Phylogenomic insights into the diversity and evolution of Palearctic vipers.

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of âˆ¼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research.

When carapace governs size: variation among age classes and individuals in a free-ranging ectotherm with delayed maturity.

Juvenile growth strongly impacts life-history traits during adulthood. Yet, in juveniles with delayed maturity, elusiveness has hindered age-specific studies of growth, precluding any detailed research on its consequences later in life. Different complex growth patterns have been extracted from captive animals, suggesting species-specific trajectories occur in free-ranging animals. How pronounced are growth and body size variation (VBS) throughout a long-lived ectotherm's life? Is VBS constant among age classes prior to maturity, or do compensatory and/or cumulative effects driven by long-lived-animal-specific strategies create distinct VBS cohorts, to ensure survival? To tackle the issue, we modelled growth data from continuous and dense annual capture-mark-recapture sampling (5096 body measurements of 1134 free-ranging individuals) of both immature and mature, long-lived Hermann's tortoises. We analysed population, cohort, and individual-based growth and VBS. Growth ring inferred ages were cross validated with annual recaptures in 289 juveniles. Analyses unravelled an S-shaped growth curve and identified three age cohorts across which VBS increases in a step-wise manner. Neonate-specific constraints and compensatory effects seem to control VBS until 4 years of age, possibly promoting survival with size. Subsequently, a hardened carapace takes over and cumulative effects fuelled by faster growth progressively increase VBS. Whereas ungulates are in a hurry to attain adult size before growth ceases (minimizing VBS), indeterminately growing tortoises can shape individual asymptotic sizes even after growth decelerates. Tortoise size is clearly shaped by age-specific ecological constraints; interestingly, it is likely the carapace that conducts the strategy, rather than maturity per se.

Oxidative stress biomarkers, cholinesterase activity and biotransformation enzymes in the liver of dice snake (Natrix tessellata Laurenti) during pre-hibernation and post-hibernation: A possible correlation with heavy metals in the environment.

We investigated in the liver of dice snakes during pre- and post-hibernation changes in the following antioxidant parameters: total, manganese and copper zinc containing superoxide dismutases (Tot SOD, MnSOD, CuZn SOD, respectively), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the concentrations of total glutathione (GSH) and sulfhydryl groups (-SH). In addition, we examined the expression of phase I biotransformation enzyme cytochrome P4501A (CYP1A) and the activity of phase II biotransformation enzyme glutathioneS-transferase (GST), the level of lipid peroxidation (by measuring the thiobarbituric acid-reactive substances (TBARS)), cholinesterase activity (ChE) and metallothionein expression (MT). We also measured the concentrations of heavy metals, including Al, Cd, As, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb and Zn in the water and snake liver during both periods. During the post-hibernation period, the activities of Tot SOD, CuZn SOD and GST and the concentration of GSH were significantly decreased, while GSH-Px and GR activities, the concentrations of -SH groups and TBARS were significantly increased. The activities of Mn SOD, CAT and ChE, and the relative amounts of CYP1A and MT did not significantly change during the investigated periods. The observed differences in the examined parameters probably represent adaptive physiological responses to sudden changes in tissue oxygenation during arousal from hibernation. Our findings also indicate that the accumulated metals modulated the responses of the examined parameters during the investigated periods.

Genetic variation and population structure in the endangered Hermann's tortoise: the roles of geography and human-mediated processes.

The Hermann's tortoise (Testudo hermanni) is an endangered land tortoise distributed in disjoint populations across Mediterranean Europe. We investigated its genetic variation by typing 1 mitochondrial locus and 9 nuclear microsatellites in approximately 300 individuals from 22 localities. Our goal was to understand the relative impact of natural and human-mediated processes in shaping the genetic structure and to identify the genetic priorities for the conservation of this species. We found that 1) all geographic areas are highly differentiated, mainly as a function of their distance but with a clear genetic discontinuity (F st values larger than 0.4) between the Eastern and the Western subspecies; 2) the contact zone between subspecies is located farthest to the west than previously believed, and it probably coincides with the delta of the largest Italian river; 3) extinction events due to climatic conditions in the Upper Palaeolithic and subsequent human-mediated translocations in the Neolithic possibly explain the unexpected similarity among Spain, Sicily, and Corsica. For conservation purposes, the large majority of genetic pools appears native although hybridization among subspecies, related to extensive 20th century trade of tortoises across Europe, is observed in Spain and some Italian samples. Most populations do not seem at immediate risk of low genetic variation, except the French population, which has very low nuclear genetic diversity (heterozygosity = 0.25) and where 50 out of 51 sampled animals shared the same mitochondrial sequence. In general, restocking and reintroduction plans should carefully consider the genetic background of the individuals.

Ontogeny, not prey availability, underlies allopatric venom variability in insular and mainland populations of Vipera ammodytes.

Allopatric populations living under distinct ecological conditions are excellent systems to infer factors underlying intraspecific venom variation. The venom composition of two populations of Vipera ammodytes, insular with a diet based on ectotherms and mainland with a diet based on ectotherms and endotherms, was compared considering the sex and age of individuals. Ten toxin families, dominated by PLA2, svMP, svSP, and DI, were identified through a bottom-up approach. The venom profiles of adult females and males were similar. Results from 58 individual SDS-PAGE profiles and venom pool analysis revealed significant differences between juveniles compared to subadults and adults. Two venom phenotypes were identified: a juvenile svMP-dominated and KUN-lacking phenotype and an adult PLA2/svMP-balanced and KUN-containing phenotype. Despite differences in prey availability (and, therefore, diet) between populations, no significant differences in venom composition were found. As the populations are geographically isolated, the lack of venom diversification could be explained by insufficient time for natural selection and/or genetic drift to act on the venom composition of island vipers. However, substantial differences in proteomes were observed when compared to venoms from geographically distant populations inhabiting different conditions. These findings highlight the need to consider ecological and evolutionary processes when studying venom variability. SIGNIFICANCE: This study provides the first comprehensive analysis of the venom composition of two allopatric populations of Vipera ammodytes, living under similar abiotic (climate) but distinct biotic (prey availability) conditions. The ontogenetic changes in venom composition, coupled with the lack of differences between sex and between populations, shed light on the main determinants of venom evolution in this medically important snake. Seven new proteomes may facilitate future comparative studies of snake venom evolution. This study highlights the importance of considering ecological and evolutionary factors to understand snake venom variation.

Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity.

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

Inside the shell: body composition of free-ranging tortoises (Testudo hermanni).

Body condition indices (BCI - mass scaled by size) are widely used in ecological studies. They presumably reflect variations of endogenous fat reserves in free-ranging animals. In the field, however, accurately quantifying internal body reserves is a difficult task. This is especially true in armoured animals where convenient clues that may guide BCI assessment (e.g. visible subcutaneous fat deposits) remain inaccessible. Alternatively, inclusive dissections may provide anatomical abacuses to estimate body reserves in living individuals. Sacrificing animals for this purpose is not acceptable. We opportunistically tested the ability of BCI to estimate body reserves in 13 free-ranging Hermann's tortoises (Gmelin, 1789) dissected soon after they died from natural causes. On average, BCI values were lower in dissected tortoises relative to living individuals (N > 10,000 measurements), but they remained within the range of variation of the studied populations. Shell mass relative to body mass was high and showed considerable inter-individual variation (33.5% to 52.3%). Stomach and digestive tract content represented another important and variable part of total body mass (4.4% to 14.5%). The contribution of fat bodies was negligible (0.0% to 0.5%). Overall, in the studied tortoises, variations of body condition are weakly determined by variations of fat stores. Other endogenous (e.g. muscles, visceral tissues, liver) and "exogenous" (e.g. digestive tract content, clutch) elements should be considered to better understand age and sex specific life-history trade-offs faced by chelonians.

Do different diets affect oxidative stress biomarkers and metal bioaccumulation in two snake species?

In this study we examined possible differences in heavy metal accumulation and oxidative stress parameters in the liver and muscle of two semi-aquatic snakes: grass snake (Natrix natrix) and dice snake (N. tessellata), that inhabit the same environment but differ in prey diversity. The obtained results revealed some interspecies, inter-tissue, prey-snake and prey-prey differences in heavy metal concentrations. Grass snakes pray contained significantly higher concentrations of Al, Cr and Fe as compared to food of dice snakes. Both investigated snakes accumulated generally lower concentrations of metals than their prey, indicating that they are not at risk of contaminant biomagnification. A significant interspecies difference in accumulation was observed only for Cu and Mn concentrations. On the other hand, analysis of oxidative stress biomarkers showed clear differences between the investigated snake species and the two investigated tissues. The liver of grass snake had increased superoxide dismutase, glutathione reductase and glutathione-S-transferase activities in comparison to dice snake. In muscle, a reverse trend was observed for the activities of these three enzymes, as well as for glutathione peroxidase activity. The higher number of significant correlations observed between oxidative stress biomarkers and heavy metal concentrations in grass snake points to upregulation of the antioxidative system (AOS), which resulted in a lower TBARS concentration. Results show that while the investigated snake species did not differ significantly in the accumulated metals, their defense mechanisms were different. This reveals the complexity of the AOS and points to the cooperation of different AOS components in individuals from natural populations.