Facultative colour change is widespread in the animal kingdom, and has been documented in many distantly related amphibians. However, experimental data testing the extent of facultative colour change, and associated physiological and morphological implications are comparatively scarce. Background matching in the face of spatial and temporal environmental variation is thought to be an important proximate function of colour change in aquatic amphibian larvae. This is particularly relevant for species with long larval periods such as the western spadefoot toad, Pelobates cultripes, whose tadpoles spend up to six months developing in temporary waterbodies with temporally variable vegetation. By rearing tadpoles on different coloured backgrounds, we show that P. cultripes larvae can regulate pigmentation to track fine-grained differences in background brightness, but not hue or saturation. We found that colour change is rapid, reversible, and primarily achieved through changes in the quantity of eumelanin in the skin. We show that this increased eumelanin production and/or maintenance is also correlated with changes in morphology and oxidative stress, with more pigmented tadpoles growing larger tail fins and having an improved redox status., We would also like to thank the funding bodies that supported this project, namely the Ministerio de Ciencia e Innovación (Grant/Award Numbers: IJC2018-035780-I and PID2020-119517GB-I00)., Peer reviewed

Alien predators are a major cause of decline and extinction of species worldwide, since native organisms are rarely equipped with specific antipredatory strategies to cope with them. However, phenotypic plasticity and learned predator recognition may help prey populations to survive novel predators. Here we examine geographical variation in the learning ability of larval spadefoot toads (Pelobates cultripes) to recognize invasive predatory crayfish (Procambarus clarkii). We compare the learning-mediated behavioural responses of tadpoles from six populations across two regions in Spain (central and southern), with different histories of exposure to the presence of the invasive species. Two of the populations showed innate recognition of chemical cues from the invasive crayfish, whereas three of them learned to recognize such cues as a threat after conditioning with conspecific alarm cues. Learning abilities did not differ among southern populations, but they did among central populations. We assessed patterns of genetic variation within and among these two regions through microsatellite markers and found low genetic divergence among the southern populations but greater differentiation among the central ones. We hypothesize that similar responses to the invasive crayfish in southern populations may have arisen from a combination of extended historical exposure to this introduced predator (~ 50 y) and higher levels of gene flow, as they inhabit a highly interconnected pond network. In contrast, populations from central Spain show lower connectivity, have been exposed to the invasive crayfish for a shorter period of time, and are more divergent in their plastic responses., Financial support was provided by the Spanish Ministry of Science and Innovation (MICINN), Grants CGL2015-68670-R and PID2020-119517GB-I00. CC-D acknowledges the Comunidad de Madrid/European Social Fund for the predoctoral contract PEJD-2019-PRE/AMB-14950 funded by the Youth Employment Initiative (YEI)., Peer reviewed

Global change exposes wildlife to a variety of environmental stressors and is affecting biodiversity worldwide, with amphibian population declines being at the forefront of the global biodiversity crisis. The use of non-invasive methods to determine the physiological state in response to environmental stressors is therefore an important advance in the field of conservation physiology. The glucocorticoid hormone corticosterone (CORT) is one useful biomarker to assess physiological stress in amphibians, and sampling water-borne (WB) CORT is a novel, non-invasive collection technique. Here, we tested whether WB CORT can serve as a valid proxy of organismal levels of CORT in larvae of the common frog (Rana temporaria). We evaluated the association between tissue and WB CORT levels sampled from the same individuals across ontogenetic stages, ranging from newly hatched larvae to froglets at 10 days after metamorphosis. We also investigated how both tissue and WB CORT change throughout ontogeny. We found that WB CORT is a valid method in pro-metamorphic larvae as values for both methods were highly correlated. In contrast, there was no correlation between tissue and WB CORT in newly hatched, pre-metamorphic larvae, metamorphs or post-metamorphic froglets probably due to ontogenetic changes in respiratory and skin morphology and physiology affecting the transdermal CORT release. Both collection methods consistently revealed a non-linear pattern of ontogenetic change in CORT with a peak at metamorphic climax. Thus, our results indicate that WB CORT sampling is a promising, non-invasive conservation tool for studies on late-stage amphibian larvae. However, we suggest considering that different contexts might affect the reliability of WB CORT and consequently urge future studies to validate this method whenever it is used in new approaches. We conclude proposing some recommendations and perspectives on the use of WB CORT that will aid in broadening its application as a non-invasive tool in amphibian conservation physiology., Peer reviewed

Developmental plasticity allows organisms to adjust life-history traits to varying environmental conditions, which can have concomitant effects across life stages. Many amphibians are suitable model systems to study plasticity because their larvae can adjust growth and differentiation under fluctuating environments. It is unknown, however, whether somatic and gonadal differentiation are equally affected by environmentally induced plasticity or whether their decoupling alters gonadal maturation postmetamorphosis, which may affect fitness. We tested if developmental acceleration in response to warming and desiccation risk results in shifts in gonadal maturation during metamorphosis and postmetamorphic growth in western spadefoot toads (Pelobates cultripes). We found additive effects of increased temperature and desiccation risk on development and growth at metamorphosis, which largely constrained gonadal maturation in metamorphic and postmetamorphic individuals of both sexes. Furthermore, the conditions experienced by larvae incurred sex-specific carryover effects on the gonadal maturation of juveniles 5 months after metamorphosis. In females, high temperature delayed ovarian maturation regardless of the water level. In males, exposure to high temperature and high water levels slightly delayed the testes’ maturation.These results highlight the relevance of larval plasticity in the gonadal maturation of species undergoing metamorphosis, which may have implications for population demographics and the evolution of life histories., Peer reviewed

Developmental plasticity evolves in heterogeneous environmental conditions as long as individuals can accurately perceive them. A paradigmatic example of developmental plasticity is the ability of amphibian larvae to alter growth and development in response to the risk of pond drying. Such responses are often found in amphibian species breeding in highly dynamic water bodies with high desiccation risk. The inselbergs of the Guianan Shield are rocky outcrops with extremely high and fluctuating temperatures and a marked seasonality in precipitation. During the rainy season, eroded depressions form precipitation-dependent pools with a high and variable risk of desiccation within the timeframe of a few days. The frog Leptodactylus lithonaetes specializes in breeding in this extreme environment, and its tadpoles are thus forced to cope with desiccation risk by adjusting their developmental trajectories and physiological performance. We experimentally assessed the effect of different levels of desiccation risk, under controlled temperature conditions, on developmental rate, growth, and temperature-dependent locomotor performance in tadpoles of Leptodactylus lithonaetes. We did not find an effect of desiccation risk on developmental rate, but under simulated drying conditions, tadpoles showed larger body size, greater body mass, and enhanced locomotor performance compared to constant (high or low) water levels. These results suggest that drying pools offer cues that trigger developmental and behavioral changes in these tadpoles, enabling them to enhance growth over a short time span without accelerating development. We discuss the potential compensatory mechanisms behind these responses and highlight the need for further investigations in species with semiterrestrial life histories in extreme environments., This study was supported by Facultad de Ciencias from Universidad de Los Andes-Colombia (grants to Delgadillo Méndez years 2019, 2022, 2023), National Program for Women in Science UNESCO -L’ORÉAL- MINCIENCIAS-ICETEX, 2021, Colombian Ministry of Science, Technology and Innovation (grant # 80740-201-2019 code 120480863597), Plan Nacional I+D of the Spanish Ministry of Science and Innovation (grant # PID2020-119517GB-I00) and Programa QUALIFICA (Junta de Andalucía, Consejería de Universidad, Investigación e Innovación -project QUAL21 020 EBD)., Peer reviewed

Organisms have often evolved developmental plasticity to cope with environmental heterogeneity, but natural systems are often quite complex and organisms have to assess and respond to variation in multiple environmental factors simultaneously, which at times can elicit opposing or synergistic phenotypic responses. Amphibian larvae can often respond to temperature, desiccation risk, and predation risk altering their behaviour, shape, coloration, physiology or life histories. However, our understanding of how species respond to fluctuations in multiple factors in complex environments is limited and primarily restricted to temperate species.
Leptodactylus fuscus is a tropical anuran that constructs foam nests in excavated subterranean chambers, where embryos develop, and nest-dwelling larvae can enter developmental arrest until the chambers are flooded and tadpoles are washed off to a neighbouring pond. Its breeding ponds are highly dynamic, and to understand how its larvae respond to multiple factors in complex environments, we experimentally exposed them to variations in temperature, water level, and predation cues.
We found that tadpoles did not modulate developmental rates in response to any of the factors studied. At high temperatures, larvae achieved greater body mass, larger size, increased fat bodies, and improved locomotor performance. In contrast, reduced water level negatively affected body mass, size, and snout length at metamorphosis. Presence of predation cues had the least effect on tadpoles, only showing an antagonistic interaction with temperature on locomotor performance. Of all three ecological factors, temperature had the greatest (and positive) impact on growth, and the effects of each factor were predominantly additive or antagonistic.
The reproductive mode of L. fuscus may have relaxed selection for plasticity in developmental rate, allowing its canalization. However, the limited capacity for developmental rate plasticity in response to temperature or desiccation risk could limit the species' ability to respond to future climate change scenarios of increased temperature and reduced hydroperiod.
Our results highlight the need to continue studying phenotypic plasticity in complex, more realistic scenarios, and expand the array of species studied to include tropical species and species with different reproductive modes., This study was supported by the Facultad de Ciencias, Universidad de Los Andes-Colombia (grants: INV-2019-68-1736, INV-2022-137-2387, INV-2022-150-2598), the National Program for Women in Science UNESCO–L'Oréal–MinCiencias–ICETEX (2021), the Colombian Ministry of Science, Technology and Innovation (grant #80740-201-2019, code 120480863597), Plan Nacional I+D of the Spanish Ministry of Science and Innovation (grant # PID2020-119517GB-I00) and Programa QUALIFICA (Junta de Andalucía, Consejería de Universidad, Investigación e Innovación -project QUAL21 020EBD)., Peer reviewed