Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

Heritability and developmental plasticity of growth in an oviparous lizard.

Selective processes act on phenotypic variation although the evolutionary potential of a trait relies on the underlying heritable variation. Developmental plasticity is an important source of phenotypic variation, but it can also promote changes in genetic variation, yet we have a limited understanding of how they are both impacted. Here, we quantified the influence of developmental temperature on growth in delicate skinks (Lampropholis delicata) and partitioned total phenotypic variance using an animal model fitted with a genomic relatedness matrix. We measured mass for 261 individuals (nhot = 125, ncold = 136) over 16 months (nobservations = 3002) and estimated heritability and maternal effects over time. Our results show that lizards reared in cold developmental temperatures had consistently higher mass across development compared to lizards that were reared in hot developmental temperatures. However, developmental temperature did not impact the rate of growth. On average, additive genetic variance, maternal effects and heritability were higher in the hot developmental temperature treatment; however, these differences were not statistically significant. Heritability increased with age, whereas maternal effects decreased upon hatching but increased again at a later age, which could be driven by social competition or intrinsic changes in the expression of variation as an individual's growth. Our work suggests that the evolutionary potential of growth is complex, age-dependent and not overtly affected by extremes in natural nest temperatures.

Mitochondrial function and sexual selection: can physiology resolve the 'lek paradox'?

Across many taxa, males use elaborate ornaments or complex displays to attract potential mates. Such sexually selected traits are thought to signal important aspects of male 'quality'. Female mating preferences based on sexual traits are thought to have evolved because choosy females gain direct benefits that enhance their lifetime reproductive success (e.g. greater access to food) and/or indirect benefits because high-quality males contribute genes that increase offspring fitness. However, it is difficult to explain the persistence of female preferences when males only provide genetic benefits, because female preferences should erode the heritable genetic variation in fitness that sexually selected traits signal. This 'paradox of the lek' has puzzled evolutionary biologists for decades, and inspired many hypotheses to explain how heritable variation in sexually selected traits is maintained. Here, we discuss how factors that affect mitochondrial function can maintain variation in sexually selected traits despite strong female preferences. We discuss how mitochondrial function can influence the expression of sexually selected traits, and we describe empirical studies that link the expression of sexually selected traits to mitochondrial function. We explain how mothers can affect mitochondrial function in their offspring by (a) influencing their developmental environment through maternal effects and (b) choosing a mate to increase the compatibility of mitochondrial and nuclear genes (i.e. the 'mitonuclear compatibility model of sexual selection'). Finally, we discuss how incorporating mitochondrial function into models of sexual selection might help to resolve the paradox of the lek, and we suggest avenues for future research.

Publication bias impacts on effect size, statistical power, and magnitude (Type M) and sign (Type S) errors in ecology and evolutionary biology.

Collaborative efforts to directly replicate empirical studies in the medical and social sciences have revealed alarmingly low rates of replicability, a phenomenon dubbed the 'replication crisis'. Poor replicability has spurred cultural changes targeted at improving reliability in these disciplines. Given the absence of equivalent replication projects in ecology and evolutionary biology, two inter-related indicators offer the opportunity to retrospectively assess replicability: publication bias and statistical power. This registered report assesses the prevalence and severity of small-study (i.e., smaller studies reporting larger effect sizes) and decline effects (i.e., effect sizes decreasing over time) across ecology and evolutionary biology using 87 meta-analyses comprising 4,250 primary studies and 17,638 effect sizes. Further, we estimate how publication bias might distort the estimation of effect sizes, statistical power, and errors in magnitude (Type M or exaggeration ratio) and sign (Type S). We show strong evidence for the pervasiveness of both small-study and decline effects in ecology and evolution. There was widespread prevalence of publication bias that resulted in meta-analytic means being over-estimated by (at least) 0.12 standard deviations. The prevalence of publication bias distorted confidence in meta-analytic results, with 66% of initially statistically significant meta-analytic means becoming non-significant after correcting for publication bias. Ecological and evolutionary studies consistently had low statistical power (15%) with a 4-fold exaggeration of effects on average (Type M error rates = 4.4). Notably, publication bias reduced power from 23% to 15% and increased type M error rates from 2.7 to 4.4 because it creates a non-random sample of effect size evidence. The sign errors of effect sizes (Type S error) increased from 5% to 8% because of publication bias. Our research provides clear evidence that many published ecological and evolutionary findings are inflated. Our results highlight the importance of designing high-power empirical studies (e.g., via collaborative team science), promoting and encouraging replication studies, testing and correcting for publication bias in meta-analyses, and adopting open and transparent research practices, such as (pre)registration, data- and code-sharing, and transparent reporting.

A robust and readily implementable method for the meta-analysis of response ratios with and without missing standard deviations.

The log response ratio, lnRR, is the most frequently used effect size statistic for meta-analysis in ecology. However, often missing standard deviations (SDs) prevent estimation of the sampling variance of lnRR. We propose new methods to deal with missing SDs via a weighted average coefficient of variation (CV) estimated from studies in the dataset that do report SDs. Across a suite of simulated conditions, we find that using the average CV to estimate sampling variances for all observations, regardless of missingness, performs with minimal bias. Surprisingly, even with missing SDs, this simple method outperforms the conventional approach (basing each effect size on its individual study-specific CV) with complete data. This is because the conventional method ultimately yields less precise estimates of the sampling variances than using the pooled CV from multiple studies. Our approach is broadly applicable and can be implemented in all meta-analyses of lnRR, regardless of 'missingness'.

Metabolic consequences of sex reversal in two lizard species: a test of the like-genotype and like-phenotype hypotheses.

Vertebrate sex is typically determined genetically, but in many ectotherms sex can be determined by genes (genetic sex determination, GSD), temperature (temperature-dependent sex determination, TSD), or interactions between genes and temperature during development. TSD may involve GSD systems with either male or female heterogamety (XX/XY or ZZ/ZW) where temperature overrides chromosomal sex determination to cause a mismatch between genetic sex and phenotypic sex (sex reversal). In these temperature-sensitive lineages, phylogenetic investigations point to recurrent evolutionary shifts between genotypic and temperature-dependent sex determination. These evolutionary transitions in sex determination can occur rapidly if selection favours the reversed sex over the concordant phenotypic sex. To investigate the consequences of sex reversal on offspring phenotypes, we measured two energy-driven traits (metabolism and growth) and 6 month survival in two species of reptile with different patterns of temperature-induced sex reversal. Male sex reversal occurs in Bassiana duperreyi when chromosomal females (female XX) develop male phenotypes (maleSR XX), while female sex reversal occurs in Pogona vitticeps when chromosomal males (male ZZ) develop female phenotypes (femaleSR ZZ). We show metabolism in maleSR XX was like that of male XY; that is, reflective of phenotypic sex and lower than genotypic sex. In contrast, for Pogona vitticeps, femaleSR ZZ metabolism was intermediate between male ZZ and female ZW metabolic rate. For both species, our data indicate that differences in metabolism become more apparent as individuals become larger. Our findings provide some evidence for an energetic advantage from sex reversal in both species but do not exclude energetic processes as a constraint on the distribution of sex reversal in nature.

Developmental environments do not affect thermal physiological traits in reptiles: an experimental test and meta-analysis.

On a global scale, organisms face significant challenges due to climate change and anthropogenic disturbance. In many ectotherms, developmental and physiological processes are sensitive to changes in temperature and resources. Developmental plasticity in thermal physiology may provide adaptive advantages to environmental extremes if early environmental conditions are predictive of late-life environments. Here, we conducted a laboratory experiment to test how developmental temperature and maternal resource investment influence thermal physiological traits (critical thermal maximum: CTmax and thermal preference: Tpref) in a common skink (Lampropholis delicata). We then compared our experimental findings more broadly across reptiles (snakes, lizards and turtles) using meta-analysis. In both our experimental study and meta-analysis, we did not find evidence that developmental environments influence CTmax or Tpref. Furthermore, the effects of developmental environments on thermal physiology did not vary by age, taxon or climate zone (temperate/tropical). Overall, the magnitude of developmental plasticity on thermal physiology appears to be limited across reptile taxa suggesting that behavioural or evolutionary processes may be more important. However, there is a paucity of information across most reptile taxa, and a broader focus on thermal performance curves themselves will be critical in understanding the impacts of changing thermal conditions on reptiles in the future.

Developmental plasticity in thermal tolerance: Ontogenetic variation, persistence, and future directions.

Understanding the factors affecting thermal tolerance is crucial for predicting the impact climate change will have on ectotherms. However, the role developmental plasticity plays in allowing populations to cope with thermal extremes is poorly understood. Here, we meta-analyse how thermal tolerance is initially and persistently impacted by early (embryonic and juvenile) thermal environments by using data from 150 experimental studies on 138 ectothermic species. Thermal tolerance only increased by 0.13°C per 1°C change in developmental temperature and substantial variation in plasticity (~36%) was the result of shared evolutionary history and species ecology. Aquatic ectotherms were more than three times as plastic as terrestrial ectotherms. Notably, embryos expressed weaker but more heterogenous plasticity than older life stages, with numerous responses appearing as non-adaptive. While developmental temperatures did not have persistent effects on thermal tolerance overall, persistent effects were vastly under-studied, and their direction and magnitude varied with ontogeny. Embryonic stages may represent a critical window of vulnerability to changing environments and we urge researchers to consider early life stages when assessing the climate vulnerability of ectotherms. Overall, our synthesis suggests that developmental changes in thermal tolerance rarely reach levels of perfect compensation and may provide limited benefit in changing environments.

Impact of fluctuating developmental temperatures on phenotypic traits in reptiles: a meta-analysis.

During the vulnerable stages of early life, most ectothermic animals experience hourly and diel fluctuations in temperature as air temperatures change. While we know a great deal about how different constant temperatures impact the phenotypes of developing ectotherms, we know remarkably little about the impacts of temperature fluctuations on the development of ectotherms. In this study, we used a meta-analytic approach to compare the mean and variance of phenotypic outcomes from constant and fluctuating incubation temperatures across reptile species. We found that fluctuating temperatures provided a small benefit (higher hatching success and shorter incubation durations) at cool mean temperatures compared with constant temperatures, but had a negative effect at warm mean temperatures. In addition, more extreme temperature fluctuations led to greater reductions in embryonic survival compared with moderate temperature fluctuations. Within the limited data available from species with temperature-dependent sex determination, embryos had a higher chance of developing as female when developing in fluctuating temperatures compared with those developing in constant temperatures. With our meta-analytic approach, we identified average mean nest temperatures across all taxa where reptiles switch from receiving benefits to incurring costs when incubation temperatures fluctuate. More broadly, our study indicates that the impact of fluctuating developmental temperature on some phenotypes in ectothermic taxa are likely to be predictable via integration of developmental temperature profiles with thermal performance curves.

Meta-analytic approaches and effect sizes to account for 'nuisance heterogeneity' in comparative physiology.

Meta-analysis is a powerful tool used to generate quantitatively informed answers to pressing global challenges. By distilling data from broad sets of research designs and study systems into standardised effect sizes, meta-analyses provide physiologists with opportunities to estimate overall effect sizes and understand the drivers of effect variability. Despite this ambition, research designs in the field of comparative physiology can appear, at the outset, as being vastly different to each other because of 'nuisance heterogeneity' (e.g. different temperatures or treatment dosages used across studies). Methodological differences across studies have led many to believe that meta-analysis is an exercise in comparing 'apples with oranges'. Here, we dispel this myth by showing how standardised effect sizes can be used in conjunction with multilevel meta-regression models to both account for the factors driving differences across studies and make them more comparable. We assess the prevalence of nuisance heterogeneity in the comparative physiology literature - showing it is common and often not accounted for in analyses. We then formalise effect size measures (e.g. the temperature coefficient, Q10) that provide comparative physiologists with a means to remove nuisance heterogeneity without the need to resort to more complex statistical models that may be harder to interpret. We also describe more general approaches that can be applied to a variety of different contexts to derive new effect sizes and sampling variances, opening up new possibilities for quantitative synthesis. By using effect sizes that account for components of effect heterogeneity, in combination with existing meta-analytic models, comparative physiologists can explore exciting new questions while making results from large-scale data sets more accessible, comparable and widely interpretable.

Plastic responses to novel environments are biased towards phenotype dimensions with high additive genetic variation.

Environmentally induced phenotypes have been proposed to initiate and bias adaptive evolutionary change toward particular directions. The potential for this to happen depends in part on how well plastic responses are aligned with the additive genetic variance and covariance in traits. Using meta-analysis, we demonstrate that plastic responses to novel environments tend to occur along phenotype dimensions that harbor substantial amounts of additive genetic variation. This suggests that selection for or against environmentally induced phenotypes typically will be effective. One interpretation of the alignment between the direction of plasticity and the main axis of additive genetic variation is that developmental systems tend to respond to environmental novelty as they do to genetic mutation. This makes it challenging to distinguish if the direction of evolution is biased by plasticity or genetic "constraint." Our results therefore highlight a need for new theoretical and empirical approaches to address the role of plasticity in evolution.

Heightened among-individual variation in life history but not morphology is related to developmental temperature in reptiles.

Increases in phenotypic variation under extreme (e.g. novel or stressful) environmental conditions are emerging as a crucial process through which evolutionary adaptation can occur. Lack of prior stabilizing selection, as well as potential instability of developmental processes in these environments, may lead to a release of phenotypic variation that can have important evolutionary consequences. Although such patterns have been shown in model study organisms, we know little about the generality of trait variance across environments for non-model organisms. Here, we test whether extreme developmental temperatures increase the phenotypic variation across diverse reptile taxa. We find that the among-individual variation in a key life-history trait (post-hatching growth) increases at extreme cold and hot temperatures. However, variations in two measures of hatchling morphology and in hatchling performance were not related to developmental temperature. Although extreme developmental temperatures may increase the variation in growth, our results suggest that plastic responses to stressful incubation conditions do not generally make more extreme phenotypes available to selection. We discuss the reasons for the general lack of increased variability at extreme incubation temperatures and the implications this has for local adaptation in hatchling morphology and physiology.

Sexual selection on performance traits in an Australian lizard with alternative reproductive tactics.

Sexual selection shapes the adaptive landscape in complex ways that lead to trait integration. Much of our understanding of selection comes from studies of morphological traits. However, few studies attempt to quantify the form and direction of selection on performance even though it is predicted to be a more direct target of selection in nature. We measured sexual selection on performance traits (bite force, sprint speed and endurance) in an Australian lizard, the Eastern water skink (Eulamprus quoyii). We first staged 123 contests between size-matched males to investigate whether performance traits were important in determining contest outcome. In a second experiment, we established six breeding populations in large replicate semi-natural enclosures to estimate whether performance traits predicted reproductive success. Our results show that none of the performance measures were important in predicting contest outcome and were not generally strong predictors of reproductive success. However, our analyses suggest a complex fitness landscape driven by males adopting different alternative reproductive tactics (ARTs). We provide a rare test of the role performance plays in sexual selection and highlight the need to test common assumptions regarding the link between maximal performance and fitness. Our results suggest that performance traits may not necessarily be direct targets of sexual selection, but rather indirect targets through their integration with morphological and/or behavioural traits, highlighting a need for more explicit tests of the predicted links between performance and fitness.

Diving in hot water: a meta-analytic review of how diving vertebrate ectotherms will fare in a warmer world.

Diving ectothermic vertebrates are an important component of many aquatic ecosystems, but the threat of climate warming is particularly salient to this group. Dive durations typically decrease as water temperatures rise; yet, we lack an understanding of whether this trend is apparent in all diving ectotherms and how this group will fare under climate warming. We compiled data from 27 studies on 20 ectothermic vertebrate species to quantify the effect of temperature on dive durations. Using meta-analytic approaches, we show that, on average, dive durations decreased by 11% with every 1°C increase in water temperature. Larger increases in temperature (e.g. +3°C versus +8-9°C) exerted stronger effects on dive durations. Although species that respire bimodally are projected to be more resilient to the effects of temperature on dive durations than purely aerial breathers, we found no significant difference between these groups. Body mass had a weak impact on mean dive durations, with smaller divers being impacted by temperature more strongly. Few studies have examined thermal phenotypic plasticity (N=4) in diving ectotherms, and all report limited plasticity. Average water temperatures in marine and freshwater habitats are projected to increase between 1.5 and 4°C in the next century, and our data suggest that this magnitude of warming could translate to substantial decreases in dive durations, by approximately 16-44%. Together, these data shed light on an overlooked threat to diving ectothermic vertebrates and suggest that time available for underwater activities, such as predator avoidance and foraging, may be shortened under future warming.

The jury is still out regarding the generality of adaptive 'transgenerational' effects.

A recent meta-analysis concluded, 'transgenerational effects are widespread, strong and persistent'. We identify biases in the literature search, data and analyses, questioning that conclusion. Re-analyses indicate few studies actually tested transgenerational effects - making it challenging to disentangle condition-transfer from anticipatory parental effects, and providing little insight into the underlying mechanisms.

Context-specific response inhibition and differential impact of a learning bias in a lizard.

Response inhibition (inhibiting prepotent responses) is needed for reaching a more favourable goal in situations where reacting automatically would be detrimental. Inhibiting prepotent responses to resist the temptation of a stimulus in certain situations, such as a novel food item, can directly affect an animal's survival. In humans and dogs, response inhibition varies between contexts and between individuals. We used two contextually different experiments to investigate response inhibition in the eastern water skink (Eulamprus quoyii): reversal of a visual two-choice discrimination and a cylinder detour task. During the two-choice task, half of our lizards were able to reach an initial learning criterion, but, thereafter, did not show consistent performance. Only two individuals reached a more stringent criterion, but subsequently failed during reversals. Furthermore, half of our animals were not able to inhibit a pre-existing side preference which affected their ability to learn during the two-choice task. Skinks were, however, able to achieve a detour around a cylinder performing at levels comparable to brown lemurs, marmosets, and some parrot species. A comparison between the tasks showed that reaching the initial criterion was associated with low success during the detour task, indicating that response inhibition could be context-specific in the water skink. To the best of our knowledge, this is the first study to examine inhibitory control and motor self-regulation in a lizard species.

Testing domain general learning in an Australian lizard.

A key question in cognition is whether animals that are proficient in a specific cognitive domain (domain specific hypothesis), such as spatial learning, are also proficient in other domains (domain general hypothesis) or whether there is a trade-off. Studies testing among these hypotheses are biased towards mammals and birds. To understand constraints on the evolution of cognition more generally, we need broader taxonomic and phylogenetic coverage. We used Australian eastern water skinks (Eulamprus quoyii) with known spatial learning ability in three additional tasks: an instrumental and two discrimination tasks. Under domain specific learning we predicted that lizards that were good at spatial learning would perform less well in the discrimination tasks. Conversely, we predicted that lizards that did not meet our criterion for spatial learning would likewise perform better in discrimination tasks. Lizards with domain general learning should perform approximately equally well (or poorly) in these tasks. Lizards classified as spatial learners performed no differently to non-spatial learners in both the instrumental and discrimination learning tasks. Nevertheless, lizards were proficient in all tasks. Our results reveal two patterns: domain general learning in spatial learners and domain specific learning in non-spatial learners. We suggest that delineating learning into domain general and domain specific may be overly simplistic and we need to instead focus on individual variation in learning ability, which ultimately, is likely to play a key role in fitness. These results, in combination with previously published work on this species, suggests that this species has behavioral flexibility because they are competent across multiple cognitive domains and are capable of reversal learning.

Maternal effects impact decision-making in a viviparous lizard.

Stressful conditions experienced during early development can have deleterious effects on offspring morphology, physiology and behaviour. However, few studies have examined how developmental stress influences an individual's cognitive phenotype. Using a viviparous lizard, we show that the availability of food resources to a mother during gestation influences a key component of her offspring's cognitive phenotype: their decision-making. Offspring from females who experienced low resource availability during gestation did better in an anti-predatory task that relied on spatial associations to guide their decisions, whereas offspring from females who experienced high resource availability during gestation did better in a foraging task that relied on colour associations to inform their decisions. This shows that the prenatal environment can influence decision-making in animals, a cognitive trait with functional implications later in life.

Mate familiarity and social learning in a monogamous lizard.

Social learning is thought to be advantageous as it allows an animal to gather information quickly without engaging in costly trial-and-error learning. However, animals should be selective about when and whom they learn from. Familiarity is predicted to positively influence an animal's reliance on social learning; yet, few studies have empirically tested this theory. We used a lizard (Liopholis whitii) that forms long-term monogamous pair bonds to examine the effects of partner familiarity on social learning in two novel foraging tasks, an association and a reversal task. We allowed female lizards to observe trained conspecifics that were either familiar (social mate) or unfamiliar execute these tasks and compared these two groups with control females that did not receive social information. Lizards preferentially relied on trial-and-error learning in the association task. In the reversal task, lizards that were demonstrated by familiar partners learnt in fewer trials compared to control lizards and made more correct choices. Our results provide some evidence for context-dependent learning with lizards differentiating between when they utilize social learning, and, to a limited degree, whom they learnt from. Understanding the role of the social context in which learning occurs provides important insights into the benefits of social learning and sociality more generally.

Meta-evaluation of meta-analysis: ten appraisal questions for biologists.

Meta-analysis is a statistical procedure for analyzing the combined data from different studies, and can be a major source of concise up-to-date information. The overall conclusions of a meta-analysis, however, depend heavily on the quality of the meta-analytic process, and an appropriate evaluation of the quality of meta-analysis (meta-evaluation) can be challenging. We outline ten questions biologists can ask to critically appraise a meta-analysis. These questions could also act as simple and accessible guidelines for the authors of meta-analyses. We focus on meta-analyses using non-human species, which we term 'biological' meta-analysis. Our ten questions are aimed at enabling a biologist to evaluate whether a biological meta-analysis embodies 'mega-enlightenment', a 'mega-mistake', or something in between.