Piecing the barcoding puzzle of Palearctic water frogs (Pelophylax) sheds light on amphibian biogeography and global invasions.

Palearctic water frogs (genus Pelophylax) are an outstanding model in ecology and evolution, being widespread, speciose, either threatened or threatening to other species through biological invasions, and capable of siring hybrid offspring that escape the rules of sexual reproduction. Despite half a century of genetic research and hundreds of publications, the diversity, systematics and biogeography of Pelophylax still remain highly confusing, in no small part due to a lack of correspondence between studies. To provide a comprehensive overview, we gathered >13,000 sequences of barcoding genes from >1700 native and introduced localities and built multigene mitochondrial (~17 kb) and nuclear (~10 kb) phylogenies. We mapped all currently recognized taxa and their phylogeographic lineages (>40) to get a grasp on taxonomic issues, cyto-nuclear discordances, the genetic makeup of hybridogenetic hybrids, and the origins of introduced populations. Competing hypotheses for the molecular calibration were evaluated through plausibility tests, implementing a new approach relying on predictions from the anuran speciation continuum. Based on our timetree, we propose a new biogeographic paradigm for the Palearctic since the Paleogene, notably by attributing a prominent role to the dynamics of the Paratethys, a vast paleo-sea that extended over most of Europe. Furthermore, our results show that distinct marsh frog lineages from Eastern Europe, the Balkans, the Near East, and Central Asia (P. ridibundus ssp.) are naturally capable of inducing hybridogenesis with pool frogs (P. lessonae). We identified 14 alien lineages (mostly of P. ridibundus) over ~20 areas of invasions, especially in Western Europe, with genetic signatures disproportionally pointing to the Balkans and Anatolia as the regions of origins, in line with exporting records of the frog leg industry and the stocks of pet sellers. Pelophylax thus emerges as one of the most invasive amphibians worldwide, and deserves much higher conservation concern than currently given by the authorities fighting biological invasions.

Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

Encouraging news for in situ conservation: Translocation of salamander larvae has limited impacts on their skin microbiota.

The key role of symbiotic skin bacteria communities in amphibian resistance to emerging pathogens is well recognized, but factors leading to their dysbiosis are not fully understood. In particular, the potential effects of population translocations on the composition and diversity of hosts' skin microbiota have received little attention, although such transfers are widely carried out as a strategy for amphibian conservation. To characterize the potential reorganization of the microbiota over such a sudden environmental change, we conducted a common-garden experiment simulating reciprocal translocations of yellow-spotted salamander larvae across three lakes. We sequenced skin microbiota samples collected before and 15 days after the transfer. Using a database of antifungal isolates, we identified symbionts with known function against the pathogen Batrachochytrium dendrobatidis, a major driver of amphibian declines. Our results indicate an important reorganization of bacterial assemblages throughout ontogeny, with strong changes in composition, diversity and structure of the skin microbiota in both control and translocated individuals over the 15 days of monitoring. Unexpectedly, the diversity and community structure of the microbiota were not significantly affected by the translocation event, thus suggesting a strong resilience of skin bacterial communities to environmental change-at least across the time-window studied here. A few phylotypes were more abundant in the microbiota of translocated larvae, but no differences were found among pathogen-inhibiting symbionts. Taken together, our results support amphibian translocations as a promising strategy for this endangered animal class, with limited impact on their skin microbiota.

What makes a great invader? Anatomical traits as predictors of locomotor performance and metabolic rate in an invasive frog.

Invasive species are characterized by their ability to establish and spread in a new environment. In alien populations of anurans, dispersal and fitness-related traits such as endurance, burst performance and metabolism are key to their success. However, few studies have investigated inter-individual variation in these traits and more specifically have attempted to understand the drivers of variation in these traits. Associations of anatomical features may be excellent predictors of variation in performance and could be targets for selection or subject to trade-offs during invasions. In this study, we used marsh frogs (Pelophylax ridibundus), a species that has been introduced in many places outside its native range and which is now colonizing large areas of Western Europe. We first measured the inter-individual variation in resting metabolism, the time and distance they were able to jump until exhaustion, and their peak jump force, and then measured the mass of specific organs and lengths of body parts suspected to play a role in locomotion and metabolism. Among the 5000 bootstrap replicates on body size-corrected variables, our statistical models most often selected the stomach (75.42%), gonads (71.46%) and the kidneys (67.26%) as predictors of inter-individual variation in metabolism, and the gluteus maximus muscle (97.24%) mass was the most frequently selected predictor of jump force. However, endurance was poorly associated with the anatomical traits (R2distance=0.42, R2time=0.37). These findings suggest that selection on these predictors may lead to physiological changes that may affect the colonization, establishment and dispersal of these frogs.

May future climate change promote the invasion of the marsh frog? An integrative thermo-physiological study.

Climate change and invasive species are two major drivers of biodiversity loss and their interaction may lead to unprecedented further loss. Invasive ectotherms can be expected to tolerate temperature variation because of a broad thermal tolerance and may even benefit from warmer temperatures in their new ranges that better match their thermal preference. Multi-trait studies provide a valuable approach to elucidate the influence of temperature on the invasion process and offer insights into how climatic factors may facilitate or hinder the spread of invasive ectotherms. We here used marsh frogs, Pelophylax ridibundus, a species that is invading large areas of Western Europe but whose invasive potential has been underestimated. We measured the maximal and minimal temperatures to sustain physical activity, the preferred temperature, and the thermal dependence of their stamina and jumping performance in relation to the environmental temperatures observed in their invasive range. Our results showed that marsh frogs can withstand body temperatures that cover 100% of the annual temperature variation in the pond they live in and 77% of the observed current annual air temperature variation. Their preferred body temperature and performance optima were higher than the average temperature in their pond and the average air temperature experienced under the shade. These data suggest that invasive marsh frogs may benefit from a warmer climate. Broad thermal tolerances, combined with high thermal preferences and traits maximised at high temperatures, may allow this species to expand their activity period and colonise underexploited shaded habitat, thereby promoting their invasion success.

Repeated ecological and life cycle transitions make salamanders an ideal model for evolution and development.

Observations on the ontogeny and diversity of salamanders provided some of the earliest evidence that shifts in developmental trajectories have made a substantial contribution to the evolution of animal forms. Since the dawn of evo-devo there have been major advances in understanding developmental mechanisms, phylogenetic relationships, evolutionary models, and an appreciation for the impact of ecology on patterns of development (eco-evo-devo). Molecular phylogenetic analyses have converged on strong support for the majority of branches in the Salamander Tree of Life, which includes 764 described species. Ancestral reconstructions reveal repeated transitions between life cycle modes and ecologies. The salamander fossil record is scant, but key Mesozoic species support the antiquity of life cycle transitions in some families. Colonization of diverse habitats has promoted phenotypic diversification and sometimes convergence when similar environments have been independently invaded. However, unrelated lineages may follow different developmental pathways to arrive at convergent phenotypes. This article summarizes ecological and endocrine-based causes of life cycle transitions in salamanders, as well as consequences to body size, genome size, and skeletal structure. Salamanders offer a rich source of comparisons for understanding how the evolution of developmental patterns has led to phenotypic diversification following shifts to new adaptive zones.

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.

How to recover from a bad start: size at metamorphosis affects growth and survival in a tropical amphibian.

BACKGROUND: In species with complex life cycles, size at metamorphosis is a key life-history trait which reflects the complex interactions between costs and benefits of life in the aquatic and terrestrial environments. Whereas the effects of a deteriorating larval habitat (e.g. pond desiccation) on triggering an early metamorphosis have been extensively investigated in amphibians, the consequences of the resulting reduced size at metamorphosis on fitness in the post-metamorphic terrestrial stage remain poorly understood. We tested the hypothesis that a smaller size at metamorphosis negatively affects performance and survival in the ensuing terrestrial stage. Using as model a tropical amphibian (Ceratophrys stolzmanni) showing a large phenotypic plasticity in metamorphosing traits, we evaluated the effects of size at metamorphosis on fitness-related trophic and locomotor performance traits, as well as on growth and survival rates. RESULTS: Our results support the hypothesis that a larger size at metamorphosis is correlated with better survival and performance. The survival rate of large metamorphosing individuals was 95%, compared to 60% for those completing metamorphosis at a small size. Locomotor performance and gape size were positively correlated with body size, larger animals being more mobile and capable to ingest larger prey. However, smaller individuals achieved higher growth rates, thus reducing the size gap. CONCLUSIONS: Overall, size at metamorphosis affected profoundly the chances of survival in the short term, but smaller surviving individuals partly compensated their initial disadvantages by increasing growth rates.

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates).

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.

Fifteen shades of green: The evolution of Bufotes toads revisited.

The radiation of Palearctic green toads (Bufotes) holds great potential to evaluate the role of hybridization in phylogeography at multiple stages along the speciation continuum. With fifteen species representing three ploidy levels, this model system is particularly attractive to examine the causes and consequences of allopolyploidization, a prevalent yet enigmatic pathway towards hybrid speciation. Despite substantial efforts, the evolutionary history of this species complex remains largely blurred by the lack of consistency among the corresponding literature. To get a fresh, comprehensive view on Bufotes phylogeography, here we combined genome-wide multilocus analyses (RAD-seq) with an extensive compilation of mitochondrial, genome size, niche modelling, distribution and phenotypic (bioacoustics, morphometrics, toxin composition) datasets, representing hundreds of populations throughout Eurasia. We provide a fully resolved nuclear phylogeny for Bufotes and highlight exceptional cyto-nuclear discordances characteristic of complete mtDNA replacement (in 20% of species), mitochondrial surfing during post-glacial expansions, and the formation of homoploid hybrid populations. Moreover, we traced the origin of several allopolyploids down to species level, showing that all were exclusively fathered by the West Himalayan B. latastii but mothered by several diploid forms inhabiting Central Asian lowlands, an asymmetry consistent with hypotheses on mate choice and Dobzhansky-Muller incompatibilities. Their intermediate call phenotypes potentially allowed for rapid reproductive isolation, while toxin compositions converged towards the ecologically-closest parent. Across the radiation, we pinpoint a stepwise progression of reproductive isolation through time, with a threshold below which hybridizability is irrespective of divergence (<6My), above which species barely admix and eventually evolve different mating calls (6-10My), or can successfully cross-breed through allopolyploidization (>15My). Finally, we clarified the taxonomy of Bufotes (including genetic analyses of type series) and formally described two new species, B. cypriensis sp. nov. (endemic to Cyprus) and B. perrini sp. nov. (endemic to Central Asia). Embracing the genomic age, our framework marks the advent of a new exciting era for evolutionary research in these iconic amphibians.

Reproductive fitness consequences of progenesis: Sex-specific pay-offs in safe and risky environments.

Progenesis is considered to have an important role in evolution because it allows the retention of both a larval body size and shape in an adult morphology. However, the cost caused by the adoption of a progenetic process in both males and females remains to be explored to explain the success of progenesis and particularly its biased prevalence across the sexes and environments. Here, through an experimental approach, we used a facultative progenetic species, the palmate newt (Lissotriton helveticus) that can either mature at a small size and retain gills or mature after metamorphosis, to test three hypotheses for sex-specific pay-offs of progenesis in safe versus risky habitats. Goldfish were used because they caused a higher decline in progenetic than metamorphic newts. We determined that progenetic newts have a lower reproductive fitness than metamorphic newts. We also found that, when compared to metamorphs, progenetic males have lower reproductive activity than progenetic females and that predatory risk affects more progenetic than metamorphic newts. By identifying ultimate causes of the female-biased sex ratios found in nature, these results support the male escape hypothesis, that is the higher metamorphosis rate of progenetic males. They also highlight that although progenesis is advantageous in advancing the age at first reproduction, it also brings an immediate fitness cost and this, particularly, in hostile predatory environments. This means that whereas some environmental constraints could favour facultative progenesis, some others, such as predation, can ultimately counter-select progenesis. Altogether, these results improve our understanding of how developmental processes can affect the sexes differently and how species invasions can impair the success of alternative developmental phenotypes.

The 'male escape hypothesis': sex-biased metamorphosis in response to climatic drivers in a facultatively paedomorphic amphibian.

Paedomorphosis is a major evolutionary process that bypasses metamorphosis and allows reproduction in larvae. In newts and salamanders, it can be facultative with paedomorphs retaining gills and metamorphs dispersing. The evolution of these developmental processes is thought to have been driven by the costs and benefits of inhabiting aquatic versus terrestrial habitats. In this context, we aimed at testing the hypothesis that climatic drivers affect phenotypic transition and the difference across sexes because sex-ratio is biased in natural populations. Through a replicated laboratory experiment, we showed that paedomorphic palmate newts (Lissotriton helveticus) metamorphosed at a higher frequency when water availability decreased and metamorphosed earlier when temperature increased in these conditions. All responses were sex-biased, and males were more prone to change phenotype than females. Our work shows how climatic variables can affect facultative paedomorphosis and support theoretical models predicting life on land instead of in water. Moreover, because males metamorphose and leave water more often and earlier than females, these results, for the first time, give an experimental explanation for the rarity of male paedomorphosis (the 'male escape hypothesis') and suggest the importance of sex in the evolution of paedomorphosis versus metamorphosis.

Morphological Integration and Alternative Life History Strategies: A Case Study in a Facultatively Paedomorphic Newt.

Tetrapod limbs are serially homologous structures that represent a particularly interesting model for studies on morphological integration, i.e. the tendency of developmental systems to produce correlated variation. In newts, limbs develop at an early larval stage and grow continuously, including after the habitat transition from water to land following metamorphosis. However, aquatic and terrestrial environments impose different constraints and locomotor modes that could affect patterns of morphological integration and evolvability. We hypothesize that this would be the case for alternative heterochronic morphs in newts, i.e. aquatic paedomorphs that keep gills at the adult stage and adult metamorphs that are able to disperse on land. To this end, we analyzed patterns and strengths of correlations between homologous skeletal elements of the fore- and hindlimbs as well as among skeletal elements within limbs in both phenotypes in the alpine newt, Ichthyosaura alpestris. Our results showed that metamorphs and paedomorphs had similar, general patterns of limb integration. Partial correlations between homologous limb elements and within limb elements were higher in paedomorphs when compared to metamorphs. A decrease in partial correlation between homologous limb elements in metamorphs is accompanied with a higher evolvability of the terrestrial morph. All these results indicate that environmental demands shaped the patterns of morphological integration of alpine newt limbs and that the observed diversity in correlation structure could be related to a qualitative difference in the modes of locomotion between the morphs.

Using kernels and ecological niche modeling to delineate conservation areas in an endangered patch-breeding phenotype.

Efficient delineation of conservation areas is a great challenge in maintaining biodiversity. Kernel density estimators (KDEs) are a powerful tool in this perspective, but they have not been applied at the population level on patch-distributed organisms. This would be particularly worthy for species that need broad habitats beyond those where they can be sampled; such as terrestrial lands for pond-breeding amphibians. The aim of this study was to compare different approaches for the identification of suitable areas for conservation: KDE, ecological niche modelling, and a combination of KDE and niche models. Paedomorphosis was chosen as a model system because this is an important form of intraspecific variation that is present in numerous taxa, but geographically localized within species and globally endangered. 277 ponds were sampled in one of the hotspots of paedomorphosis to determine the abundance and distribution of paedomorphs (i.e., individuals retaining gills at the adult stage) of the palmate newt (Lissotriton helveticus), with emphasis on the connections between the most valuable populations. KDEs gave insights into the surface areas required to balance the maintenance of certain number of connected ponds and the respective number of disjoint areas in which the whole population is divided. The inclusion of barriers in the models helped in accurately designing the limits of the areas to protect. Alone, habitat models were not able to successfully delineate the area to protect, but the integration between terrestrial suitable areas or barriers and KDE allowed an objective identification of areas required for conservation. Overall, the best performance was observed by the KDE integrating ecological barriers, and by the combination between KDE and niche modelling. In a broader perspective, KDEs are thus a pertinent tool in providing quantitative spatial measurements to delineate conservation areas based on patch-abundance data with a specific focus to connectivity.

Heterochrony in a complex world: disentangling environmental processes of facultative paedomorphosis in an amphibian.

Heterochrony, the change in the rate or timing of development between ancestors and their descendants, plays a major role in evolution. When heterochrony produces polymorphisms, it offers the possibility to test hypotheses that could explain its success across environments. Amphibians are particularly suitable to exploring these questions because they express complex life cycles (i.e. metamorphosis) that have been disrupted by heterochronic processes (paedomorphosis: retention of larval traits in adults). The large phenotypic variation across populations suggests that more complex processes than expected are operating, but they remain to be investigated through multivariate analyses over a large range of natural populations across time. In this study, we compared the likelihood of multiple potential environmental determinants of heterochrony. We gathered data on the proportion of paedomorphic and metamorphic palmate newts (Lissotriton helveticus) across more than 150 populations during 10 years and used an information-theoretic approach to compare the support of multiple potential processes. Six environmental processes jointly explained the proportion of paedomorphs in populations: predation, water availability, dispersal limitation, aquatic breathing, terrestrial habitat suitability and antipredator refuges. Analyses of variation across space and time supported models based on the advantage of paedomorphosis in favourable aquatic habitats. Paedomorphs were favoured in deep ponds, in conditions favourable to aquatic breathing (high oxygen content), with lack of fish and surrounded by suitable terrestrial habitat. Metamorphs were favoured by banks allowing easy dispersal. These results indicate that heterochrony relies on complex processes involving multiple ecological variables and exemplifies why heterochronic patterns occur in contrasted environments. On the other hand, the fast selection of alternative morphs shows that metamorphosis and paedomorphosis developmental modes could be easily disrupted in natural populations.

Endosulfan effects on Rana dalmatina tadpoles: quantitative developmental and behavioural analysis.

Endosulfan is an organochlorine pesticide that was recently labeled as a persistent organic pollutant, but it is still widely employed, particularly in developing countries. The goal of this study is to evaluate the acute (LC(50)) and chronic effects (developmental and behavioural traits) of this insecticide on Rana dalmatina tadpoles after exposure to ecologically relevant concentrations (0.005, 0.01, and 0.05 mg/L) by applying video-tracking techniques to evaluate the quantitative effect of endosulfan on amphibian behavioural patterns. The 96 h LC(50) value was 0.074 mg endosulfan/L. Tadpoles chronically exposed to 0.01 and 0.05 mg endosulfan/L underwent high mortality rate, decreased larval growth, delayed development, and increased incidence of malformations, and they did not reach metamorphosis by the end of the experiment. Moreover, tadpoles exposed to these concentrations exhibited several abnormalities in swimming patterns, such as shorter distance moved, swirling, resting, and unusual use of space. The exposure to 0.005 mg endosulfan/L did not cause any significant effects on behaviour, larval growth, or development, but we observed a significant decrease in both survival and time to metamorphosis. We showed that developmental abnormalities are dose-dependent and that the pesticide effects could differ depending on the endosulfan concentration and the species tested. We also validated the hypothesis that behavioural analysis, along with the use of new analytical methods, could be a useful tool in amphibian ecotoxicological studies.

Strong restructuration of skin microbiota during captivity challenges ex-situ conservation of amphibians.

In response to the current worldwide amphibian extinction crisis, conservation instances have encouraged the establishment of ex-situ collections for endangered species. The resulting assurance populations are managed under strict biosecure protocols, often involving artificial cycles of temperature and humidity to induce active and overwintering phases, which likely affect the bacterial symbionts living on the amphibian skin. However, the skin microbiota is an important first line of defense against pathogens that can cause amphibian declines, such as the chytrid Batrachochytrium dendrobatidis (Bd). Determining whether current husbandry practices for assurance populations might deplete amphibians from their symbionts is therefore essential to conservation success. Here, we characterize the effect of the transitions from the wild to captivity, and between aquatic and overwintering phases, on the skin microbiota of two newt species. While our results confirm differential selectivity of skin microbiota between species, they underscore that captivity and phase-shifts similarly affect their community structure. More specifically, the translocation ex-situ is associated with rapid impoverishment, decrease in alpha diversity and strong species turnover of bacterial communities. Shifts between active and overwintering phases also cause changes in the diversity and composition of the microbiota, and on the prevalence of Bd-inhibitory phylotypes. Altogether, our results suggest that current husbandry practices strongly restructure the amphibian skin microbiota. Although it remains to be determined whether these changes are reversible or have deleterious effects on their hosts, we discuss methods to limit microbial diversity loss ex-situ and emphasize the importance of integrating bacterial communities to applied amphibian conservation.

Using sets of behavioral biomarkers to assess short-term effects of pesticide: a study case with endosulfan on frog tadpoles.

Pesticides and other chemicals often have detrimental effects at environmental concentrations. Many amphibian species are particularly threatened because of their susceptibility but also because wetlands are often polluted. Behavioral assessments of toxicity have the advantage of showing sublethal effects but quantitative measures at varied scales of integrations are rarely considered together. In this study, we aimed at showing that these behavioral endpoints could be differently affected across time and concentrations, and be biomarkers of toxicity. To this end, we tested the effects of an organochlorine pesticide (endosulfan) on amphibians during a standard 96 h test. We evaluated possible lag effects in continuing the analyses after removal of the pesticide. The study was based on 240 tadpoles (4 pesticide treatments: 0.4, 3, 22, and 282 µg/l, 1 control and 1 solvent-control). Abnormal behaviors such as lying and swirling rapidly were exhibited only in the presence of the pesticide. Essential functions such as breathing and feeding were deeply affected by the pesticide: contaminated tadpoles breathed and fed less than control tadpoles. They also moved less and occupied a more central position in the aquariums in the presence of the pesticide. A higher mortality was only found at the highest concentration. These results suggest that endosulfan is toxic to amphibians at environmental concentrations. Behavioral markers showed potential as early warning systems. They should thus be used in complement to other markers to detect sublethal effects only a few days after application of the pesticide and at concentrations where mortality does not occur.

The use of visual and automatized behavioral markers to assess methodologies: a study case on PIT-tagging in the Alpine newt.

In various research fields, biomarkers are now widely used as tools for assessing individual integrity. The recent advances in quantification methods for behavioral patterns, such as computerized video-tracking procedures, make them valuable biomarkers. However, the corollary of these novelties is that they remain relatively unknown and unused. In this study, we show that such tools can assess the validity of research methods, such as individual recognition. To demonstrate this, we employed, as a model, a marking method (passive integrated transponder [PIT] tagging) widely used in amphibians. Detailed visual observations and video-tracking methods were complementary in highlighting components at different behavioral scales: locomotion, feeding, and breeding. We illustrate the scientific and ethical adequacy of the targeted marking method but also suggest that more studies should integrate behavioral analyses. Such biomarkers are a powerful tool for assessing conservation concerns when other techniques cannot detect detrimental effects.

Consumptive and nonconsumptive effects of cannibalism in fluctuating age-structured populations.

Theory and empirical studies suggest that cannibalism in age-structured populations can regulate recruitment depending on the intensity of intraspecific competition between cannibals and victims and the nature of the cannibalism window, i.e., which size classes interact as cannibals and victims. Here we report on a series of experiments that quantify that window for age-structured populations of salamander larvae and paedomorphic adults. We determined body size limits on cannibalism in microcosms and then the consumptive and nonconsumptive (injuries, foraging and activity, diet, growth) effects on victims in mesocosms with seminatural levels of habitat complexity and alternative prey. We found that cannibalism by the largest size classes (paedomorphs and > or = age 3+ yr larvae) occurs mainly on young-of-the-year (YOY) victims. Surviving YOY and other small larvae had increased injuries, reduced activity levels, and reduced growth rates in the presence of cannibals. Data on YOY survival in an experiment in which we manipulated the density of paedomorphs combined with historical data on the number of cannibals in natural populations indicate that dominant cohorts of paedomorphs can cause observed recruitment failures. Dietary data indicate that ontogenetic shifts in diet should preclude strong intraspecific competition between YOY and cannibals in this species. Thus our results are consistent with previous empirical and theoretical work that suggests that recruitment regulation by cannibalism is most likely when YOY are vulnerable to cannibalism but have low dietary overlap with cannibals. Understanding the role of cannibalism in regulating recruitment in salamander populations is timely, given the widespread occurrences of amphibian decline. Previous studies have focused on extrinsic (including anthropogenic) factors that affect amphibian population dynamics, whereas the data presented here combined with long-term field observations suggest the potential for intrinsically driven population cycles.