Embryonic and juvenile snakes (Natrix maura, Linnaeus 1758) compensate for high elevation hypoxia via shifts in cardiovascular physiology and metabolism.

The colonization of novel environments requires a favorable response to conditions never, or rarely, encountered in recent evolutionary history. For example, populations colonizing upslope habitats must cope with lower atmospheric pressure at elevation, and thus reduced oxygen availability. The embryo stage in oviparous organisms is particularly susceptible, given its lack of mobility and limited gas exchange via diffusion through the eggshell and membranes. Especially little is known about responses of Lepidosaurian reptiles to reduced oxygen availability. To test the role of physiological plasticity during early development in response to high elevation hypoxia, we performed a transplant experiment with the viperine snake (Natrix maura, Linnaeus 1758). We maintained gravid females originating from low elevation populations (432 m above sea level [ASL]-normoxia) at both the elevation of origin and high elevation (2877 m ASL-extreme high elevation hypoxia; approximately 72% oxygen availability relative to sea level), then incubated egg clutches at both low and high elevation. Regardless of maternal exposure to hypoxia during gestation, embryos incubated at extreme high elevation exhibited altered developmental trajectories of cardiovascular function and metabolism across the incubation period, including a reduction in late-development egg mass. This physiological response may have contributed to the maintenance of similar incubation duration, hatching success, and hatchling body size compared to embryos incubated at low elevation. Nevertheless, after being maintained in hypoxia, juveniles exhibit reduced carbon dioxide production relative to oxygen consumption, suggesting altered energy pathways compared to juveniles maintained in normoxia. These findings highlight the role of physiological plasticity in maintaining rates of survival and fitness-relevant phenotypes in novel environments.

High-elevation hypoxia impacts perinatal physiology and performance in a potential montane colonizer.

Climate change is generating range shifts in many organisms, notably along the elevational gradient in mountainous environments. However, moving up in elevation exposes organisms to lower oxygen availability, which may reduce the successful reproduction and development of oviparous organisms. To test this possibility in an upward-colonizing species, we artificially incubated developing embryos of the viperine snake (Natrix maura) using a split-clutch design, in conditions of extreme high elevation (hypoxia at 2877 m above sea level; 72% sea-level equivalent O2 availability) or low elevation (control group; i.e. normoxia at 436 m above sea level). Hatching success did not differ between the two treatments. Embryos developing at extreme high elevation had higher heart rates and hatched earlier, resulting in hatchlings that were smaller in body size and slower swimmers compared to their siblings incubated at lower elevation. Furthermore, post-hatching reciprocal transplant of juveniles showed that snakes which developed at extreme high elevation, when transferred back to low elevation, did not recover full performance compared to their siblings from the low elevation incubation treatment. These results suggest that incubation at extreme high elevation, including the effects of hypoxia, will not prevent oviparous ectotherms from producing viable young, but may pose significant physiological challenges on developing offspring in ovo. These early-life performance limitations imposed by extreme high elevation could have negative consequences on adult phenotypes, including on fitness-related traits.

Multiple glacial refugia and contemporary dispersal shape the genetic structure of an endemic amphibian from the Pyrenees.

Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species' present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

Isolation and characterization of fourteen polymorphic microsatellite markers in the viperine snake Natrix maura.

Nineteen polymorphic microsatellite loci were identified and developed for Natrix maura. Polymorphism was assessed for 120 individuals sampled across four sampling sites from the French Pyrenees Mountains. The number of alleles per locus ranged from 3 to 15, and expected heterozygosity per locus ranged from 0.227 to 0.863. We tested for deviation from Hardy-Weinberg equilibrium and linkage disequilibrium and assessed the presence of null alleles for all loci, resulting in a selection of 14 high-quality polymorphic markers. These markers will be extremely useful in identifying fine-scale genetic structures and providing insight into conservation management plans of this species.

Variation of preferred body temperatures along an altitudinal gradient: A multi-species study.

Temperature affects the physiological functions of ectotherms. To maintain optimal body temperature and ensure physiological performance, these organisms can use behavioral adjustments to keep the body temperature in their specific temperature range, so-called preferred temperature (Tpref). It is therefore crucial to describe and understand how Tpref vary within and amongst populations to predict the effects of climate change of altitudinal range shifts in organisms. We aimed at determining the altitudinal variations in Tpref in three ectothermic species (the Pyrenean brook salamander - a semi-aquatic and thigmothermic amphibian - the European common lizard and the wall lizard - both heliothermic species). Using an experimental approach where Tpref were measured along a temperature gradient in laboratory conditions, we used a cross-sectional approach to compare the variation of Tpref measured in populations sampled along the altitudinal gradient in the Pyrenees. We hypothesized a complex and highly variable intra-specific response of Tpref along geographical clines, with a positive relationship between Tpref and altitude (as predicted by the countergradient variation), the reverse pattern (referring to the adaptation of local optima hypothesis), or no relationship at all. Our results corroborated partially the countergradient hypothesis in the salamander (middle to high elevation part). At high altitude level, individuals may compensate for lower opportunities of favorable conditions by choosing a high temperature which maximizes their activities. However, populations from low elevation level hence better supported the adaptation of local optima hypothesis, such as both lizard species, for which Tpref tended instead to decrease with altitude. Lizards from cold climates may be physiologically adapted to low temperature, with the ability to reach optimal functioning at lower temperature than individuals from low altitude. Our findings suggest that predicting future niche models should therefore take into account the variability of Tpref, where species might be differently affected by global warming.