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.

Plastic responses to warmer climates: a semi-natural experiment on lizard populations.

Facing warming environments, species can exhibit plastic or microevolutionary changes in their thermal physiology to adapt to novel climates. Here, using semi-natural mesocosms, we experimentally investigated over two successive years whether a 2°C-warmer climate produces selective and inter- and intragenerational plastic changes in the thermal traits (preferred temperature and dorsal coloration) of the lizard Zootoca vivipara. In a warmer climate, the dorsal darkness, dorsal contrast, and preferred temperature of adults plastically decreased and covariances between these traits were disrupted. While selection gradients were overall weak, selection gradients for darkness were slightly different between climates and in the opposite direction to plastic changes. Contrary to adults, male juveniles were darker in warmer climates either through plasticity or selection and this effect was strengthened by intergenerational plasticity when juveniles' mothers also experienced warmer climates. While the plastic changes in adult thermal traits alleviate the immediate overheating costs of warming, its opposite direction to selective gradients and to juveniles' phenotypic responses may slow down evolutionary shifts toward phenotypes that are better adapted to future climates. Our study demonstrates the importance of considering inter- and intragenerational plasticity along with selective processes to better understand adaptation and population dynamics in light of climate change.

Investigating the genetic basis of vertebrate dispersal combining RNA-seq, RAD-seq and quantitative genetics.

Although animal dispersal is known to play key roles in ecological and evolutionary processes such as colonization, population extinction and local adaptation, little is known about its genetic basis, particularly in vertebrates. Untapping the genetic basis of dispersal should deepen our understanding of how dispersal behaviour evolves, the molecular mechanisms that regulate it and link it to other phenotypic aspects in order to form the so-called dispersal syndromes. Here, we comprehensively combined quantitative genetics, genome-wide sequencing and transcriptome sequencing to investigate the genetic basis of natal dispersal in a known ecological and evolutionary model of vertebrate dispersal: the common lizard, Zootoca vivipara. Our study supports the heritability of dispersal in semi-natural populations, with less variation attributable to maternal and natal environment effects. In addition, we found an association between natal dispersal and both variation in the carbonic anhydrase (CA10) gene, and in the expression of several genes (TGFB2, SLC6A4, NOS1) involved in central nervous system functioning. These findings suggest that neurotransmitters (serotonin and nitric oxide) are involved in the regulation of dispersal and shaping dispersal syndromes. Several genes from the circadian clock (CRY2, KCTD21) were also differentially expressed between disperser and resident lizards, supporting that the circadian rhythm, known to be involved in long-distance migration in other taxa, might affect dispersal as well. Since neuronal and circadian pathways are relatively well conserved across vertebrates, our results are likely to be generalisable, and we therefore encourage future studies to further investigate the role of these pathways in shaping dispersal in vertebrates.

Adaptive Maternal Effects Shape Offspring Phenotype and Survival in Natal Environments.

AbstractMaternal effects can give newborns a head start in life by adjusting natal phenotypes to natal environments, yet their strength and adaptiveness are often difficult to investigate in natural populations. Here, we studied anticipatory maternal effects and their adaptiveness in common lizards in a seminatural experimental system. Specifically, we investigated how maternal environments (i.e., vegetation cover) and maternal phenotype (i.e., activity levels and body length) can shape offspring phenotype. We further studied whether such maternal effects influenced offspring survival in natal environments varying with respect to vegetation cover, conspecific density, and, consequently, maternal fitness. More active females from dense vegetation habitats produced bigger offspring than their less active counterparts, the contrary being true for sparse vegetation habitats. Moreover, females from dense vegetation habitats produced more active offspring and more active offspring survived better in dense vegetation habitats, resulting in greater maternal fitness through maternal effects. These results suggest adaptive anticipatory maternal effects, induced by vegetation structure and mediated by activity levels that may shape early-life prospects in natal 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.

Mobility affects copulation and oviposition dynamics in Pieris brassicae in seminatural cages.

When, how often and for how long organisms mate can have strong consequences for individual fitness and are crucial aspects of evolutionary ecology. Such determinants are likely to be of even greater importance in monandrous species and species with short adult life stages. Previous work suggests that mobility, a key dispersal-related trait, may affect the dynamics of copulations, but few studies have investigated the impact of individual mobility on mating latency, copulation duration and oviposition latency simultaneously. In this paper, we monitored the copulation dynamics of 40 males and 40 females, as well as the oviposition dynamics of the females of the Large White butterfly Pieris brassicae, a facultative long-distance disperser butterfly. Individuals from a breeding were selected to create a uniform distribution of mobility and we recorded the timing, number and duration of all copulations in a semiexperimental system. We showed that mobility, measured as the time spent in flight under stressful conditions (a proxy of dispersal tendency), correlates with all aspects of copulation dynamics: mobile males and females mated earlier and for shorter periods than less mobile individuals. In turn, late mating females increased the time between copulation and oviposition. These results feed the previously described mobility syndrome of P. brassicae, involving morphological and physiological characters, with life-history traits. We suggest that the reduction of mating latency and copulation duration has an adaptive value in dispersing individuals, as their life expectancy might be shorter than that of sedentary individuals.

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.

Evolution of a butterfly dispersal syndrome.

The existence of dispersal syndromes contrasting disperser from resident phenotypes within populations has been intensively documented across taxa. However, how such suites of phenotypic traits emerge and are maintained is largely unknown, although deciphering the processes shaping the evolution of dispersal phenotypes is a key in ecology and evolution. In this study, we created artificial populations of a butterfly, in which we controlled for individual phenotypes and measured experimentally the roles of selection and genetic constraints on the correlations between dispersal-related traits: flight performance and wing morphology. We demonstrate that (i) trait covariations are not due to genetic correlations, (ii) the effects of selection are sex-specific, and (iii) both divergent and stabilizing selection maintain specific flight performance phenotypes and wing morphologies. Interestingly, some trait combinations are also favoured, depending on sex and fitness components. Moreover, we provide evidence for the role of (dis)assortative mating in the evolution of these dispersal-related traits. Our results suggest that dispersal syndromes may have high evolutionary potential, but also that they may be easily disrupted under particular environmental conditions.

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.

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.

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.