many disadvantages and the need for quick regeneration is imperative. To provide the required energy and materials for caudal tissue building, lizards are expected to undergo a number of physiological and biochemical adjustments. Previous research showed that tail regeneration induces changes in the digestive process. Here, we investigated if and how tail regeneration affects the digestive performance in five wall lizard species deriving from mainland and island sites and questioned whether the association of tail regeneration and digestion is affected by species relationships or environmental features, including predation pressure. We expected that lizards from high predation environments would regenerate their tail faster and modify accordingly their digestive efficiency, prioritizing the digestion of proteins; the main building blocks for tissue repair. Second, we anticipated that the general food shortage on islands would inhibit the process. Our findings showed that all species shifted their digestive efficiency, as predicted. Elongation rate was higher in sites with stronger predation regime and this was also applied to the rate with which protein digestion raised. Gut passage time increases during regeneration so as to improve the nutrient absorbance, but among the islanders, the pace was more intense. The deviations between species should be attributed to the different ecological conditions prevailing on islands rather than to their phylogenetic relationships.

Experimental Parasite Infection Causes Genome-Wide Changes in DNA Methylation.

Parasites are arguably among the strongest drivers of natural selection, constraining hosts to evolve resistance and tolerance mechanisms. Although, the genetic basis of adaptation to parasite infection has been widely studied, little is known about how epigenetic changes contribute to parasite resistance and eventually, adaptation. Here, we investigated the role of host DNA methylation modifications to respond to parasite infections. In a controlled infection experiment, we used the three-spined stickleback fish, a model species for host-parasite studies, and their nematode parasite Camallanus lacustris. We showed that the levels of DNA methylation are higher in infected fish. Results furthermore suggest correlations between DNA methylation and shifts in key fitness and immune traits between infected and control fish, including respiratory burst and functional trans-generational traits such as the concentration of motile sperm. We revealed that genes associated with metabolic, developmental, and regulatory processes (cell death and apoptosis) were differentially methylated between infected and control fish. Interestingly, genes such as the neuropeptide FF receptor 2 and the integrin alpha 1 as well as molecular pathways including the Th1 and Th2 cell differentiation were hypermethylated in infected fish, suggesting parasite-mediated repression mechanisms of immune responses. Altogether, we demonstrate that parasite infection contributes to genome-wide DNA methylation modifications. Our study brings novel insights into the evolution of vertebrate immunity and suggests that epigenetic mechanisms are complementary to genetic responses against parasite-mediated selection.

Partitioning thermal habitat on a vertical rock, a herculean task.

Species occurring in sympatry have to effectively segregate their niche in order to co-exist. In the case of ectotherms in particular, the very important parameter of thermal biology has to be taken into account. Here we investigated the thermoregulatory effectiveness (E) of two endemic Greek lizards (Hellenolacerta graeca and Podarcis peloponnesiacus) that live syntopically on a rocky cliff in the Peloponnese. We presumed that the two species would select different microhabitats, to avoid interspecific competition, and follow a similar thermoregulation pattern as they experience the same conditions. We also expected that E values for both species would differ depending on the season. Overall, we found that the two species had similar E values for each season but differentiated partial thermoregulatory attributes. Though they both occurred in the same types of microhabitat, H. graeca selected higher sites (average 99cm above ground) than P. peloponnesiacus (average 44cm). Also, the latter achieved higher preferred temperatures during summer and winter. Finally, the effectiveness of thermoregulation for both species varied interseasonally and received its highest values during summer, in response to the lowest thermal quality that was observed then. Similar studies stress the importance of thermal shifts for ectotherm co-existence.

Living in sympatry: The effect of habitat partitioning on the thermoregulation of three Mediterranean lizards.

The ability for effective, accurate and precise thermoregulation is of paramount importance for ectotherms. Sympatric lizards often partition their niche and select different microhabitats. These microhabitats, however, usually differ in their thermal conditions and lizards have to adapt their thermoregulation behavior accordingly. Here, we evaluated the impact of habitat partitioning on the thermal biology of three syntopic, congeneric lacertids (Podarcis peloponnesiacus, P. tauricus and P. muralis) from central Peloponnese, Greece. We assessed thermoregulation effectiveness (E) using the three standard thermal parameters: body (Tb), operative (Te) and preferred (Tpref) temperatures. We hypothesized that the microhabitats used by each species would differ in thermal quality. We also predicted that all species would effectively thermoregulate, as they inhabit a thermally challenging mountain habitat. As expected, the partition of the habitat had an effect on the thermoregulation of lizards since microhabitats had different thermal qualities. All three species were effective and accurate thermoregulators but one of them achieved smaller E values as a result of the lower Tb in the field. This discrepancy could be attributed to the cooler (but more benign) thermal microhabitats that this species occupies.

Tail regeneration affects the digestive performance of a Mediterranean lizard.

In caudal autotomy, lizards shed their tail to escape from an attacking predator. Since the tail serves multiple functions, caudal regeneration is of pivotal importance. However, it is a demanding procedure that requires substantial energy and nutrients. Therefore, lizards have to increase energy income to fuel the extraordinary requirements of the regenerating tail. We presumed that autotomized lizards would adjust their digestion to acquire this additional energy. To clarify the effects of tail regeneration on digestion, we compared the digestive performance before autotomy, during regeneration, and after its completion. Tail regeneration indeed increased gut passage time but did not affect digestive performance in a uniform pattern: though protein income was maximized, lipid and sugar acquisition remained stable. This divergence in proteins may be attributed to their particular role in tail reconstruction, as they are the main building blocks for tissue formation.

The particularities of a remote islet shape the thermoregulatory profile of an endemic Mediterranean lizard.

Environmental temperatures considerably affect the reptilian ability for thermoregulation and harsh climatic conditions may impose a highly effective body temperature regulation to lizards. Such demanding conditions are more common to extreme mainland habitats (e.g. deserts or mountains). To the contrary, islands have more benign climate conditions thanks to the thermal buffering effect of the surrounding sea. However, this favorable effect may be eliminated in small size islets where the scarcity of thermal shelters and exposure to high winds create challenging conditions. Here we investigate the impact of a tough islet habitat on the thermoregulation of Podarcis levendis, a lacertid lizard endemic to two rocky islets in the north Cretan Sea, Greece. To evaluate the thermoregulatory effectiveness of P. levendis we measured operative and body temperatures in the field and the preferred body temperatures in the lab. Analyses of the thermal data revealed an accurate, precise, and effective thermoregulator, achieving very high thermoregulation values (E =0.91, de¯-db¯ =7.6). This high effectiveness comes to compensate living in an inhospitable habitat as the operative temperatures denote (de =7.79). Our findings, together with the limited published literature, suggested the lack of a general pattern for all insular lizards and indicated a possible deviation for islet habitats.

Immune responses of a wall lizard to whole-body exposure to radiofrequency electromagnetic radiation.

PURPOSE: During the last three decades, the number of devices that emit non-ionizing electromagnetic radiation (EMR) at the wireless communication spectrum has rapidly increased and possible effects on living organisms have become a major concern. The purpose of this study was to investigate the effects of radiofrequency EMR emitted by a widely used wireless communication device, namely the Digital Enhanced Communication Telephony (DECT) base, on the immune responses of the Aegean wall lizard (Podarcis erhardii). MATERIALS AND METHODS: Adult male lizards were exposed 24 h/day for 8 weeks to 1880-1900 MHz DECT base radiation at average electric field intensity of 3.2 V/m. Immune reactivity was assessed using the phytohemagglutinin (PHA) skin swelling and mixed lymphocyte reaction (MLR) tests. RESULTS: Our results revealed a noticeable suppression (approximately 45%) of inflammatory responses in EMR-exposed lizards compared to sham-exposed animals. T cell-mediated responses were marginally affected. CONCLUSION: Daily radiofrequency EMR exposure seems to affect, at least partially, the immunocompetence of the Aegean wall lizard.

Ecological explanations to island gigantism: dietary niche divergence, predation, and size in an endemic lizard.

Although rapid evolution of body size on islands has long been known, the ecological mechanisms behind this island phenomenon remain poorly understood. Diet is an important selective pressure for morphological divergence. Here we investigate if selection for novel diets has contributed to the multiple independent cases of island gigantism in the Skyros wall lizard (Podarcis gaigeae) and if diet, predation, or both factors best explain island gigantism. We combined data on body size, shape, bite force, and realized and available diets to address this. Several lines of evidence suggest that diet has contributed to the island gigantism. The larger islet lizards have relatively wider heads and higher bite performance in relation to mainland lizards than would be expected from size differences alone. The proportions of consumed and available hard prey are higher on islets than mainland localities, and lizard body size is significantly correlated with the proportion of hard prey. Furthermore, the main axis of divergence in head shape is significantly correlated with dietary divergence. Finally, a model with only diet and one including diet and predation regime explain body size divergence equally well. Our results suggest that diet is an important ecological factor behind insular body size divergence, but could be consistent with an additional role for predation.

Effects of insularity on digestion: living on islands induces shifts in physiological and morphological traits in island reptiles.

Living on islands entails numerous challenges for animals, among which resource scarcity stands out. In order to survive, animals have to optimize energy acquisition. We examined the impact of insularity on digestion comparing a series of physiological and morphological traits of adult males between insular and mainland populations of the Balkan green lizard. Island lizards had longer gastrointestinal tracts and gut passage times and higher digestive efficiencies. The dissection of the hindgut revealed an unexpected finding, the presence of cecal valves that were more frequent in island lizards. Thanks to all above islanders retain food for longer periods and thus maximize energy income and increase the amount of the extracted nutrients. That way, they secure energy income from the limited, in time and quantity, food resources of the islands.

Molecular systematics and historical biogeography of the green lizards (Lacerta) in Greece: insights from mitochondrial and nuclear DNA.

The green lizards of the genus Lacerta (Sauria, Lacertidae) comprise nine recognized species, which in Europe are mainly restricted to the southern peninsulas. Four of them (L. trilineata, L. viridis, L. bilineata and L. agilis) occur in Greece. The uncertainty of morphological diversification renders the taxonomic assignment into species and subspecies problematic. In this study sequence data derived from two mitochondrial (cytochrome b and 16S rRNA) genes and one nuclear (NKTR) gene were used to (a) evaluate the taxonomic status of the genus Lacerta in Greece with emphasis on L. trilineata group and (b) investigate the evolutionary history of the genus through the application of phylogenetic and phylogeographic analyses, using Gallotia and Timon as outgroups. The phylogenetic analyses revealed the existence of four major clades. The first clade corresponds to L. trilineata group, the second to L. media, the third to L. agilis and the fourth to a complex of L. viridis and L. bilineata. However, the produced phylogenetic relationships are not congruent with the current taxonomy, especially in the first clade in which L. trilineata appeared to be paraphyletic in regard to L. pamphylica. Six distinct lineages were inferred within L. trilineata, despite the current recognition of nine morphological subspecies, the genetic differentiation of which exceeds that of other Lacerta species, imposing a thorough taxonomic revision of the species. Our results suggested a rapid diversification of L. trilineata group during the late Miocene. We believe that the present distribution of the genus in Greece is the result of several dispersal and vicariant events that took place during the late Miocene and early Pliocene.