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.

Impacts of temperature on O2 consumption of the Pyrenean brook newt (Calotriton asper) from populations along an elevational gradient.

Global warming impacts biodiversity worldwide, leading to species' adaptation, migration, or extinction. The population's persistence depends on the maintenance of essential activities, which is notably driven by phenotypic adaptation to local environments. Metabolic rate - that increases with temperature in ectotherms - is a key physiological proxy for the energy available to fuel individuals' activities. Cold-adapted ectotherms can exhibit a higher resting metabolism than warm-adapted ones to maintain functionality at higher elevations or latitudes, known as the metabolic cold-adaptation hypothesis. How climate change will affect metabolism in species inhabiting contrasting climates (cold or warm) is still a debate. Therefore, it is of high interest to assess the pace of metabolic responses to global warming among populations adapted to highly different baseline climatic conditions. Here, we conducted a physiological experiment in the endemic Pyrenean brook newt (Calotriton asper). We measured a proxy of standard metabolic rate (SMR) along a temperature gradient in individuals sampled among 6 populations located from 550 to 2189 m a.s.l. We demonstrated that SMR increased with temperature, but significantly diverged depending on populations' origins. The baseline and the slope of the relationship between SMR and temperature were both higher for high-elevation populations than for low-elevation populations. We discussed the stronger metabolic response observed in high-elevation populations suggesting a drop of performance in essential life activities for these individuals under current climate change. With the increase of metabolism as the climate warms, the metabolic-cold adaptation strategy selected in the past could compromise the sustainability of cold-adapted populations if short-term evolutionary responses do not allow to offset this evolutionary legacy.

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.

Influence of substrate types and morphological traits on movement behavior in a toad and newt species.

BACKGROUND: Inter-patch movements may lead to genetic mixing, decreasing both inbreeding and population extinction risks, and is hence a crucial aspect of amphibian meta-population dynamics. Traveling through heterogeneous landscapes might be particularly risky for amphibians. Understanding how these species perceive their environment and how they move in heterogeneous habitats is an essential step in explaining metapopulation dynamics and can be important for predicting species' responses to climate change and for conservation policy and management. METHODS: Using an experimental approach, the present study focused on the movement behavior (crossing speed and number of stops) on different substrates mimicking landscape components (human-made and natural substrates) in two amphibian species contrasting in locomotion mode: the common toad (Bufo bufo), a hopping and burrowing anuran and the marbled newt (Triturus marmoratus), a walking salamander. We tested the hypothesis that species reaction to substrate is dependent on specific ecological requirements or locomotion modes because of morphological and behavioral differences. RESULTS: In both species, substrate type influenced individual crossing speed, with individuals moving faster on soil than on concrete substrate. We also demonstrated that long-legged individuals moved faster than individuals with short legs. In both species, the number of stops was higher in females than in males. In common toads, the number of stops did not vary between substrates tested, whereas in marbled newts the number of stops was higher on concrete than on soil substrate. DISCUSSION: We highlighted that concrete substrate (mimicking roads) negatively affect the crossing speed of both studied species, with an effect potentially higher in marbled newts. Our findings corroborate negative effects of such heterogeneous landscapes on movement behavior of two amphibian species, which may have implications for the dynamics of metapopulations.

Intra-specific variability of hindlimb length in the palmate newt: an indicator of population isolation induced by habitat fragmentation?

Habitat fragmentation is one of the main drivers of global amphibian decline. Anthropogenic landscape elements can act as barriers, hindering the dispersal that is essential for maintaining gene flow between populations. Dispersal ability can be influenced by locomotor performance, which in turn can depend on morphological traits, such as hindlimb length (HLL) in amphibians. Here, we tested relationships between HLL and environmental variables--road types, forests and agricultural lands--among 35 sub-populations of palmate newts (Lissotriton helveticus) in southwestern France. We expected roads to select for short-legged newts due to a higher mortality of more mobile individuals (long-legged newts) when crossing roads. Accordingly, short-legged newts were found in the vicinity of roads, whereas long-legged newts were found closer to forests and in ponds close geographically to another water body. HLL in newts was hence influenced by habitat types in a heterogeneous landscape, and could therefore be used as an indicator of population isolation in a meta-population system.