Hydric physiology and ecology of a federally endangered desert lizard.

Animals can respond to extreme climates by behaviourally avoiding it or by physiologically coping with it. We understand behavioural and physiological thermoregulation, but water balance has largely been neglected. Climate change includes both global warming and changes in precipitation regimes, so improving our understanding of organismal water balance is increasingly urgent. We assessed the hydric physiology of US federally endangered blunt-nosed leopard lizards (Gambelia sila) by measuring cutaneous evaporative water loss (CEWL), plasma osmolality and body condition. Measurements were taken throughout their active season, the short period of year when these lizards can be found aboveground. Compared to a more mesic species, G. sila had low CEWL which is potentially desert-adaptive, and high plasma osmolality that could be indicative of dehydration. We hypothesized that throughout the G. sila active season, as their habitat got hotter and drier, G. sila would become more dehydrated and watertight. Instead, CEWL and plasma osmolality showed minimal change for females and non-linear change for males, which we hypothesize is connected to sex-specific reproductive behaviours and changes in food availability. We also measured thermoregulation and microhabitat use, expecting that more dehydrated lizards would have lower body temperature, poorer thermoregulatory accuracy and spend less time aboveground. However, we found no effect of CEWL, plasma osmolality or body condition on these thermal and behavioural metrics. Finally, G. sila spends considerable time belowground in burrows, and burrows may serve not only as essential thermal refugia but also hydric refugia.

Hydration and evaporative water loss of lizards change in response to temperature and humidity acclimation.

Testing acclimation plasticity informs our understanding of organismal physiology and applies to conservation management amidst our rapidly changing climate. Although there is a wealth of research on the plasticity of thermal and hydric physiology in response to temperature acclimation, there is a comparative gap for research on acclimation to different hydric regimes, as well as the interaction between water and temperature. We sought to fill this gap by acclimating western fence lizards (Sceloporus occidentalis) to experimental climate conditions (crossed design of hot or cool, dry or humid) for 8 days, and measuring cutaneous evaporative water loss (CEWL), plasma osmolality, hematocrit and body mass before and after acclimation. CEWL changed plastically in response to the different climates, with lizards acclimated to hot humid conditions experiencing the greatest increase in CEWL. Change in CEWL among individuals was negatively related to treatment vapor pressure deficit and positively related to treatment water vapor pressure. Plasma osmolality, hematocrit and body mass all showed greater changes in response to temperature than to humidity or vapor pressure deficit. CEWL and plasma osmolality were positively related across treatment groups before acclimation and within treatment groups after acclimation, but the two variables showed different responses to acclimation, suggesting that they are interrelated but governed by different mechanisms. This study is among few that assess more than one metric of hydric physiology and that test the interactive effects of temperature and humidity. Such measurements will be essential for predictive models of activity and survival for animals under climate change.

Oxygen environment and metabolic oxygen demand predictably interact to affect thermal behavior in a lizard, Sceloporus occidentalis.

The climate crisis necessitates predicting how organisms respond to changing environments, but this requires understanding the mechanisms underlying thermal tolerance. The Hierarchical Mechanisms of Thermal Limitation (HMTL) hypothesis proposes that respiratory capacity and marginal stability of proteins and membranes interact hierarchically to determine thermal performance and limits. An untested prediction of the HMTL hypothesis is that behavioral anapyrexia (i.e., reduced body temperature in hypoxia) is exacerbated when metabolic demand is high. We tested this prediction by manipulating western fence lizards' (Sceloporus occidentalis) metabolic demand and oxygen environment, then measuring selected body temperatures. Lizards with elevated metabolic demand selected lower body temperatures at higher oxygen concentrations than resting lizards, but this occurred only at oxygen concentrations <12% O2 , suggesting thermal limits are unaffected by naturally-occurring oxygen variation. Given our results and the ubiquity of behavioral anapyrexia, the HMTL hypothesis may generally explain how oxygen and temperature interactively affect reptile performance.

High-Elevation Populations of Montane Grasshoppers Exhibit Greater Developmental Plasticity in Response to Seasonal Cues.

Populations of insects can differ in how sensitive their development, growth, and performance are to environmental conditions such as temperature and daylength. The environmental sensitivity of development can alter phenology (seasonal timing) and ecology. Warming accelerates development of most populations. However, high-elevation and season-limited populations can exhibit developmental plasticity to either advance or prolong development depending on conditions. We examine how diurnal temperature variation and daylength interact to shape growth, development, and performance of several populations of the montane grasshopper, Melanoplus boulderensis, along an elevation gradient. We then compare these experimental results to observed patterns of development in the field. Although populations exhibited similar thermal sensitivities of development under long-day conditions, development of high-elevation populations was more sensitive to temperature under short-day conditions. This developmental plasticity resulted in rapid development of high elevation populations in short-day conditions with high temperature variability, consistent with their observed capacity for rapid development in the field when conditions are permissive early in the season. Notably, accelerated development generally did not decrease body size or alter body shape. Developmental conditions did not strongly influence thermal tolerance but altered the temperature dependence of performance in difficult-to-predict ways. In sum, the high-elevation and season-limited populations exhibited developmental plasticity that enables advancing or prolonging development consistent with field phenology. Our results suggest these patterns are driven by the thermal sensitivity of development increasing when days are short early in the season compared to when days are long later in the season. Developmental plasticity will shape phenological responses to climate change with potential implications for community and ecosystem structure.

A chromosome-level genome assembly for the eastern fence lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology.

BACKGROUND: High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. Lizards in the genus Sceloporus have a long history as important ecological, evolutionary, and physiological models, making them a valuable target for the development of genomic resources. FINDINGS: We present a high-quality chromosome-level reference genome assembly, SceUnd1.0 (using 10X Genomics Chromium, HiC, and Pacific Biosciences data), and tissue/developmental stage transcriptomes for the eastern fence lizard, Sceloporus undulatus. We performed synteny analysis with other snake and lizard assemblies to identify broad patterns of chromosome evolution including the fusion of micro- and macrochromosomes. We also used this new assembly to provide improved reference-based genome assemblies for 34 additional Sceloporus species. Finally, we used RNAseq and whole-genome resequencing data to compare 3 assemblies, each representing an increased level of cost and effort: Supernova Assembly with data from 10X Genomics Chromium, HiRise Assembly that added data from HiC, and PBJelly Assembly that added data from Pacific Biosciences sequencing. We found that the Supernova Assembly contained the full genome and was a suitable reference for RNAseq and single-nucleotide polymorphism calling, but the chromosome-level scaffolds provided by the addition of HiC data allowed synteny and whole-genome association mapping analyses. The subsequent addition of PacBio data doubled the contig N50 but provided negligible gains in scaffold length. CONCLUSIONS: These new genomic resources provide valuable tools for advanced molecular analysis of an organism that has become a model in physiology and evolutionary ecology.

Introduction to the special issue-Beyond CTMAX and CTMIN : Advances in studying the thermal limits of reptiles and amphibians.

Two themes emerging from the special issue "Beyond CTMAX and CTMIN : Advances in Studying the Thermal Limits of Reptiles and Amphibians" are: (1) the need to identify mechanisms that determine the shape of thermal performance curves and (2) how these curves can be best used predictively.

Thermoregulation and thermal performance of crested geckos (Correlophus ciliatus) suggest an extended optimality hypothesis for the evolution of thermoregulatory set-points.

Crested geckos (Correlophus ciliatus, formerly Rhacodactylus ciliatus) were rediscovered in New Caledonia 25 years ago and despite being common in the pet trade, there is no published information on their physiology. We measured thermoregulation (preferred body temperature, thermal set-point range, and voluntary limits) and performance (thermal performance curves [TPC] for 25 cm sprint speed and 1 m running speed) of adult and juvenile crested geckos in the laboratory to describe their thermal tolerances, differences among life stages, correlations between behavior and performance, and correlations with natural temperatures. Despite lacking special lighting or heating requirements in captivity, crested geckos displayed typical thermal biology for a lizard with no difference among life stages. They thermoregulated to a narrow set-point range (TSET , 24-28°C), that broadly overlaps natural air temperatures in New Caledonia, during activity. Somewhat surprisingly, the optimal temperature for performance (TOPT , 32°C) was substantially above preferred body temperatures and approximated the average maximum temperature voluntarily experienced (VTMAX , 33°C). Preferred body temperatures, by contrast, corresponded to the lower threshold temperature (Td ) where the TPC deviated from exponential, which we suggest is the temperature where performance is optimized after accounting for the costs of metabolic demand and overheating risk. Our results demonstrate that despite their lack of specific requirements when housed in human dwellings, crested geckos actively thermoregulate to temperatures that facilitate performance, and have thermal biology typical of other nocturnal or shade-dwelling species. Additionally, crested geckos appear at little risk of direct climate change-induced decline because increased temperatures should allow increased activity.

The thermal ecology and physiology of reptiles and amphibians: A user's guide.

Research on the thermal ecology and physiology of free-living organisms is accelerating as scientists and managers recognize the urgency of the global biodiversity crisis brought on by climate change. As ectotherms, temperature fundamentally affects most aspects of the lives of amphibians and reptiles, making them excellent models for studying how animals are impacted by changing temperatures. As research on this group of organisms accelerates, it is essential to maintain consistent and optimal methodology so that results can be compared across groups and over time. This review addresses the utility of reptiles and amphibians as model organisms for thermal studies by reviewing the best practices for research on their thermal ecology and physiology, and by highlighting key studies that have advanced the field with new and improved methods. We end by presenting several areas where reptiles and amphibians show great promise for further advancing our understanding of how temperature relations between organisms and their environments are impacted by global climate change.

Analyzing Stress as a Multivariate Phenotype.

The stress phenotype is multivariate. Recent advances have broadened our understanding beyond characterizing the stress response in a single dimension. Simultaneously, the toolbox available to ecophysiologists has expanded greatly in recent years, allowing the measurement of multiple biomarkers from an individual at a single point in time. Yet these advances-in our conceptual understanding and available methodologies-have not yet been combined in a unifying multivariate statistical framework. Here, we offer a brief review of the multivariate stress phenotype and describe a general statistical approach for analysis using nonparametric multivariate analysis of variance with residual randomization in permutation procedures (RRPP) implemented using the "RRPP" package in R. We also provide an example illustrating the novel insights that can be gained from a holistic multivariate approach to stress and provide a tutorial for how we analyzed these data, including annotated R code and a guide to interpretation of outputs (Online Appendix 1). We hope that this statistical methodology will provide a quantitative framework facilitating the unification of our theoretical understanding and empirical observations into a quantitative, multivariate theory of stress.

Contrasting Responses of Lizards to Divergent Ecological Stressors Across Biological Levels of Organization.

It is frequently hypothesized that animals employ a generalized "stress response," largely mediated by glucocorticoid (GC) hormones, such as corticosterone, to combat challenging environmental conditions. Under this hypothesis, diverse stressors are predicted to have concordant effects across biological levels of an organism. We tested the generalized stress response hypothesis in two complementary experiments with juvenile and adult male Eastern fence lizards (Sceloporus undulatus). In both experiments, animals were exposed to diverse, ecologically-relevant, acute stressors (high temperature or red imported fire ants, Solenopsis invicta) and we examined their responses at three biological levels: behavioral; physiological (endocrine [plasma corticosterone and blood glucose concentrations] and innate immunity [complement and natural antibodies]); and cellular responses (gene expression of a panel of five heat-shock proteins in blood and liver) at 30 or 90 min post stress initiation. In both experiments, we observed large differences in the cellular response to the two stressors, which contrasts the similar behavioral and endocrine responses. In the adult experiment for which we had innate immune data, the stressors affected immune function independently, and they were correlated with CORT in opposing directions. Taken together, these results challenge the concept of a generalized stress response. Rather, the stress response was context specific, especially at the cellular level. Such context-specificity might explain why attempts to link GC hormones with life history and fitness have proved difficult. Our results emphasize the need for indicators at multiple biological levels and whole-organism examinations of stress.

High Temperature, Oxygen, and Performance: Insights from Reptiles and Amphibians.

Much recent theoretical and empirical work has sought to describe the physiological mechanisms underlying thermal tolerance in animals. Leading hypotheses can be broadly divided into two categories that primarily differ in organizational scale: 1) high temperature directly reduces the function of subcellular machinery, such as enzymes and cell membranes, or 2) high temperature disrupts system-level interactions, such as mismatches in the supply and demand of oxygen, prior to having any direct negative effect on the subcellular machinery. Nonetheless, a general framework describing the contexts under which either subcellular component or organ system failure limits organisms at high temperatures remains elusive. With this commentary, we leverage decades of research on the physiology of ectothermic tetrapods (amphibians and non-avian reptiles) to address these hypotheses. Available data suggest both mechanisms are important. Thus, we expand previous work and propose the Hierarchical Mechanisms of Thermal Limitation (HMTL) hypothesis, which explains how subcellular and organ system failures interact to limit performance and set tolerance limits at high temperatures. We further integrate this framework with the thermal performance curve paradigm commonly used to predict the effects of thermal environments on performance and fitness. The HMTL framework appears to successfully explain diverse observations in reptiles and amphibians and makes numerous predictions that remain untested. We hope that this framework spurs further research in diverse taxa and facilitates mechanistic forecasts of biological responses to climate change.

Measuring behavioral thermal tolerance to address hot topics in ecology, evolution, and conservation.

Understanding the impacts of anthropogenic climate change requires knowing how animals avoid heat stress, and the consequences of failing to do so. Animals primarily use behavior to avoid overheating, but biologists' means for measuring and interpreting behavioral signs of stress require more development. Herein, we develop the measurement of behavioral thermal tolerance using four species of lizards. First, we adapt the voluntary thermal maximum concept (VTM) to facilitate its measurement, interpretation, and comparison across species. Second, we evaluate the sensitivity of the VTM to diverse measurement options (warming rate, time of day, etc) across four species with highly different life histories. Finally, we clarify the interpretation of VTM in two ways. First, we show the effects of exposure to the VTM on panting behavior, mass loss, and locomotor function loss of two species. Second, we compared the VTM with the preferred body temperatures (PBT) and critical thermal maximum (CTMAX) intraspecifically. We found that the VTM can be consistently estimated through different methods and methodological options, only very slow warming rates affected its estimates in one species. Exposure to the VTM caused panting between 5 and 50 min and induced exceptionally high mass loss rates. Loss of locomotion function started after 205 min. Further, the VTM did not show intraspecific correlations with the PBT and CTMAX. Our study suggests the VTM is a robust and flexible measure of thermal tolerance and highlights the need for multispecies evaluations of thermal indices. The lack of correlation between the VTM, the PBT and CTMAX suggests the VTM may evolve relatively free between the other parameters. We make reccommendations for understanding and using the VTM in studies of ecology, evolution, and conservation.

Oxygen supply did not affect how lizards responded to thermal stress.

Zoologists rely on mechanistic niche models of behavioral thermoregulation to understand how animals respond to climate change. These models predict that species will need to disperse to higher altitudes to persist in a warmer world. However, thermal stress and, thus, thermoregulatory behavior may depend on atmospheric oxygen as well as environmental temperatures. Severe hypoxia causes animals to prefer lower body temperatures, which could be interpreted as evidence that oxygen supply limits heat tolerance. Such a constraint could prevent animals from successfully dispersing to high elevations during climate change. Still, an effect of oxygen supply on preferred body temperature has only been observed when oxygen concentrations fall far below levels experienced in nature. To see whether animals perceive greater thermal stress at an ecologically relevant level of hypoxia, we studied the thermoregulatory behavior of lizards (Sceloporus tristichus) exposed to oxygen concentrations of 13% and 21% (equivalent to PO2 at 4000 m and 0 m, respectively). In addition, we exposed lizards to 29% oxygen to see whether they would accept a higher body temperature at hyperoxia than at normoxia. At each oxygen level, we measured a behavioral response to heat stress known as the voluntary thermal maximum: the temperature at which a warming animal sought a cool refuge. Oxygen concentration had no discernable effect on the voluntary thermal maximum, suggesting that lizards experience thermal stress similarly at all 3 levels of oxygen (13%, 12% and 29%). Future research should focus on thermoregulatory behaviors under ecologically relevant levels of hypoxia.

Reptile embryos are not capable of behavioral thermoregulation in the egg.

Reptile embryos have recently been observed moving within the egg in response to temperature, raising the exciting possibility that embryos might behaviorally thermoregulate analogous to adults. However, the conjecture that reptile embryos have ample opportunity and capacity to adaptively control their body temperature warrants further discussion. Using turtles as a model, we discuss the spatiotemporal constraints to movement in reptile embryos. We demonstrate that, as embryos grow, the internal egg space rapidly diminishes such that the temporal window for appreciable displacement is confined to stages that feature incomplete neuromuscular differentiation. During this time, muscles are insufficiently developed to actively and consistently control movement. These constraints are well illustrated by the Chinese softshelled turtle (Pelodiscus sinensis), the first reptile reported to behaviorally thermoregulate. Furthermore, sporadic embryo activity peaks after the temperature-sensitive period in species with temperature-dependent sex determination, thus nullifying the opportunity for embryos to exhibit control over this important phenotype. These embryonic constraints add to previously-identified environmental constraints on behavioral thermoregulation by reptile embryos. We discuss alternative hypotheses to explain previously reported patterns of behavioral thermoregulation. Based on a holistic consideration of embryonic limitations, we conclude that reptile embryos are generally unable to adaptively behaviorally thermoregulate within the egg.

Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment.

The mechanisms that mediate the interaction between the thermal environment and species ranges are generally uncertain. Thermal environments may directly restrict species when environments exceed tolerance limits (i.e. the fundamental niche). However, thermal environments might also differentially affect relative performance among species prior to fundamental tolerances being met (i.e. the realized niche). We examined stress physiology (plasma glucose and corticosterone), mitochondrial performance and the muscle metabolome of congeneric lizards that naturally partition the thermal niche, Elgaria multicarinata (southern alligator lizards; SALs) and Elgaria coerulea (northern alligator lizards; NALs), in response to a thermal challenge to quantify variation in physiological performance and tolerance. Both NAL and SAL displayed physiological stress in response to high temperature, but neither showed signs of irreversible damage. NAL displayed a higher baseline mitochondrial respiration rate than SAL. Moreover, NAL substantially adjusted their physiology in response to thermal challenge, whereas SAL did not. For example, the metabolite profile of NAL shifted with changes in key energetic molecules, whereas these were unaffected in SAL. Our results indicate that near-critical high temperatures should incur greater energetic cost in NAL than SAL via an elevated metabolic rate and changes to the metabolome. Thus, SAL displace NAL in warm environments that are within NAL's fundamental thermal niche, but relatively costly. Our results suggest that subcritical thermal events can contribute to biogeographic patterns via physiological differences that alter the relative costs of living in warm or cool environments.

Lizards fail to plastically adjust nesting behavior or thermal tolerance as needed to buffer populations from climate warming.

Although observations suggest the potential for phenotypic plasticity to allow adaptive responses to climate change, few experiments have assessed that potential. Modeling suggests that Sceloporus tristichus lizards will need increased nest depth, shade cover, or embryonic thermal tolerance to avoid reproductive failure resulting from climate change. To test for such plasticity, we experimentally examined how maternal temperatures affect nesting behavior and embryonic thermal sensitivity. The temperature regime that females experienced while gravid did not affect nesting behavior, but warmer temperatures at the time of nesting reduced nest depth. Additionally, embryos from heat-stressed mothers displayed increased sensitivity to high-temperature exposure. Simulations suggest that critically low temperatures, rather than high temperatures, historically limit development of our study population. Thus, the plasticity needed to buffer this population has not been under selection. Plasticity will likely fail to compensate for ongoing climate change when such change results in novel stressors.

Hormonal and metabolic responses to upper temperature extremes in divergent life-history ecotypes of a garter snake.

Extreme temperatures constrain organismal physiology and impose both acute and chronic effects. Additionally, temperature-induced hormone-mediated stress response pathways and energetic trade-offs are important drivers of life-history variation. This study employs an integrative approach to quantify acute physiological responses to high temperatures in divergent life-history ecotypes of the western terrestrial garter snake (Thamnophis elegans). Using wild-caught animals, we measured oxygen consumption rate and physiological markers of hormonal stress response, energy availability and anaerobic respiration in blood plasma across five ecologically relevant temperatures (24, 28, 32, 35 and 38°C; 3 h exposure). Corticosterone, insulin and glucose concentrations all increased with temperature, but with different thermal response curves, suggesting that high temperatures differently affect energy-regulation pathways. Additionally, oxygen consumption rate increased without plateau and lactate concentration did not increase with temperature, challenging the recent hypothesis that oxygen limitation sets upper thermal tolerance limits. Finally, animals had similar physiological thermal responses to high-temperature exposure regardless of genetic background, suggesting that local adaptation has not resulted in fixed differences between ecotypes. Together, these results identify some of the mechanisms by which higher temperatures alter hormonal-mediated energy balance in reptiles and potential limits to the flexibility of this response.

Life in the Frequency Domain: the Biological Impacts of Changes in Climate Variability at Multiple Time Scales.

Over the last few decades, biologists have made substantial progress in understanding relationships between changing climates and organism performance. Much of this work has focused on temperature because it is the best kept of climatic records, in many locations it is predicted to keep rising into the future, and it has profound effects on the physiology, performance, and ecology of organisms, especially ectothermic organisms which make up the vast majority of life on Earth. Nevertheless, much of the existing literature on temperature-organism interactions relies on mean temperatures. In reality, most organisms do not directly experience mean temperatures; rather, they experience variation in temperature over many time scales, from seconds to years. We propose to shift the focus more directly on patterns of temperature variation, rather than on means per se, and present a framework both for analyzing temporal patterns of temperature variation and for incorporating those patterns into predictions about organismal biology. In particular, we advocate using the Fourier transform to decompose temperature time series into their component sinusoids, thus allowing transformations between the time and frequency domains. This approach provides (1) standardized ways of visualizing the contributions that different frequencies make to total temporal variation; (2) the ability to assess how patterns of temperature variation have changed over the past half century and may change into the future; and (3) clear approaches to manipulating temporal time series to ask "what if" questions about the potential effects of future climates. We first summarize global patterns of change in temperature variation over the past 40 years; we find meaningful changes in variation at the half day to yearly times scales. We then demonstrate the utility of the Fourier framework by exploring how power added to different frequencies alters the overall incidence of long-term waves of high and low temperatures, and find that power added to the lowest frequencies greatly increases the probability of long-term heat and cold waves. Finally, we review what is known about the time scales over which organismal thermal performance curves change in response to variation in the thermal environment. We conclude that integrating information characterizing both the frequency spectra of temperature time series and the time scales of resulting physiological change offers a powerful new avenue for relating climate, and climate change, to the future performance of ectothermic organisms.

Reptile Embryos Lack the Opportunity to Thermoregulate by Moving within the Egg.

Historically, egg-bound reptile embryos were thought to passively thermoconform to the nest environment. However, recent observations of thermal taxis by embryos of multiple reptile species have led to the widely discussed hypothesis that embryos behaviorally thermoregulate. Because temperature affects development, such thermoregulation could allow embryos to control their fate far more than historically assumed. We assessed the opportunity for embryos to behaviorally thermoregulate in nature by examining thermal gradients within natural nests and eggs of the common snapping turtle (Chelydra serpentina; which displays embryonic thermal taxis) and by simulating thermal gradients within nests across a range of nest depths, egg sizes, and soil types. We observed little spatial thermal variation within nests, and thermal gradients were poorly transferred to eggs. Furthermore, thermal gradients sufficiently large and constant for behavioral thermoregulation were not predicted to occur in our simulations. Gradients of biologically relevant magnitude have limited global occurrence and reverse direction twice daily when they do exist, which is substantially faster than embryos can shift position within the egg. Our results imply that reptile embryos will rarely, if ever, have the opportunity to behaviorally thermoregulate by moving within the egg. We suggest that embryonic thermal taxis instead represents a play behavior, which may be adaptive or selectively neutral, and results from the mechanisms for behavioral thermoregulation in free-living stages coming online prior to hatching.

Oxygen supply limits the heat tolerance of lizard embryos.

The mechanisms that set the thermal limits to life remain uncertain. Classically, researchers thought that heating kills by disrupting the structures of proteins or membranes, but an alternative hypothesis focuses on the demand for oxygen relative to its supply. We evaluated this alternative hypothesis by comparing the lethal temperature for lizard embryos developing at oxygen concentrations of 10-30%. Embryos exposed to normoxia and hyperoxia survived to higher temperatures than those exposed to hypoxia, suggesting that oxygen limitation sets the thermal maximum. As all animals pass through an embryonic stage where respiratory and cardiovascular systems must develop, oxygen limitation may limit the thermal niches of terrestrial animals as well as aquatic ones.