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.

Reduction in the metabolic levels due to phenotypic plasticity in the Pyrenean newt, Calotriton asper, during cave colonization.

According to theories on cave adaptation, cave organisms are expected to develop a lower metabolic rate compared to surface organisms as an adaptation to food scarcity in the subterranean environments. To test this hypothesis, we compared the oxygen consumption rates of the surface and subterranean populations of a surface-dwelling species, the newt Calotriton asper, occasionally found in caves. In this study, we designed a new experimental setup in which animals with free movement were monitored for several days in a respirometer. First, we measured the metabolic rates of individuals from the surface and subterranean populations, both maintained for eight years in captivity in a natural cave. We then tested individuals from these populations immediately after they were caught and one year later while being maintained in the cave. We found that the surface individuals that acclimated to the cave significantly reduced their oxygen consumption, whereas individuals from the subterranean population maintained in the cave under a light/dark cycle did not significantly modify their metabolic rates. Second, we compared these metabolic rates to those of an obligate subterranean salamander (Proteus anguinus), a surface aquatic Urodel (Ambystoma mexicanum), and a fish species (Gobio occitaniae) as references for surface organisms from different phyla. As predicted, we found differences between the subterranean and surface species, and the metabolic rates of surface and subterranean C. asper populations were between those of the obligate subterranean and surface species. These results suggest that the plasticity of the metabolism observed in surface C. asper was neither directly due to food availability in our experiments nor the light/dark conditions, but due to static temperatures. Moreover, we suggest that this adjustment of the metabolic level at a temperature close to the thermal optimum may further allow individual species to cope with the food limitations of the subterranean environment.

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.

Author Correction: Agricultural landscapes and the Loire River influence the genetic structure of the marbled newt in Western France.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Agricultural landscapes and the Loire River influence the genetic structure of the marbled newt in Western France.

Amphibians are particularly sensitive to landscape fragmentation. Potential barriers between breeding sites can negatively influence the dispersal of individuals and increase genetic structure between populations. In this study, we genotyped 10 microsatellites for 334 marbled newts (Triturus marmoratus) at 11 different locations in Western France. Samples were collected in different regions with contrasting agricultural landscapes (low and high proportion of arable land in the north and south, respectively). We found a strong genetic structure between the northern and southern sampling sites. Isolation by distance was recorded after 62 km, but within the northern region, little or no genetic structure was detected over large distances (up to 114 km). Genetic structure at shorter distance (43 km) was found between sites situated in landscapes with larger amounts of arable lands. A significant positive relationship was found between the pairwise genetic distance (Fst) between sites and the amount of arable land together with the distance between sites. Our results suggest that the Loire River might act as a corridor for the marbled newt, while arable land might act as a barrier. Finally, although a large city is located between sampling sites, no effect was detected on population structure.

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.

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.

Evolution of Sex-Biased Dispersal.

Dispersal is central in ecology and evolution because it influences population regulation, adaptation, and speciation. In many species, dispersal is different between genders, leading to sex-biased dispersal. Several theoretical hypotheses have been proposed to explain the evolution of this bias: the resource competition hypothesis proposed by Greenwood, the local mate competition hypothesis, and the inbreeding avoidance hypothesis. Those hypotheses argued that the mating system should be the major factor explaining the direction of such bias. Sociality and the presence of handicap in genders (exaggerated sexual characters or parental care) have recently been proposed to be linked with the direction of this bias. We tested these expected coevolutions using a database of 257 species. Based on phylogenetic approaches, our findings marginally corroborated Greenwood's hypothesis by showing relationships between the direction of sex-biased dispersal, mating systems, and territoriality. More importantly, our results highlighted that the evolution of this bias was more linked to parental care and sexual dimorphism. These traits were also found to be associated with mating systems, suggesting that sexual asymmetry in morphology and parental care might be the main determinant of the evolution of sex-biased dispersal across species and not mating systems per se, as proposed in Greenwood's hypothesis.

A database of life-history traits of European amphibians.

In the current context of climate change and landscape fragmentation, efficient conservation strategies require the explicit consideration of life history traits. This is particularly true for amphibians, which are highly threatened worldwide, composed by more than 7400 species, which is constitute one of the most species-rich vertebrate groups. The collection of information on life history traits is difficult due to the ecology of species and remoteness of their habitats. It is therefore not surprising that our knowledge is limited, and missing information on certain life history traits are common for in this species group. We compiled data on amphibian life history traits from literature in an extensive database with morphological and behavioral traits, habitat preferences and movement abilities for 86 European amphibian species (50 Anuran and 36 Urodela species). When it were available, we reported data for males, females, juveniles and tadpoles. Our database may serve as an important starting point for further analyses regarding amphibian conservation.

A comparative analysis of dispersal syndromes in terrestrial and semi-terrestrial animals.

Dispersal, the behaviour ensuring gene flow, tends to covary with a number of morphological, ecological and behavioural traits. While species-specific dispersal behaviours are the product of each species' unique evolutionary history, there may be distinct interspecific patterns of covariation between dispersal and other traits ('dispersal syndromes') due to their shared evolutionary history or shared environments. Using dispersal, phylogeny and trait data for 15 terrestrial and semi-terrestrial animal Orders (> 700 species), we tested for the existence and consistency of dispersal syndromes across species. At this taxonomic scale, dispersal increased linearly with body size in omnivores, but decreased above a critical length in herbivores and carnivores. Species life history and ecology significantly influenced patterns of covariation, with higher phylogenetic signal of dispersal in aerial dispersers compared with ground dwellers and stronger evidence for dispersal syndromes in aerial dispersers and ectotherms, compared with ground dwellers and endotherms. Our results highlight the complex role of dispersal in the evolution of species life-history strategies: good dispersal ability was consistently associated with high fecundity and survival, and in aerial dispersers it was associated with early maturation. We discuss the consequences of these findings for species evolution and range shifts in response to future climate change.

Dispersal syndromes and the use of life-histories to predict dispersal.

Due to its impact on local adaptation, population functioning or range shifts, dispersal is considered a central process for population persistence and species evolution. However, measuring dispersal is complicated, which justifies the use of dispersal proxies. Although appealing, and despite its general relationship with dispersal, body size has however proven unsatisfactory as a dispersal proxy. Our hypothesis here is that, given the existence of dispersal syndromes, suites of life-history traits may be alternative, more appropriate proxies for dispersal. We tested this idea by using butterflies as a model system. We demonstrate that different elements of the dispersal process (i.e., individual movement rates, distances, and gene flow) are correlated with different suites of life-history traits: these various elements of dispersal form separate syndromes and must be considered real axes of a species' niche. We then showed that these syndromes allowed accurate predictions of dispersal. The use of life-history traits improved the precision of the inferences made from wing size alone by up to five times. Such trait-based predictions thus provided reliable dispersal inferences that can feed simulation models aiming at investigating the dynamics and evolution of butterfly populations, and possibly of other organisms, under environmental changes, to help their conservation.

Intra- and inter-individual variation in flight direction in a migratory butterfly co-vary with individual mobility.

Flight direction is a major component of an animal's migratory success. However, few studies have focused on variation in flight direction both between and within individuals, which is likely to be correlated with other traits implied in migration processes. We report patterns of intra- and inter-individual variation in flight direction in the large white butterfly Pieris brassicae. The presence of inter-individual variation in flight direction for individuals tested in the same conditions suggests that this trait is inherited in P. brassicae and we propose that a rapid loss of migratory skills may exist in the absence of selection for migration. The magnitude of intra-individual variation was negatively correlated to two surrogates of the potential for migration: mobility and wing length. Highly mobile and longed-winged individuals within the same family were found to fly in similar directions, whereas less mobile and short-winged individuals displayed divergent flight direction compared with the average direction of their kin. There was also a negative correlation between the variance to the mean flight direction of a family and its average mobility, but no correlation with wing length. We discuss these issues in terms of the evolution of traits potentially implied in both migration and dispersal in P. brassicae.

Population sex ratio and dispersal in experimental, two-patch metapopulations of butterflies.

1. Sex-biased dispersal, that is, the difference in dispersal between males and females, is thought to be the consequence of any divergent evolutionary responses between sexes. In anisogamous species, asymmetry in parental investment may lead to sexual conflict, which entails male-male competition (for sexual partner access), female-female competition (for feeding or egg-laying habitat patches) and/or male-female competition (antagonistic co-evolution). 2. As competition is one of the main causes of dispersal evolution, intra- and intersexual competition should have strong consequences on sex-biased dispersal. However, very few experimental studies, if any, have simultaneously addressed the effect of biased sex ratio on (i) each dispersal stage (emigration, transience, immigration), (ii) the dispersal phenotype and (iii) the colonization success of new habitat in order to fully separate the effects of varying male and female density. 3. Here, we used the Metatron, a unique experimental system composed of 48 interconnected enclosed patches dedicated to the study of dispersal in meta-ecosystems, to investigate the effect of sex ratio on dispersal in a butterfly. We created six populations with three different sex ratios in pairs of patches and recorded individual movements in these simple metapopulations. 4. Emigration was higher when the proportion of males was higher, and individuals reached the empty patch at a higher rate when the sex ratio in the departure patch was balanced. Males had a better dispersal success than females, which had a lower survival rate during dispersal and after colonization. We also showed that sex and wing size are major components of the dispersal response. 5. We did not observe sex-biased dispersal; our results thus suggest that female harassment by males and male-male competition might be more important mechanisms for the dispersal of females and males, than the search for a mating partner. Furthermore, the demonstration of a differential mortality between males and females during dispersal provides causal hypotheses of the evolution of sex-biased dispersal.

The Metatron: an experimental system to study dispersal and metaecosystems for terrestrial organisms.

Dispersal of organisms generates gene flow between populations. Identifying factors that influence dispersal will help predict how species will cope with rapid environmental change. We developed an innovative infrastructure, the Metatron, composed of 48 interconnected patches, designed for the study of terrestrial organism movement as a model for dispersal. Corridors between patches can be flexibly open or closed. Temperature, humidity and illuminance can be independently controlled within each patch. The modularity and adaptability of the Metatron provide the opportunity for robust experimental design for the study of 'meta-systems'. We describe a pilot experiment on populations of the butterfly Pieris brassicae and the lizard Zootoca vivipara in the Metatron. Both species survived and showed both disperser and resident phenotypes. The Metatron offers the opportunity to test theoretical models in spatial ecology.

How is dispersal integrated in life histories: a quantitative analysis using butterflies.

As dispersal plays a key role in gene flow among populations, its evolutionary dynamics under environmental changes is particularly important. The inter-dependency of dispersal with other life history traits may constrain dispersal evolution, and lead to the indirect selection of other traits as a by-product of this inter-dependency. Identifying the dispersal's relationships to other life-history traits will help to better understand the evolutionary dynamics of dispersal, and the consequences for species persistence and ecosystem functioning under global changes. Dispersal may be linked to other life-history traits as their respective evolutionary dynamics may be inter-dependent, or, because they are mechanistically related to each other. We identify traits that are predicted to co-vary with dispersal, and investigated the correlations that may constrain dispersal using published information on butterflies. Our quantitative analysis revealed that (1) dispersal directly correlated with demographic traits, mostly fecundity, whereas phylogenetic relationships among species had a negligible influence on this pattern, (2) gene flow and individual movements are correlated with ecological specialisation and body size, respectively and (3) routine movements only affected short-distance dispersal. Together, these results provide important insights into evolutionary dynamics under global environmental changes, and are directly applicable to biodiversity conservation.