Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

Natural and anthropogenic disturbances cause profound alterations in organisms, inducing physiological adjustments to avoid, reduce, or remedy the impact of disturbances. In vertebrates, the stress response is regulated via neuroendocrine pathways, including the hypothalamic-pituitary-interrenal axis that regulates the secretion of glucocorticoids. Glucocorticoids have cascading effects on multiple physiological pathways, affecting the metabolic rate, reactive oxygen species production, or immune system. Determining the extent to which natural and anthropogenic environmental factors induce stress responses in vertebrates is of great importance in ecology and conservation biology. Here we study the physiological stress response in spadefoot toad tadpoles (Pelobates cultripes) against three levels of a series of natural and anthropogenic stressors common to many aquatic systems: salinity (0, 6, and 9 ppt), herbicide (0, 1, and 2 mg/L acid equivalent of glyphosate), water acidity (pH 4.5, 7.0, and 9.5), predators (absent, native, and invasive), and temperature (217, 257, and 29 degrees C). The physiological stress response was assessed examining corticosterone levels, standard metabolic rate, activity of antioxidant enzymes, oxidative cellular damage in lipids, and immunological status. We found that common stressors substantially altered the physiological state of tadpoles. In particular, salinity and herbicides cause dramatic physiological changes in tadpoles. Moreover, tadpoles reduced corticosterone levels in the presence of natural predators but did not do so against invasive predators, indicating a lack of innate recognition. Corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters to stress in this study. Anthropogenic perturbations of aquatic systems pose serious threats to larval amphibians even at nonlethal concentrations, judging from the marked physiological stress responses generated, and reveal the importance of incorporating physiological information onto conservation, ecological, and evolutionary studies.Peer Reviewe

Extreme reduction in body size and reproductive output associated with sandy substrates in two anuran species

Geographic variation in body size and reproductive traits has been reported in a wide range of organisms, including amphibians. Most studies have focused on latitudinal and/or altitudinal variation where differences in temperature and duration of the growing season are the main causes for population divergence. We describe a steep variation in body size and reproductive traits in two anuran species in southwestern Spain, associated with changes in the geological substrate. Pelobates cultripes and Bufo calamita (= Epidalea calamita) drastically reduced their size (a 71.6% and 76.1% reduction in body mass for P. cultripes and B. calamita, respectively) in just about 60 km. This extreme size reduction was more pronounced at the boundary between two different geological substrates (hercinic and sandy soil). Mean clutch mass, egg size, and clutch size were all smaller in B. calamita populations in the sandy environment. Likewise, clutch mass and egg size were both smaller in sandy P. cultripes populations. We observed a negative correlation between size-adjusted fecundity and egg size for both species, suggesting the existence of a reproductive trade-off that could explain the differences in reproductive allocation between populations and species. In P. cultripes, small-bodied populations had relatively higher fecundities and smaller eggs than large-bodied ones, whereas in B. calamita populations from the sandy area we found both populations with high fecundity and small eggs, and populations with low fecundity and large eggs. Common environmental effects associated with the sandy substrate produce a similar reduction in size in both toad species. © 2008 Koninklijke Brill NV, Leiden.Peer Reviewe

Phylogenetic analyses reveal unexpected patterns in the evolution of reproductive modes in frogs

Understanding phenotypic diversity requires not only identification of selective factors that favor origins of derived states, but also factors that favor retention of primitive states. Anurans (frogs and toads) exhibit a remarkable diversity of reproductive modes that is unique among terrestrial vertebrates. Here, we analyze the evolution of these modes, using comparative methods on a phylogeny and matched life-history database of 720 species, including most families and modes. As expected, modes with terrestrial eggs and aquatic larvae often precede direct development (terrestrial egg, no tadpole stage), but surprisingly, direct development evolves directly from aquatic breeding nearly as often. Modes with primitive exotrophic larvae (feeding outside the egg) frequently give rise to direct developers, whereas those with nonfeeding larvae (endotrophic) do not. Similarly, modes with eggs and larvae placed in locations protected from aquatic predators evolve frequently but rarely give rise to direct developers. Thus, frogs frequently bypass many seemingly intermediate stages in the evolution of direct development. We also find significant associations between terrestrial reproduction and reduced clutch size, larger egg size, reduced adult size, parental care, and occurrence in wetter and warmer regions. These associations may help explain the widespread retention of aquatic eggs and larvae, and the overall diversity of anuran reproductive modes. © 2012 The Society for the Study of Evolution.Peer Reviewe

The Amphibian Research in Sierra Norte Natural Park, sw. Spain

A nivel mundial, los anfibios están desapareciendo por causas muy diversas, algunas poco conocidas, como los efectos del cambio climático. Son, en general, especies muy sensibles a los cambios en el medio y, por tanto, resultan especialmente interesantes tanto para los investigadores como para todo aquel interesado en la conservación de la biodiversidad. Desde hace años, investigadores del CSIC y otros centros españoles (Universidad de Sevilla y Granada) y extranjeros (Universidad de Lisboa, Portugal, Universidad de Chile y la Universidad de Western Kentucky, USA) están realizando estudios sobre bioacústica, biología de la reproducción, variación geográfi ca y adaptaciones locales, morfología y dinámica poblacional de algunas especies presentes en el Parque Natural de la Sierra Norte. Los resultados más relevantes de tales estudios se resumen en estas páginas, incluyendo resultados preliminares de un proyecto nacional I+D+i, actualmente en marcha. Además, sus observaciones han permitido detectar y sugerir acciones que están permitiendo mejorar la conservación de algunas de estas poblaciones. Con ello esperamos contribuir a un mejor conocimiento y conservación de estas especies y su medio.Currently, there is a global decline of amphibians due to an array of different factors, including global warming. Amphibians are among the more sensitive vertebrate species to changes in the environment, what target them both for research and for anyone interested in biodiversity conservation. During the last decade, researchers from diff erent institutions, including CSIC, the University of Seville, the University of Granada, the University of Lisbon, the University of Chile, and Western Kentucky University, have conducted studies on population divergence and local adaptations, bioacoustics, reproductive biology, morphology, and population dynamics of some of the anuran species occurring in the Natural Park of Sierra Norte. In this paper, we summarize relevant results from these studies, including preliminary results from an ongoing I+D+i national project. These studies have already help us to detect and suggest some conservation actions for amphibians, which once implemented, will improve the conservation status of some of these amphibian populations. Our hope is to contribute to increase the knowledge and conservation of these species and their habitats

Contrasting patterns of quantitative and neutral genetic variation in locally adapted populations of the natterjack toad, Bufo calamita

The relative importance of natural selection and genetic drift in determining patterns of phenotypic diversity observed in nature is still unclear. The natterjack toad (Bufo calamita) is one of a few amphibian species capable of breeding in saline ponds, even though water salinity represents a considerable stress for them. Results from two common-garden experiments showed a pattern of geographic variation in embryonic salinity tolerance among populations from either fresh or brackish environments, consistent with the hypothesis of local adaptation. Full-sib analysis showed increased variation in survival among sibships within population for all populations as osmotic stress was increased (broad-sense heritability increased as salinity raised). Nevertheless, toads native to the brackish water environment had the highest overall survival under brackish conditions. Levels of population genetic differentiation for salinity tolerance were higher than those of neutral genetic differentiation, the latter obtained through the analysis of eight microsatellite loci. Microsatellite markers also revealed little population differentiation, lack of an isolation-by-distance pattern, and moderate gene flow connecting the populations. Therefore, environmental stress tolerance appears to have evolved in absence of geographic isolation, and consequently we reject the null hypothesis of neutral differentiation.Peer Reviewe

Risk-induced hatching timing showslow heritability and evolves independently of spontaneous hatching in red-eyed treefrogs

Plasticity in the timing of transitions between stages of complex life cycles allows organisms to adjust their growth and development to local environ- mental conditions. Genetic variation in such plasticity is common, but the evolution of context-dependent transition timing may be constrained by information reliability, lag-time and developmental constraints. We studied the genetic architecture of hatching plasticity in embryos of the red-eyed treefrog (Agalychnis callidryas) in response to simulated predator attacks using a series of paternal and maternal half-sibs from a captive breeding colony of wild-collected animals. We compared the developmental timing of induced early hatching across sibships and estimated cross-environment genetic correlations between induced and spontaneous hatching traits. Additive genetic variance for induced early hatching was very low, indicating a constraint on the short-term evolution of earlier hatching timing. This con- straint is likely related to the maturation of the hatching mechanism. The most plastic genotypes produced the most extreme spontaneous hatching phenotypes, indicating that developmental range, per se, is not constrained. Cross-environment genetic correlation in hatching timing was negligible, so the evolution of spontaneous hatching in this species has not depended on the evolution of risk-induced hatching and vice versa.Peer reviewe

Local adaptation of an anuran amphibian to osmotically stressful environments

Water salinity is an intense physiological stress for amphibians. However, some species, such as Bufo calamita, breed in both brackish and freshwater environments. Because selection under environmentally stressful conditions can promote local adaptation of populations, we examined the existence of geographic variation in water salinity tolerance among B. calamita populations from either fresh or brackish water ponds in Southern Spain. Comparisons were made throughout various ontogenetic stages. A combination of field transplant and common garden experiments showed that water salinity decreased survival probability of individuals in all populations, prolonged their larval period, and reduced their mass at metamorphosis. However, significant population x salinity interactions indicated that the population native to brackish water (Saline) had a higher salinity tolerance than the freshwater populations, suggesting local adaptation. Saline individuals transplanted to freshwater environments showed similar survival probabilities, length of larval period, and mass at metamorphosis than those native to freshwater. This indicates that increased tolerance to osmotic stress does not imply a loss of performance in freshwater, at least during the larval and juvenile phases. Despite the adaptive process apparently undergone by Saline, all populations still shared the same upper limit of embryonic stress tolerance (around 10 g/l), defining a window of salinity range within which selection can act. Significant differences in embryonic and larval survival in brackish water among sibships for all populations suggest the existence of a genetic basis for the osmotic tolerance.Peer Reviewe