Urea and glucose modulation during freezing exposure in three temperate frogs reveals specific targets in relation to climate.

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.

Declining metabolic scaling parallels an ontogenetic change from elongate to deep-bodied shapes in juvenile Brown trout.

Body shape and metabolic rate can be important determinants of animal performance, yet often their effects on influential traits are evaluated in a non-integrated way. This creates an important gap because the integration between shape and metabolism may be crucial to evaluate metabolic scaling theories. Here, we measured standard metabolic rate in 1- and 2-years old juvenile brown trout Salmo trutta, and used a geometric morphometrics approach to extricate the effects of ontogeny and size on the link between shape and metabolic scaling. We evidenced near-isometric ontogenetic scaling of metabolic rate with size, but also a biphasic pattern driven by a significant change in metabolic scaling, from positive to negative allometry. Moreover, the change in metabolic allometry parallels an ontogenetic change from elongate to deep-bodied shapes. This is consistent with the dynamic energy budget (DEB) and surface area (SA) theories, but not with the resource transport network theory which predicts increasing allometric exponents for trends towards more robust, three-dimensional bodies. In addition, we found a relationship between body shape and size independent metabolic rate, with a positive correlation between robustness and metabolic rate, which fits well within the view of Pace-of-Life Syndromes (POLS). Finally, our results align with previous studies that question the universality of metabolic scaling exponents and propose other mechanistic models explaining the diversity of metabolic scaling relationships or emphasizing the potential contribution of ecological factors.

Phenology and plasticity can prevent adaptive clines in thermal tolerance across temperate mountains: The importance of the elevation-time axis.

Critical thermal limits (CTmax and CTmin) decrease with elevation, with greater change in CTmin, 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 CTmax and CTmin, 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 PST-FST 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 CTmax and CTmin, and unexpected elevation gradients in pond temperatures. However, variation in CTmax and CTmin 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 CTmax and CTmin plasticities than lowland populations, but not more extreme CTmax and CTmin. 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.

Independent evolutionary transitions to pueriparity across multiple timescales in the viviparous genus Salamandra.

The ability to bear live offspring, viviparity, has evolved multiple times across the tree of life and is a remarkable adaptation with profound life-history and ecological implications. Within amphibians the ancestral reproductive mode is oviparity followed by a larval life stage, but viviparity has evolved independently in all three amphibian orders. Two types of viviparous reproduction can be distinguished in amphibians; larviparity and pueriparity. Larviparous amphibians deliver larvae into nearby ponds and streams, while pueriparous amphibians deliver fully developed juveniles and thus do not require waterbodies for reproduction. Among amphibians, the salamander genus Salamandra is remarkable as it exhibits both inter- and intraspecific variation in the occurrence of larviparity and pueriparity. While the evolutionary relationships among Salamandra lineages have been the focus of several recent studies, our understanding of how often and when transitions between modes occurred is still incomplete. Furthermore, in species with intraspecific variation, the reproductive mode of a given population can only be confirmed by direct observation of births and thus the prevalence of pueriparous populations is also incompletely documented. We used sequence capture to obtain 1,326 loci from 94 individuals from across the geographic range of the genus, focusing on potential reproductive mode transition zones. We also report additional direct observations of pueriparous births for 20 new locations and multiple lineages. We identify at least five independent transitions from the ancestral mode of larviparity to pueriparity among and within species, occurring at different evolutionary timescales ranging from the Pliocene to the Holocene. Four of these transitions occurred within species. Based on a distinct set of markers and analyses, we also confirm previous findings of introgression between species and the need for taxonomic revisions in the genus. We discuss the implications of our findings with respect to the evolution of this complex trait, and the potential of using five independent convergent transitions for further studies on the ecological context in which pueriparity evolves and the genetic architecture of this specialized reproductive mode.

Individual differences in dominance-related traits drive dispersal and settlement in hatchery-reared juvenile brown trout.

Effective management of exploited populations is based on an understanding of population dynamics and evolutionary processes. In spatially structured populations, dispersal is a central process that ultimately can affect population growth and viability. It can be influenced by environmental conditions, individual phenotypes, and stochastic factors. However, we have a limited knowledge of the relative contribution of these components and its interactions, and which traits can be used as reliable predictors of the dispersal ability. Here, we conducted a longitudinal field experiment aimed to identify traits which can be used as proxy for dispersal in juvenile brown trout (Salmo trutta L.). We measured body size and standard metabolic rates, and estimated body shapes for 212 hatchery-reared juvenile fish that were marked with individual codes and released in a small coastal stream in northwest Spain. We registered fish positions and distances to the releasing point after 19, 41, 60 and 158 days in the stream. We detected a high autocorrelation of dispersal distances, demonstrating that most individuals settle down relatively soon and then hold stable positions over the study period. Body size and fish shape were reliable predictors of dispersal, with bigger and more robust-set individuals being more likely to settle closer to the release site than smaller and more elongated fish. In addition, the analysis of spacing and spatial patterns indicated that the dispersal of introduced fish could affect the distribution of resident conspecifics. All together, these results suggest that stocking programs aimed to the enhancement of overexploited populations at fine spatial scales can be optimized by adjusting the size and shape of the introduced fish to specific management targets and environmental conditions.

The evolution of pueriparity maintains multiple paternity in a polymorphic viviparous salamander.

The reduction in fecundity associated with the evolution of viviparity may have far-reaching implications for the ecology, demography, and evolution of populations. The evolution of a polygamous behaviour (e.g. polyandry) may counteract some of the effects underlying a lower fecundity, such as the reduction in genetic diversity. Comparing patterns of multiple paternity between reproductive modes allows us to understand how viviparity accounts for the trade-off between offspring quality and quantity. We analysed genetic patterns of paternity and offspring genetic diversity across 42 families from two modes of viviparity in a reproductive polymorphic species, Salamandra salamandra. This species shows an ancestral (larviparity: large clutches of free aquatic larvae), and a derived reproductive mode (pueriparity: smaller clutches of larger terrestrial juveniles). Our results confirm the existence of multiple paternity in pueriparous salamanders. Furthermore, we show the evolution of pueriparity maintains, and even increases, the occurrence of multiple paternity and the number of sires compared to larviparity, though we did not find a clear effect on genetic diversity. High incidence of multiple paternity in pueriparous populations might arise as a mechanism to avoid fertilization failures and to ensure reproductive success, and thus has important implications in highly isolated populations with small broods.

Niche models at inter- and intraspecific levels reveal hierarchical niche differentiation in midwife toads.

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 roles of acclimation and behaviour in buffering climate change impacts along elevational gradients.

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.

Are glacial refugia hotspots of speciation and cytonuclear discordances? Answers from the genomic phylogeography of Spanish common frogs.

Subdivided Pleistocene glacial refugia, best known as "refugia within refugia", provided opportunities for diverging populations to evolve into incipient species and/or to hybridize and merge following range shifts tracking the climatic fluctuations, potentially promoting extensive cytonuclear discordances and "ghost" mtDNA lineages. Here, we tested which of these opposing evolutionary outcomes prevails in northern Iberian areas hosting multiple historical refugia of common frogs (Rana cf. temporaria), based on a genomic phylogeography approach (mtDNA barcoding and RAD-sequencing). We found evidence for both incipient speciation events and massive cytonuclear discordances. On the one hand, populations from northwestern Spain (Galicia and Asturias, assigned to the regional endemic R. parvipalmata), are deeply-diverged at mitochondrial and nuclear genomes (~4 My of independent evolution), and barely admix with northeastern populations (assigned to R. temporaria sensu stricto) across a narrow hybrid zone (~25 km) located in the Cantabrian Mountains, suggesting that they represent distinct species. On the other hand, the most divergent mtDNA clade, widespread in Cantabria and the Basque country, shares its nuclear genome with other R. temporaria s. s. lineages. Patterns of population expansions and isolation-by-distance among these populations are consistent with past mitochondrial capture and/or drift in generating and maintaining this ghost mitochondrial lineage. This remarkable case study emphasizes the complex evolutionary history that shaped the present genetic diversity of refugial populations, and stresses the need to revisit their phylogeography by genomic approaches, in order to make informed taxonomic inferences.

Integrating mechanistic and correlative niche models to unravel range-limiting processes in a temperate amphibian.

Insights into the causal mechanisms that limit species distributions are likely to improve our ability to anticipate species range shifts in response to climate change. For species with complex life histories, a mechanistic understanding of how climate affects different lifecycle stages may be crucial for making accurate forecasts. Here, we use mechanistic niche modeling (NicheMapR) to derive "proximate" (mechanistic) variables for tadpole, juvenile, and adult Rana temporaria. We modeled the hydroperiod, and maximum and minimum temperatures of shallow (30 cm) ponds, as well as activity windows for juveniles and adults. We then used those ("proximate") variables in correlative ecological niche models (Maxent) to assess their role in limiting the species' current distribution, and to investigate the potential effects of climate change on R. temporaria across Europe. We further compared the results with a model based on commonly used macroclimatic ("distal") layers (i.e., bioclimatic layers from WorldClim). The maximum temperature of the warmest month (a macroclimatic variable) and maximum pond temperatures (a mechanistic variable) were the most important range-limiting factors, and maximum temperature thresholds were consistent with the observed upper thermal limit of R. temporaria tadpoles. We found that range shift forecasts in central Europe are far more pessimistic when using distal macroclimatic variables, compared to projections based on proximate mechanistic variables. However, both approaches predicted extensive decreases in climatic suitability in southern Europe, which harbors a significant fraction of the species' genetic diversity. We show how mechanistic modeling provides ways to depict gridded layers that directly reflect the microenvironments experienced by organisms at continental scales, and to reconstruct those predictors without extrapolation under novel future conditions. Furthermore, incorporating those predictors in correlative ecological niche models can help shed light on range-limiting processes, and can have substantial impacts on predictions of climate-induced range shifts.

Mapping of quantitative trait loci for life history traits segregating within common frog populations.

The evolution of complex traits is often shaped by adaptive divergence. However, very little is known about the number, effect size, and location of the genomic regions influencing the variation of these traits in natural populations. Based on a dense linkage map of the common frog, Rana temporaria, we have localized, for the first time in amphibians, three significant and nine suggestive quantitative trait loci (QTLs) for metabolic rate, growth rate, development time, and weight at metamorphosis, explaining 5.6-18.9% of the overall phenotypic variation in each trait. We also found a potential pleiotropic QTL between development time and size at metamorphosis that, if confirmed, might underlie the previously reported genetic correlation between these traits. Furthermore, we demonstrate that the genetic variation linked to fitness-related larval traits segregates within Rana temporaria populations. This study provides the first insight into the genomic regions that affect larval life history traits in anurans, providing a valuable resource to delve further into the genomic basis of evolutionary change in amphibians.

Ontogenetic reduction in thermal tolerance is not alleviated by earlier developmental acclimation in Rana temporaria.

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.

A rapid rate of sex-chromosome turnover and non-random transitions in true frogs.

The canonical model of sex-chromosome evolution predicts that, as recombination is suppressed along sex chromosomes, gametologs will progressively differentiate, eventually becoming heteromorphic. However, there are numerous examples of homomorphic sex chromosomes across the tree of life. This homomorphy has been suggested to result from frequent sex-chromosome turnovers, yet we know little about which forces drive them. Here, we describe an extremely fast rate of turnover among 28 species of Ranidae. Transitions are not random, but converge on several chromosomes, potentially due to genes they harbour. Transitions also preserve the ancestral pattern of male heterogamety, in line with the 'hot-potato' model of sex-chromosome transitions, suggesting a key role for mutation-load accumulation in non-recombining genomic regions. The importance of mutation-load selection in frogs might result from the extreme heterochiasmy they exhibit, making frog sex chromosomes differentiate immediately from emergence and across their entire length.

Hindered and constrained: limited potential for thermal adaptation in post-metamorphic and adult Rana temporaria along elevational gradients.

Adaptation to warming climates could counteract the effects of global warming. Thus, understanding how species cope with contrasting climates may inform us about their potential for thermal adaptation and which processes may hamper that ability (e.g. evolutionary trade-offs, phenology or behavioural thermoregulation). In addition to temperature, time constraints may also exert important selective pressures. Here, we compare the thermal sensitivity of locomotion of metamorphic and adult European common frogs (Rana temporaria) originating from populations along an elevational gradient. We employed the template mode of variation (TMV) analysis to decompose the thermal sensitivity of locomotion and explore the existence of trade-offs ('hotter is better' and 'specialist-generalist') and the degree of local adaptation. To that end, we studied the relationship between TMV parameters and local environmental conditions. Further, we compared preferred temperatures to assess whether behavioural thermoregulation could dampen the effects of thermal variation, reducing the intensity of selection and limiting thermal adaptation (i.e. 'Bogert effect'). We suggest that behavioural thermoregulation has promoted the conservatism of thermal sensitivity in R. temporaria. Yet, we observed a trend towards narrower thermal niches shifted towards warmer temperature in populations with severe temporal constraints, conforming to the 'generalist-specialist' trade-off. Apparently, this enables time-constrained populations - especially in the case of metamorphs - to effectively exploit resources during the warmest periods. The limited potential of R. temporaria for thermal adaptation suggests that forecasts of global warming should incorporate thermoregulation and explore its potential to buffer species from rising temperatures.

Phenotypic convergence of artificially reared and wild trout is mediated by shape plasticity.

Phenotypic plasticity can be viewed as the first level of defense of organism homeostasis against environmental stress and therefore represents the potential to deal with rapid environmental changes. Transitions between low complexity, artificial environments and complex, natural habitats can promote phenotypic plasticity. Here, we conducted an experimental introduction with juvenile brown trout to evaluate the plasticity of shape in response to a transition between contrasting environments. We released 202 juvenile trout reared under hatchery conditions in a natural stream and analyzed changes in shape and morphological variability after 5 months. A geometric morphometrics approach based on 14 landmarks was used to compare changes in body shape for 37 fish recaptured at the end of the experiment. A similar number of hatchery and wild fish caught at the receptor stream were used as controls for shape in the two environments. After 5-months, fish showed significant change in shape, shifting from elongated to robust shapes, and affecting to the relative position of the caudal peduncle. These new shapes were closer to wild than to the hatchery shapes, suggesting a process of rapid phenotype change. Moreover, these changes were concomitant with a marked increase in morphological variability. Our results support the hypothesis that phenotypic plasticity is a major potential for adjustment to environmental change but not the idea that shape can be constrained by initial shapes. We confirmed the "increased" variance hypothesis and phenotype convergence with wild morphs. This has important implications because stresses the role of phenotypic plasticity as a buffer that allows organisms to cope with important environmental discontinuities at time scales that preclude the onset of adaptive adjustments. We suggest that environmental conditioning and shape plasticity can overcome both reduced morphological diversity and phenotype uncoupling with habitat characteristics resulting from initial rearing in low complexity artificial environments.

Comparative High-Density Linkage Mapping Reveals Conserved Genome Structure but Variation in Levels of Heterochiasmy and Location of Recombination Cold Spots in the Common Frog.

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis We confirmed marked heterochiasmy between sexes and detected nonrecombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.

Neighbouring populations, opposite dynamics: influence of body size and environmental variation on the demography of stream-resident brown trout (Salmo trutta).

In organisms such as fish, where body size is considered an important state variable for the study of their population dynamics, size-specific growth and survival rates can be influenced by local variation in both biotic and abiotic factors, but few studies have evaluated the complex relationships between environmental variability and size-dependent processes. We analysed a 6-year capture-recapture dataset of brown trout (Salmo trutta) collected at 3 neighbouring but heterogeneous mountain streams in northern Spain with the aim of investigating the factors shaping the dynamics of local populations. The influence of body size and water temperature on survival and individual growth was assessed under a multi-state modelling framework, an extension of classical capture-recapture models that considers the state (i.e. body size) of the individual in each capture occasion and allows us to obtain state-specific demographic rates and link them to continuous environmental variables. Individual survival and growth patterns varied over space and time, and evidence of size-dependent survival was found in all but the smallest stream. At this stream, the probability of reaching larger sizes was lower compared to the other wider and deeper streams. Water temperature variables performed better in the modelling of the highest-altitude population, explaining over a 99 % of the variability in maturation transitions and survival of large fish. The relationships between body size, temperature and fitness components found in this study highlight the utility of multi-state approaches to investigate small-scale demographic processes in heterogeneous environments, and to provide reliable ecological knowledge for management purposes.

Larval life history and anti-predator strategies are affected by breeding phenology in an amphibian.

Seasonal time constraints can pose strong selection on life histories. Time-constrained animals should prioritise fast development over predation risk to avoid unfavourable growing conditions. However, changes in phenology could alter the balance between anti-predator and developmental needs. We studied variation of anti-predator strategies in common frog (Rana temporaria) tadpoles in four populations from the two extremes of a latitudinal gradient across Sweden. We examined, under common conditions in the laboratory, the anti-predator responses and life histories of tadpoles raised with predatory Aeshna dragonfly larvae in two consecutive years with a difference of 20 days in breeding time in the north, but no difference in breeding time in the nouth. In a year with late breeding, northern tadpoles did not modify their behaviour and morphology in the presence of predators, and metamorphosed faster and smaller than tadpoles born in a year with early breeding. In the year with early breeding, northern tadpoles showed a completely different anti-predator strategy by reducing activity and developing morphological defences in the presence of predators. We discuss the possible mechanisms that could activate these responses (likely a form of environmentally-mediated parental effect). To our knowledge, this is the first study to show that a vertebrate modifies the anti-predator strategy of its offspring in response to natural variation in reproductive phenology, which highlights the need to consider phenology in studies of life-history evolution.

Time constraints and flexibility of growth strategies: geographic variation in catch-up growth responses in amphibian larvae.

1. As size is tightly associated with fitness, compensatory strategies for growth loss can be vital for restoring individual fitness. However, immediate and delayed costs of compensatory responses may prevent their generalization, and the optimal strategy may depend on environmental conditions. Compensatory responses may be particularly important in high-latitude habitats with short growing seasons, and thus, high-latitude organisms might be more efficient at compensating after periods of unfavourable growth conditions than low-latitude organisms. 2. We investigated geographical differences in catch-up growth strategies of populations of the common frog (Rana temporaria) from southern and northern Sweden in two factorial common garden experiments involving predation risk and two different causes of growth arrest (nutritional stress and low temperatures) to evaluate how the compensatory strategies can be affected by context-dependent costs of compensation. Larval and metamorphic traits, and post-metamorphic performance were used as response variables. 3. Only northern tadpoles exposed to low food completely caught up in terms of metamorphic size, mainly by extending the larval period. Low food decreased survival and post-metamorphic jumping performance in southern, but not in northern tadpoles, suggesting that northern tadpoles have a better ability to compensate after periods of restricted food. 4. Both northern and southern tadpoles were able to metamorphose at the same size as control tadpoles after being exposed to low temperatures, indicating that consequences of variation in temperature and food availability differed for tadpoles. However, the combination of low temperatures and predation risk reduced survival in both southern and northern tadpoles. Also, predation risk decreased energy storage in both experiments. 5. Our results highlight the influence of climatic variation and the type of stressor as selective factors shaping compensatory strategies.

Statistical analysis of structural compensatory growth: how can we reduce the rate of false detection?

Compensatory growth (CG) is a key issue in work aiming at a full understanding of the adaptive significance of growth plasticity and its carryover effects on life-history. The number of studies addressing evolutionary explanations for CG has increased rapidly during the last few years, but there has not been a parallel gain in our understanding of the methodological difficulties associated with the analysis of CG. We point out two features of growth that can have serious consequences for detecting CG: (1) size dependence of growth rates, which causes nonlinearity of growth trajectories, and; (2) temporal overlapping of structural growth and replenishment of energy reserves after a period of famine. We show that the currently used methods can be prone to spurious detection of CG (Type I error) under conditions of nonlinear growth, and therefore lead to the accumulation of a significant amount of false "empirical support." True and simulated growth data provided consistent results suggesting that a substantial fraction of the existing evidence for CG may be spurious. A small curvature in the growth trajectory can lead to spurious "detection" of CG when control and manipulated trajectories are compared over the same time interval (the "simultaneous" approach). We present a novel, robust method (the "asynchronous" approach) based on the accurate selection of control trajectories and comparison of control and treatment growth rates at different times. This method enables a reliable test to be performed for compensation under asymptotic growth. While the general results of our simulations do not support the application of conventional methods to the general case of nonlinear growth trajectories under the simultaneous approach, simple methods may prove valid if the experimental design allows for asynchronous comparisons. We advocate an alternative approach to deal with "safe" detection of CG that overcomes the problems associated with the occurrence of nonlinear and asymptotic growth, and provide recommendations for improving CG study designs.