Phenotypic divergence of the common toad (Bufo bufo) along an altitudinal gradient: evidence for local adaptation.

Variation in the environment can induce different patterns of genetic and phenotypic differentiation among populations. Both neutral processes and selection can influence phenotypic differentiation. Altitudinal phenotypic variation is of particular interest in disentangling the interplay between neutral processes and selection in the dynamics of local adaptation processes but remains little explored. We conducted a common garden experiment to study the phenotypic divergence in larval life-history traits among nine populations of the common toad (Bufo bufo) along an altitudinal gradient in France. We further used correlation among population pairwise estimates of quantitative trait (QST) and neutral genetic divergence (FST from neutral microsatellite markers), as well as altitudinal difference, to estimate the relative role of divergent selection and neutral genetic processes in phenotypic divergence. We provided evidence for a neutral genetic differentiation resulting from both isolation by distance and difference in altitude. We found evidence for phenotypic divergence along the altitudinal gradient (faster development, lower growth rate and smaller metamorphic size). The correlation between pairwise QSTs-FSTs and altitude differences suggested that this phenotypic differentiation was most likely driven by altitude-mediated selection rather than by neutral genetic processes. Moreover, we found different divergence patterns for larval traits, suggesting that different selective agents may act on these traits and/or selection on one trait may constrain the evolution on another through genetic correlation. Our study highlighted the need to design more integrative studies on the common toad to unravel the underlying processes of phenotypic divergence and its selective agents in the context of environmental clines.

Within- and among-population impact of genetic erosion on adult fitness-related traits in the European tree frog Hyla arborea.

Assessing in wild populations how fitness is impacted by inbreeding and genetic drift is a major goal for conservation biology. An approach to measure the detrimental effects of inbreeding on fitness is to estimate correlations between molecular variation and phenotypic performances within and among populations. Our study investigated the effect of individual multilocus heterozygosity on body size, body condition and reproductive investment of males (that is, chorus attendance) and females (that is, clutch mass and egg size) in both small fragmented and large non-fragmented populations of European tree frog (Hyla arborea). Because adult size and/or condition and reproductive investment are usually related, genetic erosion may have detrimental effects directly on reproductive investment, and also on individual body size and condition that in turn may affect reproductive investment. We confirmed that the reproductive investment was highly size-dependent for both sexes. Larger females invested more in offspring production, and larger males attended the chorus in the pond more often. Our results did not provide evidence for a decline in body size, condition and reproductive effort with decreased multilocus heterozygosity both within and among populations. We showed that the lack of heterozygosity-fitness correlations within populations probably resulted from low inbreeding levels (inferior to ca. 20% full-sib mating rate), even in the small fragmented populations. The detrimental effects of fixation load were either low in adults or hidden by environmental variation among populations. These findings will be useful to design specific management actions to improve population persistence.

Heterozygosity-fitness correlations among wild populations of European tree frog (Hyla arborea) detect fixation load.

Quantifying the impacts of inbreeding and genetic drift on fitness traits in fragmented populations is becoming a major goal in conservation biology. Such impacts occur at different levels and involve different sets of loci. Genetic drift randomly fixes slightly deleterious alleles leading to different fixation load among populations. By contrast, inbreeding depression arises from highly deleterious alleles in segregation within a population and creates variation among individuals. A popular approach is to measure correlations between molecular variation and phenotypic performances. This approach has been mainly used at the individual level to detect inbreeding depression within populations and sometimes at the population level but without consideration about the genetic processes measured. For the first time, we used in this study a molecular approach considering both the interpopulation and intrapopulation level to discriminate the relative importance of inbreeding depression vs. fixation load in isolated and non-fragmented populations of European tree frog (Hyla arborea), complemented with interpopulational crosses. We demonstrated that the positive correlations observed between genetic heterozygosity and larval performances on merged data were mainly caused by co-variations in genetic diversity and fixation load among populations rather than by inbreeding depression and segregating deleterious alleles within populations. Such a method is highly relevant in a conservation perspective because, depending on how populations lose fitness (inbreeding vs. fixation load), specific management actions may be designed to improve the persistence of populations.

Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea).

The detrimental effects of genetic erosion on small isolated populations are widely recognized contrary to their interactions with environmental changes. The ability of genotypes to plastically respond to variability is probably essential for the persistence of these populations. Genetic erosion impact may be exacerbated if inbreeding affects plastic responses or if their maintenance were at higher phenotypic costs. To understand the interplay 'genetic erosion-fitness-phenotypic plasticity', we experimentally compared, in different environments, the larval performances and plastic responses to predation of European tree frogs (Hyla arborea) from isolated and connected populations. Tadpoles from isolated populations were less performant, but the traits affected were environmental dependant. Heterosis observed in crosses between isolated populations allowed attributing their low fitness to inbreeding. Phenotypic plasticity can be maintained in the face of genetic erosion as inducible defences in response to predator were identical in all populations. However, the higher survival and developmental costs for isolated populations in harsh conditions may lead to an additional fitness loss for isolated populations.

Tracking a heterosis effect in the field: tadpole resistance to parasites in the water frog hybridogenetic complex.

Depending on the extent of evolutionary divergence among parent taxa, hybrids may suffer from a breakdown of co-adapted genes or may conversely exhibit vigour due to the heterosis effect, which confers advantages to increased genetic diversity. That last mechanism could explain the success of hybrids when hybridization zones are large and long lasting, such as in the water frog hybridization complex. In this hybridogenetic system, hybrid individuals exhibit full heterozygosity that makes it possible to investigate in situ the impact of hybridization. We have compared parasite intensity between hybrid Rana esculenta and parental R. lessonae individuals at the tadpole stage in two populations inhabiting contrasted habitats. We estimated intensity of Gyrinicola sp. (Nematoda) in the gut, Echinostome metacercariae in the kidneys and Haplometra cylindracea in the body cavity (both species belong to Trematoda). Despite high sampling effort, no variation in parasite intensity was detected between taxa, except a possible higher tolerance to H. cylindracea in hybrid tadpoles. The low effect of hybridization suggests efficient gene co-adaptation between the two genomes that could result from hemiclonal selection. Variation in infection intensity among ponds could support the Red Queen hypothesis.

Heterozygosity and parasite intensity: lung parasites in the water frog hybridization complex.

In hybridogenetic systems, hybrid individuals are fully heterozygous because one of the parental genomes is discarded from the germinal line before meiosis. Such systems offer the opportunity to investigate the influence of heterozygosity on susceptibility to parasites. We studied the intensity of lung parasites (the roundworm Rhabdias bufomis and the fluke Haplometra cylindracea) in 3 populations of water frogs of the Rana lessonae-esculenta complex in eastern France. In these mixed populations, hybrid frogs (R. esculenta) outnumbered parental ones (R. lessonae). Despite variation in parasite intensity and demographic variability among populations, the relationship between host age and intensity of parasitism suggests a higher susceptibility in parentals than in hybrids. Mortality is probably enhanced by lung parasites in parental frogs. On the other hand, while parental frogs harboured higher numbers of H. cylindracea than hybrid frogs, the latter had higher numbers of R. bufonis. Despite such discrepancies, these results support the hybrid resistance hypothesis, although other factors, such as differences in body size, age-related immunity, differential exposure risks and hemiclonal selection, could also contribute to the observed patterns of infection.

Are hybridogenetic complexes structured by habitat in water frogs?

The success and the evolutionary fate of hybridogenetic lineages are explained by both a generalistic heterosis hypothesis and an alternative hypothesis, the habitat segregation hypothesis. Because such hypotheses have rarely been tested at the level of whole habitats, our aim was to compare performances of two taxa within a hybridogenetic complex across diverse natural habitats. We took advantage of the waterfrog hybridogenetic complex (Rana esculenta and R. lessonae) by rearing tadpoles in natural contrasted habitats by means of enclosure experiments. We also monitored the frequency of each taxon in the waterfrog assemblages that naturally breed in the studied ponds. The hybridogenenetic taxon showed no evidence of broader tolerance as growth, development and physiology strongly varied in response to environmental heterogeneity. Our study reveals a differential success of the hybridogenetic taxon and its sexual host among environments. Moreover, hybridogenetic taxa rarely dominated the sexual species in natural assemblages. Consequently, our results show that the generalistic model does not explain the success of hybridogenetic lineages, but rather support the habitat segregation, among other alternative concepts.

Escape behaviour and ultimate causes of specific induced defences in an anuran tadpole.

Induced defences, such as the predator avoidance morphologies in amphibians, result from spatial or temporal variability in predation risk. One important component of this variability should be the difference in hunting strategies between predators. However, little is known about how specific and effective induced defences are to different types of predators. We analysed the impact of both pursuing (fish, Gasterosteus aculeatus) and sit-and-wait (dragonfly, Aeshna cyanea) predators on tadpole (Rana dalmatina) morphology and performance (viz locomotive performance and growth rate). We also investigated the potential benefits of the predator-induced phenotype in the presence of fish predators. Both predators induced deeper tail fins in tadpoles exposed to threat of predation, and stickleback presence also induced longer tails and deeper tail muscles. Morphological and behavioural differences resulted in better escape ability of stickleback-induced tadpoles, leading to improved survival in the face of stickleback predation. These results clearly indicate that specific morphological responses to different types of predators have evolved in R. dalmatina. The specific morphologies suggest low correlations between the traits involved in the defence. Independence of traits allows prey species to fine-tune their response according to current predation risk, so that the benefit of the defence can be maximal.

Tadpoles' responses to risk of fish introduction.

The introduction of predatory species, such as fish, in amphibian breeding sites is one of the many likely causes of amphibian population decline. The existence of inducible or constitutive (permanent) defences is expected to temper the lethal effects of fish on tadpoles. According to current theories on the evolution of phenotypic plasticity, the amphibian species that occur in habitats that are mostly or often fish-free were expected to display inducible defences, while the species that reproduce mainly in fish ponds ought to present constitutive defences. The tested species were Rana dalmatina, Hyla arborea and R. ridibunda, respectively. As expected, R. ridibunda tadpoles were less vulnerable to fish predation than the other species. Nevertheless, all three species exhibited morphological responses in the presence of fish. Their increases in tail area and decreases in body depth were similar. However, behaviour was only modified in R. dalmatina, which exhibited lower activity rates and higher refuge use in the presence of fish cues. Such lower activity rates were related to lower growth and developmental rates. While not detected in this experiment, the existence of inducible defences in the three species suggests the existence of costs of anti-predator defence. Those costs may have delayed effects so that increasing the frequency of fish ponds may exacerbate the impact of delayed costs whatever the species, suggesting that the dynamics of species that are not fish-adverse could also suffer from the introduction of fish.