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Amphibian diversity is most prominent in the warm and humid tropical and subtropical regions across the globe. Nonetheless, amphibians also inhabit high-altitude tropical mountains and regions at medium and high latitudes, exposing them to subzero temperatures and requiring behavioural or physiological adaptations to endure freezing events. While freeze tolerance has been predominantly reported in high-latitude zones where species endure prolonged freezing (several weeks or months), less is known about mid-latitudes amphibians exposed to occasional subzero temperatures. In this study, we employed a controlled ecological protocol, subjecting three frog species from the Iberian Peninsula (Rana parvipalmata, Epidalea calamita, and Pelobates cultripes) to a 2-h exposure to temperatures of -2 °C to investigate the accumulation of urea and glucose as physiological mechanisms associated with survival at freezing temperatures. Our results revealed a moderate response in the production of cryoprotectant metabolites under experimental freezing conditions, particularly urea, with notable findings in R. parvipalmata and E. calamita and no response in P. cultripes. However, no significant alterations in glucose concentrations were observed in any of the studied frog species. This relatively weak freezing tolerance response differs from the strong response exhibited by amphibians inhabiting high latitudes and enduring prolonged freezing conditions, suggesting potential reliance on behavioural adaptations to cope with occasional freezing episodes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The "climate extremes hypothesis" is a major assumption of geographic studies of heat tolerance and climatic vulnerability. However, this assumption remains vastly untested across taxa, and multiple factors may contribute to uncoupling heat tolerance estimates and geographic limits. Our dataset includes 1000 entries of heat tolerance data and maximum temperatures for each species' known geographic limits (hereafter, Tmax). We gathered this information across major animal taxa, including marine fish, terrestrial arthropods, amphibians, non-avian reptiles, birds, and mammals. We first tested if heat tolerance constrains the Tmax of sites where species could be observed. Secondly, we tested if the strength of such restrictions depends on how high Tmax is relative to heat tolerance. Thirdly, we correlated the different estimates of Tmax among them and across species. Restrictions are strong for amphibians, arthropods, and birds but often weak or inconsistent for reptiles and mammals. Marine fish describe a non-linear relationship that contrasts with terrestrial groups. Traditional heat tolerance measures in thermal vulnerability studies, like panting temperatures and the upper set point of preferred temperatures, do not predict Tmax or are inversely correlated to it, respectively. Heat tolerance restricts the geographic warm edges more strongly for species that reach sites with higher Tmax for their heat tolerance. These emerging patterns underline the importance of reliable species' heat tolerance indexes to identify their thermal vulnerability at their warm range edges. Besides, the tight correlations of Tmax estimates across on-land microhabitats support a view of multiple types of thermal challenges simultaneously shaping ranges' warm edges for on-land species. The heterogeneous correlation of Tmax estimates in the ocean supports the view that fish thermoregulation is generally limited, too. We propose new hypotheses to understand thermal restrictions on animal distribution., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Catarina Vinagre reports financial support was provided by Foundation for Science and Technology. Miguel Trefaut Rodrigues reports financial support was provided by State of Sao Paulo Research Foundation. Miguel Tejedo reports financial support was provided by Secretaría de Estado de Investigación, Desarrollo e Innovación. Agustín Camacho Guerrero reports financial support was provided by Council of the European Union., (Copyright © 2024. Published by Elsevier B.V.)

Plasticity in the critical thermal maximum (CT max ) helps ectotherms survive in variable thermal conditions. Yet, little is known about the environmental mechanisms modulating its time course. We used the larvae of three neotropical anurans (Boana platanera, Engystomops pustulosus and Rhinella horribilis) to test whether the magnitude of temperature changes and the existence of fluctuations in the thermal environment affected both the amount of change in CT max and its acclimation rate (i.e., its time course). For that, we transferred tadpoles from a pre-treatment temperature (23 °C, constant) to two different water temperatures: mean (28 °C) and hot (33 °C), crossed with constant and daily fluctuating thermal regimes, and recorded CT max values, daily during six days. We modeled changes in CT max as an asymptotic function of time, temperature, and the daily thermal fluctuation. The fitted function provided the asymptotic CT max value (CT max ∞) and CT max acclimation rate (k). Tadpoles achieved their CT max ∞ between one and three days. Transferring tadpoles to the hot treatment generated higher CT max ∞ at earlier times, inducing faster acclimation rates in tadpoles. In contrast, thermal fluctuations equally led to higher CT max ∞ values but tadpoles required longer times to achieve CT max ∞ (i.e., slower acclimation rates). These thermal treatments interacted differently with the studied species. In general, the thermal generalist Rhinella horribilis showed the most plastic acclimation rates whereas the ephemeral-pond breeder Engystomops pustulosus, more exposed to heat peaks during larval development, showed less plastic (i.e., canalized) acclimation rates. Further comparative studies of the time course of CT max acclimation should help to disentangle the complex interplay between the thermal environment and species ecology, to understand how tadpoles acclimate to heat stress., Competing Interests: Declaration of competing interest None declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Critical thermal limits (CT max and CT min ) decrease with elevation, with greater change in CT min , and the risk to suffer heat and cold stress increasing at the gradient ends. A central prediction is that populations will adapt to the prevailing climatic conditions. Yet, reliable support for such expectation is scant because of the complexity of integrating phenotypic, molecular divergence and organism exposure. We examined intraspecific variation of CT max and CT min , neutral variation for 11 microsatellite loci, and micro- and macro-temperatures in larvae from 11 populations of the Galician common frog ( Rana parvipalmata ) across an elevational gradient, to assess (1) the existence of local adaptation through a P ST -F ST comparison, (2) the acclimation scope in both thermal limits, and (3) the vulnerability to suffer acute heat and cold thermal stress, measured at both macro- and microclimatic scales. Our study revealed significant microgeographic variation in CT max and CT min , and unexpected elevation gradients in pond temperatures. However, variation in CT max and CT min could not be attributed to selection because critical thermal limits were not correlated to elevation or temperatures. Differences in breeding phenology among populations resulted in exposure to higher and more variable temperatures at mid and high elevations. Accordingly, mid- and high-elevation populations had higher CT max and CT min plasticities than lowland populations, but not more extreme CT max and CT min . Thus, our results support the prediction that plasticity and phenological shifts may hinder local adaptation, promoting thermal niche conservatism. This may simply be a consequence of a coupled variation of reproductive timing with elevation (the "elevation-time axis" for temperature variation). Mid and high mountain populations of R. parvipalmata are more vulnerable to heat and cool impacts than lowland populations during the aquatic phase. All of this contradicts some of the existing predictions on adaptive thermal clines and vulnerability to climate change in elevational gradients., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Current climate change is generating accelerated increase in extreme heat events and organismal plastic adjustments in upper thermal tolerances, (critical thermal maximum -CT max ) are recognized as the quicker mitigating mechanisms. However, current research casts doubt on the actual mitigating role of thermal acclimation to face heat impacts, due to its low magnitude and weak environmental signal. Here, we examined these drawbacks by first estimating maximum extent of thermal acclimation by examining known sources of variation affecting CT max expression, such as daily thermal fluctuation and heating rates. Second, we examined whether the magnitude and pattern of CT max plasticity is dependent of the thermal environment by comparing the acclimation responses of six species of tropical amphibian tadpoles inhabiting thermally contrasting open and shade habitats and, finally, estimating their warming tolerances (WT = CT max  - maximum temperatures) as estimator of heating risk. We found that plastic CT max responses are improved in tadpoles exposed to fluctuating daily regimens. Slow heating rates implying longer duration assays determined a contrasting pattern in CT max plastic expression, depending on species environment. Shade habitat species suffer a decline in CT max whereas open habitat tadpoles greatly increase it, suggesting an adaptive differential ability of hot exposed species to quick hardening adjustments. Open habitat tadpoles although overall acclimate more than shade habitat species, cannot capitalize this beneficial increase in CT max, because the maximum ambient temperatures are very close to their critical limits, and this increase may not be large enough to reduce acute heat stress under the ongoing global warming., (© 2022 Wiley Periodicals LLC.)

Climate change may have dramatic consequences for communities through both direct effects of peak temperatures upon individual species and through interspecific mismatches in thermal sensitivities of interacting organisms which mediate changes in interspecific interactions (i.e. predation). Despite this, there is a paucity of information on the patterns of spatial physiological sensitivity of interacting species (at both landscape and local scales) which could ultimately influence geographical variation in the effects of climate change on community processes. In order to assess where these impacts may occur, we first need to evaluate the spatial heterogeneity in the degree of mismatch in thermal tolerances between interacting organisms. We quantify the magnitude of interspecific mismatch in maximum (CT max ) and minimum (CT min ) thermal tolerances among a predator-prey system of dragonfly and anuran larvae in tropical montane (242-3,631 m) and habitat (ponds and streams) gradients. To compare thermal mismatches between predator and prey, we coined the parameters maximum and minimum predatory tolerance margins (PTM max and PTM min ), or difference in CT max and CT min of interacting organisms sampled across elevational and habitat gradients. Our analyses revealed that: (a) predators exhibit higher heat tolerances than prey (~4°C), a trend which remained stable across habitats and elevations. In contrast, we found no differences in minimum thermal tolerances between these groups. (b) Maximum and minimum thermal tolerances of both predators and prey decreased with elevation, but only maximum thermal tolerance varied across habitats, with pond species exhibiting higher heat tolerance than stream species. (c) Pond-dwelling organisms from low elevations (0-1,500 m a.s.l.) may be more susceptible to direct effects of warming than their highland counterparts because their maximum thermal tolerances are only slightly higher than their exposed maximum environmental temperatures. The greater relative thermal tolerance of dragonfly naiad predators may further increase the vulnerability of lowland tadpoles to warming due to potentially enhanced indirect effects of higher predation rates by more heat-tolerant dragonfly predators. However, further experimental work is required to establish the individual and population-level consequences of this thermal tolerance mismatch upon biotic interactions such as predator-prey. ​., (© 2021 British Ecological Society.)

Determining both the age structure and growth pattern allows to establish the causal factors, environmental and/or genetic, that eventually may be responsible for the observed pattern of divergence. We examined the variation in age structure and growth pattern across populations of two toad species, Pelobates cultripes and Epidalea calamita that exhibit a geographic variation in body size in southern Spain. For both species, populations differed in mean age but age structure did not correlate with body size variation across populations. Although the population with the youngest females found for E. calamita was the smallest in body size, the oldest males for both species were found in a small body size population. The growth pattern fit well to a von Bertalanffy growth model and interdemic divergence were found for both the asymptotic body size (Sm ) and the growth coefficients (k). As expected, Large-Bodied populations of both species attained higher Sm but, Small-Bodied population had higher, although non significantly different, k growth coefficients. Also, the Small-Bodied population attained sexual maturity sooner but had also high longevity. The observed pattern may reflect both environmental variations in resources availability affecting body size observed across populations, but also different growth and maturity pathways that may respond to contrasting selective pressures.

Communities usually possess a multitude of interconnected trophic interactions within food webs. Their regulation generally depends on a balance between bottom-up and top-down effects. However, if sensitivity to temperature varies among species, rising temperatures may change trophic interactions via direct and indirect effects. We examined the critical thermal maximum (CT max ) of 19 species from temperate wetlands (insect predators, amphibian larvae, zooplankton and amphipods) and determined if they vary in their sensitivity to warming temperatures. CT max differed between the groups, with predatory insects having higher CT max than amphibians (both herbivorous larval anurans and predatory larval salamanders), amphipods and zooplankton. In a scenario of global warming, these differences in thermal tolerance may affect top-down and bottom-up processes, particularly considering that insect predators are more likely to maintain or improve their performance at higher temperatures, which could lead to increased predation rates on the herbivores in the food web. Further studies are needed to understand how the energy flows through communities, how species' energy budgets may change and whether other physiological and behavioral responses (such as phenotypic plasticity and thermoregulation) can buffer or increase these changes in the top-down regulation of wetland food webs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Variation and population structure play key roles in the speciation process, but adaptive intraspecific genetic variation is commonly ignored when forecasting species niches. Amphibians serve as excellent models for testing how climate and local adaptations shape species distributions due to physiological and dispersal constraints and long generational times. In this study, we analysed the climatic factors driving the evolution of the genus Alytes at inter- and intraspecific levels that may limit realized niches. We tested for both differences among the five recognized species and among intraspecific clades for three of the species (Alytes obstetricans, A. cisternasii, and A. dickhilleni). We employed ecological niche models with an ordination approach to perform niche overlap analyses and test hypotheses of niche conservatism or divergence. Our results showed strong differences in the environmental variables affecting species climatic requirements. At the interspecific level, tests of equivalence and similarity revealed that sister species were non-identical in their environmental niches, although they neither were entirely dissimilar. This pattern was also consistent at the intraspecific level, with the exception of A. cisternasii, whose clades appeared to have experienced a lower degree of niche divergence than clades of the other species. In conclusion, our results support that Alytes toads, examined at both the intra- and interspecific levels, tend to occupy similar, if not identical, climatic environments.

The vulnerability of species to climate change is jointly influenced by geographic phenotypic variation, acclimation and behavioural thermoregulation. The importance of interactions between these factors, however, remains poorly understood. We demonstrate how advances in mechanistic niche modelling can be used to integrate and assess the influence of these sources of uncertainty in forecasts of climate change impacts. We explored geographic variation in thermal tolerance (i.e. maximum and minimum thermal limits) and its potential for acclimation in juvenile European common frogs Rana temporaria along elevational gradients. Furthermore, we employed a mechanistic niche model (NicheMapR) to assess the relative contributions of phenotypic variation, acclimation and thermoregulation in determining the impacts of climate change on thermal safety margins and activity windows. Our analyses revealed that high-elevation populations had slightly wider tolerance ranges driven by increases in heat tolerance but lower potential for acclimation. Plausibly, wider thermal fluctuations at high elevations favour more tolerant but less plastic phenotypes, thus reducing the risk of encountering stressful temperatures during unpredictable extreme events. Biophysical models of thermal exposure indicated that observed phenotypic and plastic differences provide limited protection from changing climates. Indeed, the risk of reaching body temperatures beyond the species' thermal tolerance range was similar across elevations. In contrast, the ability to seek cooler retreat sites through behavioural adjustments played an essential role in buffering populations from thermal extremes predicted under climate change. Predicted climate change also altered current activity windows, but high-elevation populations were predicted to remain more temporally constrained than lowland populations. Our results demonstrate that elevational variation in thermal tolerances and acclimation capacity might be insufficient to buffer temperate amphibians from predicted climate change; instead, behavioural thermoregulation may be the only effective mechanism to avoid thermal stress under future climates., (© 2020 British Ecological Society.)

The brain depends on glucose as a source of energy. This implies the presence of glucose transporters, being GLUT1 and GLUT3 the most relevant. Expression of GLUT12 is found in mouse and human brain at low levels. We previously demonstrated GLUT12 upregulation in the frontal cortex of aged subjects that was even higher in aged Alzheimer's disease (AD) patients. However, the cause and the mechanism through which this increase occurs are still unknown. Here, we aimed to investigate whether the upregulation of GLUT12 in AD is related with aging or Aβ deposition in comparison with GLUT1, GLUT3, and GLUT4. In the frontal cortex of two amyloidogenic mouse models (Tg2576 and APP/PS1) GLUT12 levels were increased. Contrary, expression of GLUT1 and GLUT3 were decreased, while GLUT4 did not change. In aged mice and the senescence-accelerated model SAMP8, GLUT12 and GLUT4 were upregulated in comparison with young animals. GLUT1 and GLUT3 did not show significant changes with age. The effect of β-amyloid (Aβ) deposition was also evaluated in Aβ peptide i.c.v. injected mice. In the hippocampus, GLUT12 expression increased whereas GLUT4 was not modified. Consistent with the results in the amyloidogenic models, GLUT3 and GLUT1 were downregulated. In summary, Aβ increases GLUT12 protein expression in the brain pointing out a central role of the transporter in AD pathology and opening new perspectives for the treatment of this neurodegenerative disease.

Climate change and infectious disease by the chytrid fungus Batrachochytrium dendrobatidis (Bd) are major drivers of amphibian extinctions, but the potential interactions of these two factors are not fully understood. Temperature is known to influence (1) the infectivity, pathogenicity and virulence of Bd; (2) host-parasite dynamics, especially when both hosts and parasites are ectothermic organisms exhibiting thermal sensitivities that may or may not differ; and (3) amphibian vulnerability to extinction depending on their heat tolerance, which may decrease with infection. Thus, in a global warming scenario, with rising temperatures and more frequent and extreme weather events, amphibians infected by Bd could be expected to be more vulnerable if temperatures approach their critical thermal maximum (CTmax). However, it is also possible that predicted high temperatures could clear the Bd infection, thus enhancing amphibian survival. We tested these hypotheses by measuring CTmax values of Bd-infected and Bd-free aquatic tadpoles and terrestrial toadlets/juveniles of the common midwife toad (Alytes obstetricans) and examining whether exposure of A. obstetricans individuals to peak temperatures reaching their CTmax clears them from Bd infection. We show that (1) Bd has a wide thermal tolerance range; (2) Bd is capable of altering the thermal physiology of A. obstetricans, which is stage-dependent, lowering CTmax in tadpoles but not in toadlets; and (3) Bd infection is not cleared after exposure of tadpoles or toadlets to CTmax. Living under climatic change with rising temperatures, the effect of Bd infection might tip the balance and lead some already threatened amphibian communities towards extinction., Competing Interests: This study was supported by Banco Santander. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Amphibians are considered one of the groups most susceptible to chemical contamination, therefore are good bio-indicators of aquatic pollution. Synergistic effects of temperature and pesticides have been found in amphibians determining amplified toxicity effect on survival and malformations with increasing temperatures. We studied the sensitivity of sublethal concentrations of chlorpyrifos in Rhinella arenarum tadpoles over on two fitness related thermal traits: locomotor swimming performance and thermal tolerance limits (CT max  = critical thermal maximum and CT min  = critical thermal minimum). Our result shows a decrease in the locomotor performance of R. arenarum tadpoles with increasing sublethal chlorpyrifos concentrations. The experimental temperature increased locomotor performance but this being only significant for the control whereas tadpoles raised at any sublethal chlorpyrifos concentration did not increase their total swimming distance with temperature (Concentration × Temperature interaction, P < 0.019). Chlorpyrifos toxicity decreases maximum swimming distance but this reduction not compensated at high temperatures that do enhance swimming performance in the control treatment. On the other hand, higher chlorpyrifos sensitivity in CT max than CT min since tadpoles exposed to all polluted treatments exhibits a significant decline in CT max but not in CT min . Current global warming and the increase of atypical climatic events, such as heat waves may put at risk the larval chlorpyrifos polluted populations of R. arenarum. Our results show that the sublethal concentrations of the chlorpyrifos pesticide may affect the fitness and survival of the larvae of R. arenarum., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Complex life-histories may promote the evolution of different strategies to allow optimal matching to the environmental conditions that organisms can encounter in contrasting environments. For ectothermic animals, we need to disentangle the role of stage-specific thermal tolerances and developmental acclimation to predict the effects of climate change on spatial distributions. However, the interplay between these mechanisms has been poorly explored. Here we study whether developmental larval acclimation to rearing temperatures affects the thermal tolerance of subsequent terrestrial stages (metamorphs and juveniles) in common frogs (Rana temporaria). Our results show that larval acclimation to warm temperatures enhances larval heat tolerance, but not thermal tolerance in later metamorphic and juvenile stages, which does not support the developmental acclimation hypothesis. Further, metamorphic and juvenile individuals exhibit a decline in thermal tolerance, which would confer higher sensitivity to extreme temperatures. Because thermal tolerance is not enhanced by larval developmental acclimation, these 'risky' stages may be forced to compensate through behavioural thermoregulation and short-term acclimation to face eventual heat peaks in the coming decades.

The current global warming scenario has led to a renewed interest in determining which species are more vulnerable to climate change. Hence, it is important to understand which factors can affect estimates of species vulnerability. We determined the critical thermal maxima (CT max ) for six species of North American anuran larvae and measured the environmental temperatures to which they are exposed during their aquatic stage to estimate their warming tolerance (WT; difference between the critical thermal maximum and the macro- and microhabitat maximum environmental temperatures). Our results indicate that these species exhibited CT max values (37.8-41.7 °C) that were similar to other temperate species and positively correlated only with environmental temperatures measured at the microclimate scale. This indicates that microclimatic variables are better predictors of CT max variation than macroclimate data. Moreover, most of the CT max variation found was associated with higher taxonomic levels, indicating that related species may show similar CT max values due to phylogenetic inertia. Studied species also exhibited high values of WT (10.3-22.6 °C), similar to temperate amphibian species from other bioregions. This indicates that there is a considerable gap between the species' critical thermal maximum and maximum environmental temperature, whether using datalogger (microclimate) or WorldClim (macroclimate) environmental data. However, WT estimates based on datalogger data were not related to those based on macroclimate environmental data. Finally, variation associated with the type of environmental data used (macro- vs. microclimate) had a profound influence on WT estimates. Hence, our perception of which species are more vulnerable to global warming changes may depend on the scale of the climate data used., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The quantification of enzyme activity in the patient treated with enzyme replacement therapy (ERT) has been suggested as a tool for dosage individualization, so we conducted a study to evaluate the relationship between glucocerebrosidase activity and clinical response in patients with Gaucher disease type I (GD1) to ERT. The study included patients diagnosed with GD1, who were being treated with ERT, and healthy individuals. Markers based on glucocerebrosidase activity measurement in patients' leucocytes were studied: enzyme activity at 15 min. post-infusion (Act 75 ) reflects the amount of enzyme that is distributed in the body post-ERT infusion, and accumulated glucocerebrosidase activity during ERT infusion (Act 75-0 ) indicates the total drug exposure during infusion. The clinical response was evaluated based on criteria established by Pastores et al. and Gaucher Severity Score Index. Statistical analysis included ROC analysis and area under the curve test. Act 75 and Act 75-0 were found to be moderate predictive markers of an optimal clinical response (area under the ROC of Act 75 was 0.733 and Act 75-0 was 0.817). Act 75-0 showed statistical significance in its discriminative capacity (p < 0.05) for obtaining an optimal response to ERT. The cut-off point was 58% (RR = 1.800; 95% CI: 1.003-3.229; p < 0.05). Moreover, Act 75 showed a significant and inverse correlation with the Gaucher Severity Score Index, and Act 75 and Act 75-0 presented a significant correlation with residual enzyme activity at diagnosis. Markers based on glucocerebrosidase activity have a good correlation with clinical response to ERT. Therefore, it could provide supporting clinical data for dose management in GD1 patients., (© 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Parsley frogs (Pelodytes) comprise the only genus in the family Pelodytidae, an ancient anuran lineage that split from their closest relatives over 140 million years ago. Pelodytes is a Palearctic group restricted to Western Eurasia including three extant species: the eastern species P. caucasicus, endemic to the Caucasus area, and two closely related species inhabiting Western Europe: the Iberian endemic P. ibericus and the more widespread P. punctatus. Previous studies based on mitochondrial and nuclear DNA markers have revealed the existence of two additional lineages of Parsley frogs in the Iberian Peninsula, which have been flagged as candidate species. Here, we integrate novel molecular, morphological and bioacoustical data to assess the differentiation of the four western Parsley frog lineages. Species trees and Bayesian population assignment analyses based on nuclear markers confirm previous studies and concordantly delineate four parapatric lineages with narrow hybrid zones. Mitochondrial divergence is low (< 2% pairwise distances in the 16S rRNA gene), in line with previously reported low mitochondrial substitution rates in non-neobatrachian frogs. Based on concordance between mitochondrial and nuclear markers, we conclude that four species of Parsley frogs occur in Western Europe: Pelodytes punctatus, distributed from northern Italy to northeastern Spain; Pelodytes ibericus, inhabiting southern Spain and southern Portugal; Pelodytes atlanticus sp. nov., from the Portuguese Atlantic coast; and Pelodytes hespericus sp. nov., occurring in central and eastern Spain. However, bioacoustical and morphological differentiation of these species is low, with no obvious and qualitative diagnostic characters allowing full species discrimination. Differences in the relative size of metacarpal tubercles exist but this character is variable. Pelodytes ibericus and Pelodytes atlanticus are smaller than the other two species, and P. ibericus has shorter limbs and various distinctive osteological characters. Bioacoustically, the pattern by which two different note types are combined in advertisement calls separates P. hespericus from the remaining species. Despite these differences, we emphasize that the taxonomic status of all four western Parsley frogs requires additional investigation, especially the patterns of genetic admixture across contact zones. While a status of separate species best conforms to the currently available data, alternative hypotheses are also discussed.

Many ectotherms employ diverse behavioral adjustments to effectively buffer the spatio-temporal variation in environmental temperatures, whereas others remain passive to thermal heterogeneity. Thermoregulatory studies are frequently performed on species living in thermally benign habitats, which complicate understanding of the thermoregulation-thermoconformity continuum. The need for new empirical data from ectotherms exposed to thermally challenging conditions requires the evaluation of available methods for quantifying thermoregulatory strategies. We evaluated the applicability of various thermoregulatory indices using fire salamander larvae, Salamandra salamandra, in two aquatic habitats, a forest pool and well, as examples of disparate thermally-constrained environments. Water temperatures in the well were lower and less variable than in the pool. Thermal conditions prevented larvae from reaching their preferred body temperature range in both water bodies. In contrast to their thermoregulatory abilities examined in a laboratory thermal gradient, field body temperatures only matched the mean and range of operative temperatures, showing thermal passivity of larvae in both habitats. Despite apparent thermoconformity, thermoregulatory indices indicated various strategies from active thermoregulation, to thermoconformity, and even thermal evasion, which revealed their limited applicability under thermally-constrained conditions. Salamander larvae abandoned behavioral thermoregulation despite varying opportunities to increase their body temperature above average water temperatures. Thermoconformity represents a favored strategy in these ectotherms living in more thermally-constrained environments than those examined in previous thermoregulatory studies. To understand thermal ecology and its impact on population dynamics, the quantification of thermoregulatory strategies of ectotherms in thermally-constrained habitats requires the careful choice of an appropriate method to avoid misleading results., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Although temperature variation is known to cause large-scale adaptive divergence, its potential role as a selective factor over microgeographic scales is less well-understood. Here, we investigated how variation in breeding pond temperature affects divergence in multiple physiological (thermal performance curve and critical thermal maximum [CTmax]) and life-history (thermal developmental reaction norms) traits in a network of Rana arvalis populations. The results supported adaptive responses to face two main constraints limiting the evolution of thermal adaptation. First, we found support for the faster-slower model, indicating an adaptive response to compensate for the thermodynamic constraint of low temperatures in colder environments. Second, we found evidence for the generalist-specialist trade-off with populations from colder and less thermally variable environments exhibiting a specialist phenotype performing at higher rates but over a narrower range of temperatures. By contrast, the local optimal temperature for locomotor performance and CTmax did not match either mean or maximum pond temperatures. These results highlight the complexity of the adaptive multiple-trait thermal responses in natural populations, and the role of local thermal variation as a selective force driving diversity in life-history and physiological traits in the presence of gene flow., (© 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.)

Species delineation is a central topic in evolutionary biology, with current efforts focused on developing efficient analytical tools to extract the most information from molecular data and provide objective and repeatable results. In this paper we use a multilocus dataset (mtDNA and two nuclear markers) in a geographically comprehensive population sample across Iberia and Western Europe to delineate candidate species in a morphologically cryptic species group, Parsley frogs (genus Pelodytes). Pelodytes is the sole extant representative of an ancient, historically widely distributed anuran clade that currently includes three species: P. caucasicus in the Caucasus; P. punctatus in Western Europe, from Portugal to North-Western Italy; and P. ibericus in Southern Iberia. Phylogenetic analyses recovered four major well-supported haplotype clades in Western Europe, corresponding to well demarcated geographical subdivisions and exhibiting contrasting demographic histories. Splitting times date back to the Plio-Pleistocene and are very close in time. Species-tree analyses recovered one of these species lineages, corresponding to P. ibericus (lineage B), as the sister taxon to the other three major species lineages, distributed respectively in: western Iberian Peninsula, along the Atlantic coast and part of central Portugal (lineage A); Central and Eastern Spain (lineage C); and North-eastern Spain, France and North-western Italy (lineage D). The latter is in turn subdivided into two sub-clades, one in SE France and NW Italy and the other one from NE Spain to NW France, suggesting the existence of a Mediterranean-Atlantic corridor along the Garonne river. An information theory-based validation approach implemented in SpedeSTEM supports an arrangement of four candidate species, suggesting the need for a taxonomic revision of Western European Pelodytes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To forecast biological responses to changing environments, we need to understand how a species's physiology varies through space and time and assess how changes in physiological function due to environmental changes may interact with phenotypic changes caused by other types of environmental variation. Amphibian larvae are well known for expressing environmentally induced phenotypes, but relatively little is known about how these responses might interact with changing temperatures and their thermal physiology. To address this question, we studied the thermal physiology of grey treefrog tadpoles (Hyla versicolor) by determining whether exposures to predator cues and an herbicide (Roundup) can alter their critical maximum temperature (CTmax) and their swimming speed across a range of temperatures, which provides estimates of optimal temperature (Topt) for swimming speed and the shape of the thermal performance curve (TPC). We discovered that predator cues induced a 0.4°C higher CTmax value, whereas the herbicide had no effect. Tadpoles exposed to predator cues or the herbicide swam faster than control tadpoles and the increase in burst speed was higher near Topt. In regard to the shape of the TPC, exposure to predator cues increased Topt by 1.5°C, while exposure to the herbicide marginally lowered Topt by 0.4°C. Combining predator cues and the herbicide produced an intermediate Topt that was 0.5°C higher than the control. To our knowledge this is the first study to demonstrate a predator altering the thermal physiology of amphibian larvae (prey) by increasing CTmax, increasing the optimum temperature, and producing changes in the thermal performance curves. Furthermore, these plastic responses of CTmax and TPC to different inducing environments should be considered when forecasting biological responses to global warming.

Amphibians are declining worldwide and pollutants have been implicated as a major contributor to these declines. To understand these declines, many studies have assessed the impact of pollutants on amphibian behaviour. However, information regarding their effect on locomotor abilities, as well as the intra-specific variation of the tolerance to pollutants, is extremely rare. Further, the majority of studies examining the impact of pollutants on amphibians have been conducted in simplified laboratory settings. Given the complexity of natural systems, determining whether amphibian responses in laboratory studies can be generalized to more realistic natural scenarios is critical. Towards this goal, this study assessed the impact of nitrogenous pollution on survival and fitness-related larval traits (growth, mass and swimming performance) for three populations of the frog Pelophylax perezi, exposed to different degrees of eutrophication in two different and complementary experiments: (1) pond mesocosms, with NH4Cl isolated or combined with NaNO2 and NaNO3, and (2) field enclosures placed in natural streams differing in their degree of pollution. For both mesocosm and field enclosure experiments, larval mortality was unaffected by nitrogenous pollution. However, in the mesocosm experiment, exposure to nitrogenous compounds reduced final larvae mass and growth. In contrast, in the enclosure experiment, polluted locations facilitated final mass and growth of surviving tadpoles. Population-level variation in the effect of pollution was observed for final larval mass in the mesocosm but not in the field enclosure experiment. In addition, although nitrogenous compounds in both mesocosm and natural conditions had no direct effect on absolute larval swimming performance, they may impact the viability of larvae by affecting the relationships between growth and the swimming abilities. The differential pattern found in the impacts of nitrogenous compounds on larvae of P. perezi when raised in different experimental venues (mesocosms and field conditions) points to the convenience of considering more realistic natural scenarios in assessing the impact of pollutants on amphibians., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Many studies have assessed the impact of different pollutants on amphibians across a variety of experimental venues (laboratory, mesocosm, and enclosure conditions). Past reviews, using vote-counting methods, have described pollution as one of the major threats faced by amphibians. However, vote-counting methods lack strong statistical power, do not permit one to determine the magnitudes of effects, and do not compare responses among predefined groups. To address these challenges, we conducted a meta-analysis of experimental studies that measured the effects of different chemical pollutants (nitrogenous and phosphorous compounds, pesticides, road deicers, heavy metals, and other wastewater contaminants) at environmentally relevant concentrations on amphibian survival, mass, time to hatching, time to metamorphosis, and frequency of abnormalities. The overall effect size of pollutant exposure was a medium decrease in amphibian survival and mass and a large increase in abnormality frequency. This translates to a 14.3% decrease in survival, a 7.5% decrease in mass, and a 535% increase in abnormality frequency across all studies. In contrast, we found no overall effect of pollutants on time to hatching and time to metamorphosis. We also found that effect sizes differed among experimental venues and among types of pollutants, but we only detected weak differences among amphibian families. These results suggest that variation in sensitivity to contaminants is generally independent of phylogeny. Some publication bias (i.e., selective reporting) was detected, but only for mass and the interaction effect size among stressors. We conclude that the overall impact of pollution on amphibians is moderately to largely negative. This implies that pollutants at environmentally relevant concentrations pose an important threat to amphibians and may play a role in their present global decline.

Anurans breed in a variety of aquatic habitats with contrasting levels of desiccation risk, which may result in selection for faster development during larval stages. Previous studies suggest that species in ephemeral ponds reduce their developmental times to minimize desiccation risks, although it is not clear how variation in desiccation risk affects developmental strategies in different species. Employing a comparative phylogenetic approach including data from published and unpublished studies encompassing 62 observations across 30 species, we tested if species breeding in ephemeral ponds (High risk) develop faster than those from permanent ponds (Low risk) and/or show increased developmental plasticity in response to drying conditions. Our analyses support shorter developmental times in High risk, primarily by decreasing body mass at metamorphosis. Plasticity in developmental times was small and did not differ between groups. However, accelerated development in High risk species generally resulted in reduced sizes at metamorphosis, while some Low risk species were able compensate this effect by increasing mean growth rates. Taken together, our results suggest that plastic responses in species breeding in ephemeral ponds are constrained by a general trade-off between development and growth rates.

Several studies have assessed the effects of nitrogenous compounds on amphibian behavior. However, few have focused on the effects of their combination with other stressors or on the variation of the response to pollutants among populations. We analyzed the effect of nitrogenous compounds (NH(4)(+); NO(2)(-); NO(3)(-), both alone and in combination) on larval behavior (activity level and location in the water column) in four populations of Pelophylax perezi naturally exposed to different levels of eutrophication. Larval activity was highest and use of the bottom of the experimental beaker was lowest at lower concentrations of nitrogenous compounds acting singly, these responses being minimal and maximal, respectively, at both control and higher concentrations. This pattern appears to fit to an hormetic response. Additionally, the combination of nitrogenous compounds affected more severely the response variables than when ammonium or nitrite acted singly according to an additive model. Populations inhabiting highly polluted aquatic habitats marginally showed higher activity level than the populations from less polluted environments, especially when larvae were exposed to ammonium or when nitrite appeared in combination with other nitrogen forms. Levels of activity correlated positively with larval final mass. Moreover, for similar levels of activity, larvae from polluted populations had higher growth rates than those coming from reference populations which suggests interdemic differences in behavioral sensitivity to nitrogenous pollutants.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii., (© 2011 Blackwell Publishing Ltd.)

Pollution by nitrogenous compounds is a putative stressful factor that may be causally linked to the decline of amphibians. One way to understand the potentially detrimental consequences of eutrophication on amphibian populations is to investigate variation among populations differing in exposure to nitrogen, this variation potentially indicating evolutionary potential to cope with this stressor. We have examined the effect of nitrogenous compounds (NH(4)(+); NO(2)(-); NO(3)(-), both alone and in combination) on fitness-related larval traits in four populations of Pelophylax perezi naturally exposed to different degrees of eutrophication. The results indicate that both survival and larval final size decrease at higher concentrations of these compounds, either singly or in combination. Additionally, the nitrogenous compounds were more lethal and larval food consumption and final mass were significantly reduced when they were exposed to combinations of compounds. Populations inhabiting highly polluted aquatic environments tolerated higher levels of nitrogenous compounds and showed higher survival rates and larger final size than the populations of less polluted environments, suggesting the potential to adapt to increased nitrogenous contamination in this species.

In an experiment carried out in the laboratory in beakers, the avoidance of ammonium chloride, isolated or combined with sodium nitrite and sodium nitrate, in aquatic habitat by froglets of Pelophylax perezi was studied. The results obtained suggest that nitrogen polluted treatments were not avoided by froglets of the studied species. However, despite the non-avoidance of the aquatic environment as a consequence of the presence of nitrogen compounds, significant inter-individual variation in treatment avoidance was detected. Although these results are not conclusive, they would suggest that froglets of P. perezi might occupy habitats which contain high levels of organic compounds and that they differ in their level of avoidance to fertilizer exposure.

The natterjack toad (Bufo calamita) shows variation in embryonic and larval salinity tolerance across populations in southern Spain. However, its aquatic/terrestrial biphasic life cycle, together with remarkable differences in salinity tolerance between Spanish and UK freshwater populations suggest an alternative hypothesis to local adaptation. Drought resistance during the terrestrial phase and salinity tolerance during the aquatic phase are both related to osmotic stress tolerance, and if there were an association between them, one could have evolved as an exaptation from the other. To test such an association, we reared B. calamita juveniles from three populations known to differ genetically in their salinity tolerance, under either dry or humid conditions. Drought decreased growth rate, enhanced burying behaviour, and decreased foraging activity and efficiency. No significant population x treatment interaction was found for any variable, i.e. populations were equally affected by drought. These results do not support the hypothesis of a genetic association between salinity and drought tolerance.

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.

Water salinity represents an environmental stress for many species. Amphibians are particularly sensitive because they are generally poor osmoregulators, and most species are completely absent from brackish and saline environments. We experimentally examined the effect of different salinity levels on larvae of the toad Bufo calamita L., a species that occupies freshwater ponds but can also breed in brackish ponds. Two independent experiments are reported here. In both experiments, tadpoles under saline conditions (ranging between 85 and 200 mOsm) showed a slower developmental rate, metamorphosing between 4 and 9 d later than the controls. Bufo calamita tadpoles reared in brackish water increased their osmolality and solute concentration (mainly sodium and chloride), decreased their levels of glucose, and decreased the total protein content, all measured from whole-animal extracts. Although most larval anurans are strictly ammoniotelic until the completion of metamorphosis, a few species exposed to dehydrating environments have evolved the ability to use urea as an osmolyte during the larval phase. The data presented here reveal that although B. calamita seems to be yet another exception to the rule of larval strict ammoniotelism, the tadpoles are not able to use urea as an osmolyte and rely on sodium-chloride balance instead. Preliminary immunoassays of thyroid hormone content suggest a possible decrease in hormone levels induced in water salinity conditions that correlate with a decreased developmental rate.

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.

A trade-off between size and number of offspring was not found for females of similar sizes of the natterjack toad (Bufo calamita). Moreover, for large females, clutches with higher number of eggs had larger eggs as well. This suggests that larger females produce more numerous and larger eggs because they potentially have more energy available for reproduction. Egg size diminished allometrically with clutch size. Egg size, however, did not increase offspring fitness. Therefore, this allometric decrease may be considered a consequence of phylogenetic constraints rather than a result of optimizing selection.