Synopsis 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 nonavian 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.

Introduction

For most animals, the proximate mechanisms that underlie reduced performance and eventual death at high temperatures, and how such mechanisms might change with ontogeny and context, are uncertain (reviewed in [START_REF] Angilletta | Thermal Adaptation: A Theoretical and Empirical Synthesis[END_REF][START_REF] Clark | Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations[END_REF][START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]. Such a mechanistic understanding would greatly enhance our ability to predict the effects of realistic high-temperature exposures on individuals or populations, which are difficult to forecast with traditional methods because responses can be highly variable (e.g. [START_REF] Gunderson | Plasticity in thermal tolerance has limited potential to buffer ectotherms from global warming[END_REF][START_REF] Seebacher | Physiological plasticity increases resilience of ectothermic animals to climate change[END_REF][START_REF] Kingsolver | Beyond thermal performance curves: Modeling timedependent effects of thermal stress on ectotherm growth rates[END_REF][START_REF] Sheldon | Beyond the mean: Biological impacts of cryptic temperature change[END_REF][START_REF] Williams | Biological impacts of thermal extremes: Mechanisms and costs of functional responses matter[END_REF]. Even so, much progress has been made toward developing mechanistic models that use knowledge of individual physiology to predict the effects of thermal environments on populations, largely inspired by growing concern over the impacts of global climate change [START_REF] Buckley | Linking traits to energetics and population dynamics to predict lizard ranges in changing environments[END_REF][START_REF] Kearney | Mechanistic niche modelling: Combining physiological and spatial data to predict species' ranges[END_REF][START_REF] Huey | Predicting organismal vulnerability to climate warming: Roles of behavior, physiology and adaptation[END_REF][START_REF] Kearney | Metabolic theory, life history and the distribution of a terrestrial ectotherm[END_REF][START_REF] Levy | Resolving the life cycle alters expected impacts of climate change[END_REF][START_REF] Malishev | An individual-based model of ectotherm movement integrating metabolic and microclimatic constraints[END_REF]. Commonly, such models employ empirically-derived thermal performance curves (TPC, [START_REF] Huey | Integrating thermal physiology and ecology of ectotherms: Discussion of approaches[END_REF] to predict the effects of thermal environments on organisms and populations (e.g. [START_REF] Colwell | Global warming, elevational range shifts, and lowland biotic attrition in the wet tropics[END_REF][START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF][START_REF] Vasseur | Increased temperature variation poses a greater risk to species than climate warming[END_REF][START_REF] Buckley | How extreme temperatures impact organisms and the evolution of their thermal tolerance[END_REF][START_REF] Dillon | Life in the frequency domain: The biological impacts of changes in climate variability at multiple time scales[END_REF]. TPCs describe how performance varies with temperature-typically performance increases with temperature above a critical minimum (CT MIN ) until an optimum is reached (T OPT ), then rapidly drops to zero at the critical maximum (CT MAX , see Fig. 1 for examples). Pejus (getting worse) temperatures above and below T OPT are described by breakpoints in physiological function (e.g. ventilation rate, heart rate, P O2 ) indicative of rapid declines in whole-organism performance (T PEJUS , [START_REF] Frederich | Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado[END_REF][START_REF] Pörtner | Climate variations and the physiological basis of temperature dependent biogeography: Systemic to molecular hierarchy of thermal tolerance in animals[END_REF][START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]. Typically TPCs are estimated under controlled laboratory conditions for a single trait and time, but TPC shape can vary with season, ontogeny, trait, and http://mc.manuscriptcentral.com/icbiol prior experience in ways that are difficult to predict [START_REF] Rezende | Tolerance landscapes in thermal ecology[END_REF][START_REF] Telemeco | Immobile and mobile life-history stages have different thermal physiologies in a lizard[END_REF][START_REF] Kingsolver | Beyond thermal performance curves: Modeling timedependent effects of thermal stress on ectotherm growth rates[END_REF][START_REF] Williams | Biological impacts of thermal extremes: Mechanisms and costs of functional responses matter[END_REF]). Thus, using empirically-derived TPCs to predict the effects of natural environments on performance is problematic because it requires extrapolating from the traits or environments originally used to estimate TPCs, thereby ignoring probable context-dependency. A mechanistic understanding of the processes that underlie thermal performance is needed to predict the effects of variable or novel environments on TPC shape, which will greatly improve models relying on TPCs to predict population responses. In particular, knowledge of the mechanisms that result in loss of function at high temperatures is needed to predict the rate at which performance will drop in response to thermal challenge, the capacity for animals to recover from sub-lethal thermal exposure, and the capacity for thermal tolerance to change via plasticity or evolution [START_REF] Helmuth | Biophysics, physiological ecology, and climate change: Does mechanism matter?[END_REF][START_REF] Buckley | How extreme temperatures impact organisms and the evolution of their thermal tolerance[END_REF][START_REF] Williams | Biological impacts of thermal extremes: Mechanisms and costs of functional responses matter[END_REF].

Potential mechanisms explaining why animals lose function at high temperatures can be divided into two major categories differing in the level of organization first affected. At lower levels of organization, subcellular components could be critically impaired when animals are exposed to temperatures above the optimum. Subcellular impairment results from either denaturation of key molecules, such as proteins and cell membranes, or reduced efficiency of these molecules to perform their biological functions (reviewed in [START_REF] Hochachka | Biochemical adaptation: Mechanism and process in physiological evolution[END_REF][START_REF] Angilletta | Thermal Adaptation: A Theoretical and Empirical Synthesis[END_REF][START_REF] Schulte | The effects of temperature on aerobic metabolism: Towards a mechanistic understanding of the responses of ectotherms to a changing environment[END_REF]. Impairment of subcellular components would result in the breakdown of higher levels of organization and lead to rapid performance loss. Alternatively, higher-levels of organization, such as organ systems, could be impaired at temperatures below those that directly affect the performance of their subcellular components if high temperatures disrupt subcellular interactions or pathways necessary for organ system function. A recent http://mc.manuscriptcentral.com/icbiol mechanistic model explaining organ-system impairment at high temperatures is the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, which proposes that oxygen demand for aerobic metabolism at high temperatures outpaces the ability of the cardiovascular and respiratory systems to provide sufficient amounts of oxygen to tissues [START_REF] Pörtner | Climate variations and the physiological basis of temperature dependent biogeography: Systemic to molecular hierarchy of thermal tolerance in animals[END_REF][START_REF] Pörtner | Climate change affects marine fishes through the oxygen limitation of thermal tolerance[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF][START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]. Under this hypothesis, oxygen diffusion and transport capacity limit metabolic rates such that resting rates converge with potential maximum rates at high temperatures thereby reducing aerobic scope to zero (i.e. zero aerobic power budget sensu [START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]. This mismatch in supply and demand will first reduce aerobic performance at T PEJUS but eventually result in basal oxygen demands not being met and thus collapse of organismal systems at CT MAX. Proponents of OCLTT argue that this mechanism integrates across levels of organization from systemic to molecular mechanisms and can explain diverse evolutionary and ecological phenomena (Portner 2002, Portner et al 2017). However, the temporal window, activity range, magnitude, and biological relevancy of reduced aerobic scope at high temperature are debated [START_REF] Clark | Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations[END_REF][START_REF] Gräns | Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO 2 in Atlantic halibut[END_REF][START_REF] Jutfelt | Response to 'How and how not to investigate the oxygen and capacity limitation of thermal tolerance (OCLTT) and aerobic scoperemarks on the article by Gräns et al[END_REF][START_REF] Pörtner | How and how not to investigate the oxygen and capacity limitation of thermal tolerance (OCLTT) and aerobic scope -remarks on the article by Gräns et al[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF][START_REF] Jutfelt | Oxygen-and capacity-limited thermal tolerance: Blurring ecology and physiology[END_REF]). Identifying the level of organization first impaired by high temperature is necessary to understand how temperature affects whole-organism function.

Recent work exploring the mechanisms underlying thermal tolerance in ectotherms focuses on aquatic taxa (e.g. [START_REF] Frederich | Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado[END_REF][START_REF] Lucassen | Mitochondrial mechanisms of cold adaptation in cod (Gadus morhua L.) populations from different climatic zones[END_REF][START_REF] Verberk | Anaerobic metabolism at thermal extremes: A metabolomic test of the oxygen limitation hypothesis in an aquatic insect[END_REF][START_REF] Gräns | Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO 2 in Atlantic halibut[END_REF][START_REF] Ern | Oxygen dependence of upper thermal limits in fishes[END_REF][START_REF] Pörtner | Impacts of climate variability and change on (marine) animals: Physiological underpinnings and evolutionary consequences[END_REF][START_REF] Verberk | Thermal limits in native and alien freshwater peracarid Crustacea: The role of habitat use and oxygen limitation[END_REF]) and terrestrial arthropods, particularly insects (reviewed in [START_REF] Mccue | Upper thermal limits of insects are not the result of insufficient oxygen delivery[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF]. These observations suggest that subcellular-level mechanisms are more important determinants of thermal tolerance than oxygen capacity limitations in terrestrial taxa, perhaps http://mc.manuscriptcentral.com/icbiol because oxygen is more abundant in air than water. However, recent work includes few studies of terrestrial vertebrate ectotherms: non-avian reptiles (hereafter, reptiles) and amphibians.

Reptiles and amphibians display varied ecology, life-history, and thermal tolerance [START_REF] Vitt | Herpetology: An introductory biology of amphibians and reptiles[END_REF][START_REF] Sunday | Global analysis of thermal tolerance and latitude in ectotherms[END_REF] and are important to a broad understanding of thermal tolerance. Additionally, these animals frequently contend with challenging temperatures, with some species inhabiting the hottest terrestrial environments [START_REF] Cowles | A preliminary study of the thermal requirements of desert reptiles[END_REF][START_REF] Vitt | Herpetology: An introductory biology of amphibians and reptiles[END_REF][START_REF] Sunday | Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation[END_REF]. Moreover, both the thermal and oxygen environment can vary throughout ontogeny (e.g. aquatic to terrestrial transition in amphibians), and modes of respiration can vary (e.g. cutaneous respiration in many amphibians and some reptiles, such as turtles; [START_REF] Hutchison | Relation of body size and surface area to gas exchange in anurans[END_REF][START_REF] Glass | Pulmonary diffusing capacity in reptiles (relations to temperature and O 2 -uptake)[END_REF][START_REF] Wang | Gas exchange in frogs and turtles: How ectothermic vertebrates contributed to solving the controversy of pulmonary oxygen secretion[END_REF]. Because of these traits, reptiles and amphibians have been important models for thermal physiology and ecology for the last century (e.g. [START_REF] Cowles | A preliminary study of the thermal requirements of desert reptiles[END_REF][START_REF] Snyder | Temperature adaptations in amphibians[END_REF][START_REF] Huey | Integrating thermal physiology and ecology of ectotherms: Discussion of approaches[END_REF][START_REF] Huey | Temperature, physiology, and the ecology of reptiles[END_REF][START_REF] Huey | Temperature, demography, and ectotherm fitness[END_REF][START_REF] Vitt | Herpetology: An introductory biology of amphibians and reptiles[END_REF][START_REF] Kearney | Metabolic theory, life history and the distribution of a terrestrial ectotherm[END_REF]. This historic work can be leveraged to address modern ideas, such as the OCLTT hypothesis. Furthermore, these taxa are at risk of high-temperature induced extinction resulting from global climate change [START_REF] Thomas | Extinction risk from climate change[END_REF][START_REF] Huey | Are lizards toast?[END_REF][START_REF] Rohr | Linking global climate and temperature variability to widespread amphibian declines putatively caused by disease[END_REF][START_REF] Sinervo | Erosion of lizard diversity by climate change and altered thermal niches[END_REF][START_REF] Kearney | Activity restriction and the mechanistic basis for extinctions under climate warming[END_REF]. Here, we apply the rich history of physiological studies in reptiles and amphibians to modern hypotheses for mechanisms underlying thermal performance and tolerance. This will complete taxonomic coverage of recent reviews, and, more importantly, further understanding of how organismal performance and survival are restricted by high temperatures.

In this commentary, we begin by synthesizing evidence for subcellular-and organsystem-level failures underlying high temperature tolerance and performance in reptiles and amphibians. Because neither framework satisfactorily explains observed patterns individually, http://mc.manuscriptcentral.com/icbiol we next build upon work in other taxa to present an integrative framework combining both subcellular and organ-system sensitivity to high temperatures and the thermal performance curve paradigm. Using data for reptiles and amphibians, we illustrate how this combined framework appears to predict a broad array of physiological and behavioral observations. We conclude by identifying future research directions to test this framework and discuss how it might be applied to other systems to predict consequences of future high-temperature exposure for organisms and populations.

Evidence for mechanisms underlying thermal tolerance and performance in reptiles and amphibians

Subcellular mechanisms

High temperatures compromise the structure and function of subcellular components, such as enzymes and cell membranes [START_REF] Fields | Review: Protein function at thermal extremes: Balancing stability and flexibility[END_REF][START_REF] Hochachka | Biochemical adaptation: Mechanism and process in physiological evolution[END_REF] but such damage will only underlie organismal thermal tolerance if it occurs at temperatures below those affecting function at higher levels of organization, such as organ systems [START_REF] Pörtner | Climate variations and the physiological basis of temperature dependent biogeography: Systemic to molecular hierarchy of thermal tolerance in animals[END_REF]. The complexities of enzymatic interactions make extending observations for individual reactions to the whole cell or organism potentially problematic [START_REF] Schulte | The effects of temperature on aerobic metabolism: Towards a mechanistic understanding of the responses of ectotherms to a changing environment[END_REF]. Nonetheless, the observation that subcellular components are frequently stable to temperatures above the critical and lethal limits of animals (CT MAX and T LETHAL hereafter), commonly maintaining function to temperatures > 50˚C and capable of evolving stability to 120˚C [START_REF] Fields | Review: Protein function at thermal extremes: Balancing stability and flexibility[END_REF], was an important motivation for the development of hypotheses such as OCLTT [START_REF] Pörtner | Climate change and temperature-dependent biogeography: Oxygen limitation of thermal tolerance in animals[END_REF]2002;[START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]. For example, CT MAX in reptiles and amphibians generally ranges from the mid 30s to low 40s ˚C http://mc.manuscriptcentral.com/icbiol (Brattstrom 1965;[START_REF] Sunday | Global analysis of thermal tolerance and latitude in ectotherms[END_REF][START_REF] Sunday | Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation[END_REF], although a few warm-adapted reptiles such as the desert iguana (Dipsosaurus dorsalis) tolerate acute exposure to 47.5 ˚C [START_REF] Cowles | A preliminary study of the thermal requirements of desert reptiles[END_REF]Brattstrom 1965). Even so, most reptile and amphibian proteins do not lose function until they experience temperatures well above CT MAX . For example, ribonucleases from three frog species were stable to 85˚C and maintained high activity at temperatures > 50˚C [START_REF] Irie | Biochemistry of frog ribonucleases[END_REF], lactate dehydrogenase in Agama stellio lizards maintained both stability and function to 70˚C (Al-Jassabi 2002), and alkaline phosphatase maintained high function up to 50˚C in four lizard species [START_REF] Licht | The temperature dependence of myosin-adenosinetriphosphatase and alkaline phosphatase in lizards[END_REF]. Similarly, acute exposure to CT MAX did not cause tissue damage or reduce function of serum glutamic-oxaloacetic or glutamic-pyruvic transaminases in great plains toads (Anaxyrus cognatus, [START_REF] Paulson | Blood changes in Bufo cognatus following acute heat stress[END_REF] and 2.5-h exposure to temperatures just below CT MAX had no effect on mitochondrial respiration or free-radical production in alligator lizards (Elgaria coerulea and E. multicarinata; [START_REF] Telemeco | Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment[END_REF], implying no subcellular damage. That said, only one key component needs to lose function for the entire organism to become compromised. For example, the activity of myosin ATPase, a key enzyme for organismal muscle function, closely resembled whole-organism thermal performance curves and denatured at relatively low temperatures in eight lizard species corresponding closely to their respective CT MAX (20% denatured between 37 and 45.2 ˚C; [START_REF] Licht | The temperature dependence of myosin-adenosinetriphosphatase and alkaline phosphatase in lizards[END_REF]. Subcellular components such as ATPase could underlie thermal tolerance even though most components maintain function to higher temperature.

Given the complexities of subcellular interactions and the paucity of data for the thermal performance of subcellular components in reptiles and amphibians, heat shock protein (HSP) production might better indicate whether subcellular-level components are challenged at subcritical high temperatures. HSPs commonly act as molecular chaperones, maintaining protein http://mc.manuscriptcentral.com/icbiol structure and preventing aggregations of denatured proteins, and inducible variants are produced in response to cellular stress or damage [START_REF] Fernando | Functional characterization of Xenopus small heat shock protein, Hsp30C: The carboxyl end is required for stability and chaperone activity[END_REF][START_REF] Kregel | Heat shock proteins: Modifying factors in physiological stress response and acquired thermotolerance[END_REF][START_REF] Daugaard | The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions[END_REF]. In diverse reptiles and amphibians, HSPs (particularly HSPA family members) are produced in response to sub-critical high temperatures, and production of HSPs can allow acclimation with increased thermal tolerance [START_REF] Ulmasov | Heat shock proteins and thermoresistance in lizards[END_REF][START_REF] Fernando | Functional characterization of Xenopus small heat shock protein, Hsp30C: The carboxyl end is required for stability and chaperone activity[END_REF][START_REF] Zatespina | m. are frogs, D.d. is a lizard, P.m. is a snake, C.s. is a turtle, and A.m. is an alligator. Experimental temperature is given to the right of the species' initials. Data are means ± s.e.m. derived from the literature. See Table S1 for data and citations[END_REF][START_REF] Mcmillan | Geographic variation in the effects of heat exposure on maximum sprint speed and Hsp70 expression in the western fence lizard Sceloporus occidentalis[END_REF][START_REF] Gao | Heat shock protein expression enhances heat tolerance of reptile embryos[END_REF][START_REF] Simoniello | Alterations in brain morphology and HSP70 expression in lizard embryos exposed to thermal stress[END_REF][START_REF] Tedeschi | Heritable variation in heat shock gene expression: A potential mechanism for adaptation to thermal stress in embryos of sea turtles[END_REF]. Moreover, patterns of HSP expression are correlated with thermal tolerance in lizards: warm-adapted species display higher constitutive HSP concentrations, and both initiate and maintain synthesis of HSPs to higher temperatures than more cold-adapted species [START_REF] Ulmasov | Heat shock proteins and thermoresistance in lizards[END_REF][START_REF] Zatespina | m. are frogs, D.d. is a lizard, P.m. is a snake, C.s. is a turtle, and A.m. is an alligator. Experimental temperature is given to the right of the species' initials. Data are means ± s.e.m. derived from the literature. See Table S1 for data and citations[END_REF]. This pattern of increased HSP production correlating with increases in thermal tolerance in reptiles and amphibians, along with observations for myosin ATPase in lizards, provides compelling evidence for subcellular-function loss playing a role in setting thermal limits, despite other subcellular components displaying little loss of function at relevant temperatures.

Organ-system mechanisms

Even though subcellular-level traits will be compromised at sufficiently high temperatures, higher-order systems might break down at lower temperatures and thus be proximally responsible for setting thermal tolerances [START_REF] Pörtner | Climate change and temperature-dependent biogeography: Oxygen limitation of thermal tolerance in animals[END_REF]2002;[START_REF] Storch | Climate sensitivity across marine domains of life: Limits to evolutionary adaptation shape species interactions[END_REF], but see Clark et al. 2013). The OCLTT hypothesis proposes that the highest organizational level in animals is the integrated cardiovascular and respiratory system because all tissues will be limited by their ability to acquire oxygen for respiration, and that this system is compromised by high temperatures prior to other systems [START_REF] Pörtner | Climate change and temperature-dependent biogeography: Oxygen limitation of thermal tolerance in animals[END_REF]2002;[START_REF] Storch | Climate sensitivity across marine domains of life: Limits to evolutionary adaptation shape species interactions[END_REF]. However, similar to http://mc.manuscriptcentral.com/icbiol other terrestrial species [START_REF] Klok | Upper thermal tolerance and oxygen limitation in terrestrial arthropods[END_REF][START_REF] Mccue | Upper thermal limits of insects are not the result of insufficient oxygen delivery[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF] and many fish [START_REF] Clark | Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations[END_REF][START_REF] Gräns | Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO 2 in Atlantic halibut[END_REF][START_REF] Norin | Aerobic scope does not predict the performance of a tropical eurythermal fish at elevated temperatures[END_REF][START_REF] Wang | Anaemia only causes a small reduction in the upper critical temperature of sea bass: Is oxygen delivery the limiting factor for tolerance of acute warming in fishes?[END_REF]Ern et al. 2016), evidence for the OCLTT mechanism underlying thermal tolerance in reptiles and amphibians is limited. Under the OCLTT hypothesis, maximal and resting rates of oxygen consumption are expected to converge as animals reach their physiological limits at high temperatures, thereby reducing aerobic scope and potentially inducing a short-term reliance on anaerobic respiration [START_REF] Frederich | Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado[END_REF][START_REF] Pörtner | Climate change affects marine fishes through the oxygen limitation of thermal tolerance[END_REF][START_REF] Elaison | Differences in thermal tolerance among sockeye salmon populations[END_REF][START_REF] Verberk | Anaerobic metabolism at thermal extremes: A metabolomic test of the oxygen limitation hypothesis in an aquatic insect[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF]; Table 1). Some evidence points to such a mechanism playing an important role in early animal evolution, notably in the transition to air breathing [START_REF] Berner | Oxygen and Evolution[END_REF][START_REF] Giomi | Improved heat tolerance in air drives the recurrent evolution of air-breathing[END_REF][START_REF] Teague | A positive genetic correlation between hypoxia tolerance and heat tolerance supports a controversial theory of heat stress[END_REF]). However, the few studies exposing animals to high temperatures and measuring indicators of aerobic and anaerobic respiration fail to find evidence for oxygen limitation in adult reptiles and amphibians [START_REF] Carey | Aerobic and anaerobic energy expenditure during rest and activity in montane Bufo b. boreas and Rana pipiens[END_REF][START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF][START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF][START_REF] Gangloff | Hormonal and metabolic responses to upper temperature extremes in divergent life-history ecotypes of a garter snake[END_REF][START_REF] Telemeco | Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment[END_REF]. For example, oxygen consumption (ܸ ሶ O 2 ) by pythons (Python regius) did not plateau at temperatures approaching CT MAX either when at rest or during periods of high metabolic demand (Fobian et al. 2014, Fig. 1), and resting oxygen consumption in garter snakes (Thamnophis elegans) increased with temperature with no apparent limit when animals experienced near-lethal temperatures [START_REF] Gangloff | Hormonal and metabolic responses to upper temperature extremes in divergent life-history ecotypes of a garter snake[END_REF]. Moreover, neither garter snakes (T. elegans) nor alligator lizards (E. coerulea and E. multicarinata) transitioned to anaerobic respiration when exposed to near-critical temperatures [START_REF] Gangloff | Hormonal and metabolic responses to upper temperature extremes in divergent life-history ecotypes of a garter snake[END_REF][START_REF] Telemeco | Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment[END_REF], despite snakes and lizards rapidly transitioning when oxygen availability is limited during exercise (reviewed in [START_REF] Gleeson | Patterns of metabolic recovery from exercise in amphibians and reptiles[END_REF]. Observations in amphibians are similar to those for reptiles. For example, oxygen consumption, arterial oxygen saturation, and the proportion of saturated hemoglobin did not plateau at high temperatures in http://mc.manuscriptcentral.com/icbiol active or resting cane toads (Rhinella marina), thus providing evidence for these toads' ability to maintain a positive aerobic power budget at near-critical temperatures [START_REF] Seebacher | Physiology of invasion: Cane toads are constrained by thermal effects on physiological mechanisms that support locomotor performance[END_REF][START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF][START_REF] Hs | Does greater thermal plasticity facilitate range expansion of an invasive terrestrial anuran into higher latitudes?[END_REF]. In both the boreal toad (Anaxyrus boreas) and leopard frog (Lithobates pipiens), aerobic scope increased with temperature to 30ºC [START_REF] Carey | Aerobic and anaerobic energy expenditure during rest and activity in montane Bufo b. boreas and Rana pipiens[END_REF]. Whole-organism lactate concentration also increases with temperature, but there is no indication that either species becomes oxygen limited up to at least 30ºC [START_REF] Carey | Aerobic and anaerobic energy expenditure during rest and activity in montane Bufo b. boreas and Rana pipiens[END_REF]). These results are in line with previous work showing that amphibians can maintain substantial aerobic scope at temperatures above active and preferred temperatures, although not necessarily at temperatures approaching T LETHAL [START_REF] Whitford | The effects of temperature on respiration in the amphibia[END_REF]. While such studies provide strong evidence for adult reptiles and amphibians maintaining aerobic scope at high temperatures, we currently lack data on tissue and cellular oxygen supply, such as venous P O 2 , which would more directly test the mechanisms described by the OCLTT hypothesis [START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]2018).

A second approach for testing the influence of oxygen capacity on thermal constraints is to manipulate oxygen availability and examine changes in thermal behavior or tolerance (Table 1). Under the OCLTT hypothesis, hypoxia is predicted to reduce thermal optima and tolerance limits [START_REF] Smith | Oxygen supply limits the heat tolerance of lizard embryos[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF], and this prediction is somewhat supported in reptiles and amphibians. For example, CT MAX , preferred body temperature (PBT), panting temperature (T PANT ), and gaping temperature (T GAPE ) are reduced when diverse species are exposed to very-low oxygen environments (< 10 kPa; mostly lizards examined; [START_REF] Hicks | Temperature regulation in lizards: Effects of hypoxia[END_REF][START_REF] Dupre | The effect of hypoxia on evaporative cooling thresholds of lizards[END_REF][START_REF] Branco | Interaction between temperature and hypoxia in the alligator[END_REF][START_REF] Cadena | Decreased precision contributes to the hypoxic thermoregulatory response in lizards[END_REF][START_REF] Shea | Oxygen concentration affects upper thermal tolerance in a terrestrial vertebrate[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF]; Fig. 2) and when hematocrit is experimentally reduced (only PBT examined; [START_REF] Hicks | Temperature regulation in lizards: Effects of hypoxia[END_REF][START_REF] Wood | Effect of hematocrit on behavioral thermoregulation of the toad Bufo marinus[END_REF]). Hypoxia-induced PBT reduction is a well-described phenomenon in ectotherms, termed "behavioral anapyrexia," that http://mc.manuscriptcentral.com/icbiol allows adaptive reduction of metabolic demand when oxygen is limited [START_REF] Hicks | Temperature regulation in lizards: Effects of hypoxia[END_REF][START_REF] Wood | Hypothermia in hypoxic animals: Mechanisms, mediators, and functional significance[END_REF][START_REF] Steiner | Hypoxia-induced anapyrexia: Implications and putative mediators[END_REF][START_REF] Hicks | Hypometabolism in reptiles: Behavioural and physiological mechanisms that reduce aerobic demands[END_REF], supporting the hypothesis that oxygen limitation influences thermal preference and possibly tolerance. Along with CT MAX , reductions in T GAPE and T PANT , which provide an indication of perceived heat stress [START_REF] Heatwole | Panting thresholds of lizards-I. Some methodological and internal influences on the panting threshold of an agamid, Amphibolurus muricatus[END_REF][START_REF] Tattersall | Respiratory cooling and thermoregulatory coupling in reptiles[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF], imply reduced thermal tolerance under hypoxia. Hypoxia also induces elevated heart rates and reduces both resting and active oxygen consumption (ܸ ሶ O 2 ) in diverse species (Fig. 3), suggesting observed shifts in thermal tolerance and behavior are related to physiological limits of oxygen capacity (including diffusion and transport). Interestingly, only extreme hypoxia had the predicted effects on thermal tolerance and behavior, with levels of hypoxia within the range generally found in terrestrial environments having no effect (Figs. 2 and3). Thus, it is not clear that the OCLTT mechanism will be generally relevant in nature.

In contrast to adult stages, naturalistic hypoxia reduces thermal performance and tolerance in larvae and eggs of reptiles and amphibians. Because amphibian eggs and larvae inhabit aqueous environments, environmental oxygen availability is reduced relative to terrestrial stages, thereby increasing the potential importance of the OCLTT mechanism similar to some fully aquatic ectotherms (e.g. [START_REF] Pörtner | Climate change affects marine fishes through the oxygen limitation of thermal tolerance[END_REF][START_REF] Verberk | Anaerobic metabolism at thermal extremes: A metabolomic test of the oxygen limitation hypothesis in an aquatic insect[END_REF][START_REF] Pörtner | Impacts of climate variability and change on (marine) animals: Physiological underpinnings and evolutionary consequences[END_REF]. Oxygen limitation is further exacerbated when species develop in small water bodies that can rapidly lose dissolved oxygen, such as ephemeral pools [START_REF] Seymour | Respiration of amphibian eggs[END_REF][START_REF] Sacerdote | Dissolved oxygen requirements for hatching success of two ambystomatid salamanders in restored ephemeral ponds[END_REF], and because amphibian eggs are commonly produced within large gelatinous masses with low oxygen diffusion potential [START_REF] Pinder | Oxygen transport in egg masses of the amphibians Rana sylvatica and Ambystoma maculatum: Convection, diffusion, and oxygen production by algae[END_REF][START_REF] Woods | Egg-mass size and cell size: Effects of temperature on oxygen distribution[END_REF][START_REF] Sacerdote | Dissolved oxygen requirements for hatching success of two ambystomatid salamanders in restored ephemeral ponds[END_REF]. Although data are limited, these constraints appear to affect early stage amphibians congruent with oxygen limiting thermal tolerances. For example, amphibians http://mc.manuscriptcentral.com/icbiol that nest in warm water produce smaller eggs and egg masses to facilitate oxygen diffusion to embryos [START_REF] Woods | Egg-mass size and cell size: Effects of temperature on oxygen distribution[END_REF][START_REF] Sacerdote | Dissolved oxygen requirements for hatching success of two ambystomatid salamanders in restored ephemeral ponds[END_REF], and aquatic larvae born into warm water (from aquatic or terrestrial eggs) are smaller and have lower metabolic demands [START_REF] Kuramoto | Adaptive significance in oxygen consumption of frog embryos in relation to the environmental temperatures[END_REF][START_REF] Rollinson | Oxygen limitation at the larval stage and the evolution of maternal investment per offspring in aquatic environments[END_REF]. In oviparous reptiles, embryonic gas exchange occurs via passive diffusion across the shell into the chorioallantoic membrane, which is much less efficient than adult respiration [START_REF] Vitt | Herpetology: An introductory biology of amphibians and reptiles[END_REF]. As predicted by the OCLTT hypothesis, thermal tolerance is reduced when developing embryos experience modest to extreme hypoxia [START_REF] Flewelling | Effects of temperature and oxygen on growth and differentiation of embryos of the ground skink, Scincella lateralis[END_REF][START_REF] Liang | Oxygen-dependent heat tolerance and developmental plasticity in turtle embryos[END_REF][START_REF] Smith | Oxygen supply limits the heat tolerance of lizard embryos[END_REF]. For example, lower environmental P O 2 experienced at high elevations reduces T LETHAL in plateau fence lizard (Sceloporus tristichus) embryos compared to P O 2 at sea level [START_REF] Smith | Oxygen supply limits the heat tolerance of lizard embryos[END_REF], although such modest hypoxia does not affect the thermal tolerance of adult congeners (S. occidentalis and S. graciosus; [START_REF] Shea | Oxygen concentration affects upper thermal tolerance in a terrestrial vertebrate[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF]). Thus, available data suggest that reptiles and amphibians experience ontogenetic shifts in the proximate mechanism underlying thermal tolerance: early stages appear more affected by organ-system-level failures, such as the OCLTT mechanism, whereas later stages are more impacted by subcellular mechanisms.

An integrative framework: Hierarchical Mechanisms of Thermal Limitation

Available evidence implies that neither failure of subcellular components nor oxygen and capacity limitation are solely responsible for loss of performance at high temperatures in reptiles and amphibians. Rather, mechanisms across levels of organization appear to play partial, context-dependent roles. We propose a unified framework that combines subcellular mechanisms, the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, and the http://mc.manuscriptcentral.com/icbiol thermal performance curve (TPC) paradigm. We call this integrative framework "Hierarchical Mechanisms of Thermal Limitation" (HMTL, Fig. 1). Like the OCLTT hypothesis, we propose that oxygen diffusion and transport capacity largely shape thermal performance at sub-critical temperatures via effects of temperature on aerobic scope. However, the HMTL framework explicitly recognizes the importance of subcellular-level mechanisms and allows them to underlie absolute thermal limits, such as CT MAX and T LETHAL . Under this hypothesis, the relative importance of subcellular-and organ-system-level mechanisms on thermal tolerance is context dependent, but predictable. We propose that the HMTL framework explains diverse empirical results and can bring together decades of physiological studies in reptiles and amphibians.

Given that numerous models of thermal performance and tolerance have been proposed, we think that it is useful to state their similarities and differences with HMTL. The HMTL framework builds upon ideas developed for the OCLTT hypothesis proposed by Pörtner and colleagues (recently reviewed in [START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF] and is similar to that put forth by [START_REF] Ern | Oxygen dependence of upper thermal limits in fishes[END_REF] for fishes (see Fig. 1 in [START_REF] Ern | Oxygen dependence of upper thermal limits in fishes[END_REF]), but more formally incorporates the TPC paradigm and explicitly describes mechanism-dependent critical temperatures. Importantly, this hypothesis predicts conditions under which subcellular and organ-system failure will set thermal tolerance limits and when transitions are likely to occur. Our framework also differs importantly from the Multiple Performances -Multiple Optima (MPMO) framework put forth by [START_REF] Clark | Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations[END_REF] for fishes. The MPMO suggests a single thermal limit set by an undefined mechanism but multiple, trait-specific optima that can dramatically differ from the temperature that maximizes aerobic scope (summarized in Fig. 7 of [START_REF] Clark | Aerobic scope measurements of fishes in an era of climate change: Respirometry, relevance and recommendations[END_REF]. By contrast, we propose multiple potential mechanisms of thermal limitation but a single whole-organism thermal optimum for http://mc.manuscriptcentral.com/icbiol performance correlated with the temperature that maximizes aerobic scope (although this will be an integration across traits).

Figure 1 illustrates the HMTL framework. In the top row of panels, solid blue and red lines represent the effect of temperature on oxygen consumption during rest and activity, respectively. Assuming activity is maximal, aerobic scope is the difference between these lines.

To illustrate this concept with empirical data, we derived these lines by fitting a generalized logistic function to data from [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF] for pythons (Python regius) assuming the shape of the response curve is the same during rest and activity. Based on available data, the general shape of these curves is qualitatively similar across diverse taxa (e.g. [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF][START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF][START_REF] Carey | Aerobic and anaerobic energy expenditure during rest and activity in montane Bufo b. boreas and Rana pipiens[END_REF][START_REF] Frederich | Oxygen limitation of thermal tolerance defined by cardiac and ventilatory performance in spider crab, Maja squinado[END_REF], Ern et al. 2016) and we think that the predictions of the HMTL framework can be generalized beyond the specific data used to generate this illustration. The dotted blue and red lines depict exponential functions fit to these data and illustrate predicted oxygen demand/use if organisms were unconstrained by capacity limitations.

The columns illustrate predicted effects of variable oxygen environments, and the bottom row displays TPCs predicted to result given relationships in the top row.

A central assumption of the HMTL framework is that critical thermal limits exist for both subcellular mechanisms (subcellular T CRIT ) and aerobic respiration (aerobic T CRIT ), and that organismal thermal limits are proximally caused by the lower T CRIT (Fig. 1). The environment and oxygen-handling capacity of the organism will co-determine the hierarchy of these critical limits and thus their relative importance. The subcellular T CRIT is the temperature at which key subcellular components begin to lose function, and should be insensitive to activity state or oxygen environment. By contrast, aerobic T CRIT is the temperature at which oxygen capacity is maximized, and is affected by activity state and oxygen environment. For example, aerobic T CRIT http://mc.manuscriptcentral.com/icbiol Downloaded from https://academic.oup.com/icb/advance-article-abstract/doi/10.1093/icb/icy005/4985721 by guest on 26 April 2018 during rest will be higher than aerobic T CRIT during activity because elevated O 2 demand during activity causes capacity limits to be reached more readily (Fig 1). Moreover, both active-and resting-aerobic T CRIT should be reduced during exposure to hypoxic environments (Figs. 1B-1C).

Any reduction in environmental oxygen availability below normoxia will reduce aerobic scope.

However, CT MAX will only be reduced if environmental hypoxia is sufficient to cause restingaerobic T CRIT to drop below subcellular T CRIT (Fig. 1C). The environmental oxygen tension where resting-aerobic T CRIT equals subcellular T CRIT is P CTMAX as defined by [START_REF] Ern | Oxygen dependence of upper thermal limits in fishes[END_REF].

By integrating T CRIT values and aerobic scope, we can derive whole-organism TPCs (Fig. 1, bottom row). Because the active-aerobic T CRIT is the lowest temperature where maximal metabolic rate can be achieved, it closely corresponds to the optimal temperature for aerobic performance (whole-organism T OPT ) where aerobic scope is maximized. At temperatures below active-aerobic T CRIT , we predict that performance increases with temperature proportional to aerobic scope. At temperatures above active-aerobic T CRIT , performance will drop until CT MAX is reached, but we predict that the shape of this drop will depend on which T CRIT underlies CT MAX .

If resting-aerobic T CRIT underlies CT MAX , the TPC should be more symmetrical with performance and aerobic scope decreasing at a rate mirroring the increase (Fig 1C). However, if subcellular T CRIT underlies CT MAX , the curve will be asymmetric with loss of performance occurring more rapidly the closer active-aerobic T CRIT is to subcellular T CRIT (Figs. 1A-1B).

Assumptions, predictions, and evidence for Hierarchical Mechanisms of Thermal Limitation in reptiles and amphibians

The HMTL framework produces numerous testable predictions, some of which can be addressed with available data, while others offer exciting avenues for future research. First, any http://mc.manuscriptcentral.com/icbiol environmental or organismal characteristics that reduce oxygen availability or capacity for oxygen utilization (e.g. aquatic respiration, reliance on cutaneous gas exchange, or life-stages such as eggs with reduced diffusion potential) should increase the probability of resting-aerobic T CRIT underlying CT MAX . This can explain oxygen capacity setting thermal limits in embryos [START_REF] Smith | Oxygen supply limits the heat tolerance of lizard embryos[END_REF] but not adults [START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF][START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF].

Moreover, the transition between aerobic and subcellular T CRIT predicted by HMTL explains extreme experimental hypoxia reducing thermal tolerance in adult reptiles and amphibians, while moderate hypoxia does not (Fig. 1C and Figs. 2B-2D). Similarly, HMTL predicts that CT MAX is lower when governed by oxygen limitation than when governed by subcellular mechanisms. Supporting this prediction, CT MAX drops in tadpoles as oxygen demand increases with growth and when respiratory structures are compromised in late-stage metamorphs, but frequently elevates again in terrestrial adults presumably as a result of increased oxygen capacity (ShermanCupp 1980;[START_REF] Sherman | Ontogenetic change in thermal tolerance of the toad Bufo woodhousii fowleri[END_REF][START_REF] Floyd | Ontogenetic change in the temperature tolerance of larval Bufo marinus (Anura: Bufonidae)[END_REF].

Another important prediction of the HMTL hypothesis is that performance can be limited by oxygen exchange capacity at high temperatures even when CT MAX and T LETHAL are proximally set by subcellular mechanisms. Some interpretations of the OCLTT hypothesis similarly highlight limits on performance at pejus rather than critical temperatures [START_REF] Pörtner | Climate change affects marine fishes through the oxygen limitation of thermal tolerance[END_REF][START_REF] Pörtner | How and how not to investigate the oxygen and capacity limitation of thermal tolerance (OCLTT) and aerobic scope -remarks on the article by Gräns et al[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF][START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF]), but other workers suggest OCLTT should explain critical limits to be useful (i.e. [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF][START_REF] Smith | Oxygen supply limits the heat tolerance of lizard embryos[END_REF][START_REF] Ern | Oxygen dependence of upper thermal limits in fishes[END_REF][START_REF] Verberk | Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence[END_REF][START_REF] Dubois | Effects of oxygen on responses to heating in two lizard species sampled along an elevational gradient[END_REF]. Under the HMTL framework, activity state is assumed to have no appreciable effect on oxygen capacity, although this may not hold in some highly aerobic taxa (e.g. [START_REF] Wang | Why savannah monitor lizards hyperventilate during activity: A comparison of model predictions and experimental data[END_REF]. The extent to which activity-induced increases in oxygen capacity affect thermal performance curves and limits is an important direction for http://mc.manuscriptcentral.com/icbiol future work. Generally however, individuals in metabolically-demanding states are expected to reach capacity limits at temperatures below those at which such limits are reached by resting individuals. Anaerobic respiration might compensate for short-term mismatches between energy demand and oxygen capacity (reviewed in Gleeson 1991; Fig. 1A, red shaded region), but aerobic constraints are predicted to reduce performance during long-term activity (e.g. digestion, reproduction, recovery; [START_REF] Jackson | Temperature and hypoxia in ectothermic tetrapods[END_REF][START_REF] Pörtner | Oxygen-and capacity-limited thermal tolerance: Bridging ecology and physiology[END_REF].

In diverse species, active metabolic rate asymptotes despite increases in cardiovascular output, implying oxygen flux becomes limited at temperatures above active-aerobic T CRIT (e.g. [START_REF] Bartholomew | Control of changes in body temperature, metabolism, and circulation by the Agamid lizard Amphibolurus barbatus[END_REF][START_REF] Bennett | Anaerobic metabolism during activity in lizards[END_REF][START_REF] Wilson | The relationship of oxygen supply for activity to body temperature in four species of lizards[END_REF][START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF][START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]. Furthermore, pulmonary diffusion capacity is limited by reduced plasma gas solubility and hemoglobin binding affinity at high temperatures [START_REF] Wood | The influence of temperature on the respiratory properties of iguana blood[END_REF][START_REF] Kinney | Cardiorespiratory effects of temperature in the turtle, Pseudemys floridana[END_REF][START_REF] Pough | Blood oxygen transport and delivery in reptiles[END_REF][START_REF] Jackson | Temperature and hypoxia in ectothermic tetrapods[END_REF][START_REF] Da Silva | Temperature and respiratory function in ectothermic vertebrates[END_REF]. Diffusion limitation of cutaneous gas exchange is also well established (reviewed in [START_REF] Burggren | Role of the central circulation in regulation of cutaneous gas exchange[END_REF][START_REF] Wang | Gas exchange in frogs and turtles: How ectothermic vertebrates contributed to solving the controversy of pulmonary oxygen secretion[END_REF] and will be important in species that spend considerable time submerged (e.g. [START_REF] Ultsch | A theoretical and experimental investigation of the relationship between metabolic rate, body size, and oxygen exchange capacity[END_REF] or are lungless (e.g. Plethodontid salamanders; [START_REF] Whitford | Gas exchange in salamanders[END_REF][START_REF] Spotilla | Role of temperature and water in the ecology of lungless salamanders[END_REF]. Finally, increases in the products of anaerobic respiration, such as lactic acid, can lead to blood acidification and thereby further declines in blood oxygen affinity [START_REF] Bennett | Blood physiology and oxygen transport during activity in two lizards Varanus gouldii and Sauromalus hispidus[END_REF]. The interaction of numerous factors at high temperatures reduces oxygen capacity, which in turn limits aerobic performance at high temperatures in reptiles and amphibians, regardless of the mechanism governing CT MAX (further reviewed in [START_REF] Jackson | Temperature and hypoxia in ectothermic tetrapods[END_REF].

The HMTL framework predicts that whole-organism T OPT is the temperature providing maximal aerobic scope, which is governed by active-aerobic T CRIT . Thus, alleviating the limits that underlie active-aerobic T CRIT should allow increased maximal performance (i.e. aerobic http://mc.manuscriptcentral.com/icbiol scope) and T OPT (Fig. 1). Unfortunately, few data are available comparing whole-organism T OPT and aerobic scope in reptiles or amphibians. In the snake P. regius, whole-organism T OPT and the temperature where aerobic scope is maximized appear to correspond [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]). In the toad R. marina, observations are more complex and suggest that the maintenance of aerobic scope to high temperatures could be independent of whole-organism T OPT , at least in some cases.

Hopping performance is maximized at ~30ºC [START_REF] Kearney | Modelling species distributions without using species distributions: The cane toad in Australia under current and future climates[END_REF]) whereas aerobic scope can plateau at 30ºC, but can also increase to at least 40ºC depending on acclimation treatment [START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF]. Additional data are needed to determine if acclimation elevates T OPT for whole-organism performance similar to maximal aerobic scope in R. marina, as would be predicted by the HMTL framework.

Observations for PBT provide additional indirect evidence that whole-organism T OPT corresponds to the temperature that maximizes aerobic scope in reptiles, but again data for amphibians are less clear. Generally, terrestrial ectotherms thermoregulate to within a narrow thermal range during activity if costs to thermoregulation are not prohibitively high [START_REF] Huey | Temperature, physiology, and the ecology of reptiles[END_REF][START_REF] Bauwens | Evolution of sprint speed in lacertid lizards: Morphological, physiological, and behavioral covariation[END_REF][START_REF] Angilletta | Thermal Adaptation: A Theoretical and Empirical Synthesis[END_REF][START_REF] Kingsolver | Climate variability slows evolutionary responses of Colias butterflies to recent climate change[END_REF][START_REF] Sears | Configuration of the thermal landscape determines thermoregulatory performance of ectotherms[END_REF]. Natural selection is predicted to shape thermal preference such that PBT corresponds to, or is slightly below, whole-organism T OPT [START_REF] Huey | Temperature, physiology, and the ecology of reptiles[END_REF][START_REF] Bauwens | Evolution of sprint speed in lacertid lizards: Morphological, physiological, and behavioral covariation[END_REF][START_REF] Angilletta | Is physiological performance optimized by thermoregulatory behavior?: A case study of the eastern fence lizard, Sceloporus undulatus[END_REF]; but see [START_REF] Huey | Phylogenetic studies of coadaptation: Preferred temperatures versus optimal performance temperatures of lizards[END_REF]. As predicted, PBT and temperature of maximum aerobic scope are highly concordant in lizards exposed to normoxic environments [START_REF] Wilson | The relationship of oxygen supply for activity to body temperature in four species of lizards[END_REF]. On the other hand, in the boreal toad (Anaxyrus boreas), aerobic scope is maximal at 30ºC whereas T PREF is 24ºC [START_REF] Carey | Factors affecting body temperature of toads[END_REF][START_REF] Carey | Aerobic and anaerobic energy expenditure during rest and activity in montane Bufo b. boreas and Rana pipiens[END_REF]. Interestingly, A. boreas and other anurans (L. pipiens, Carey 1979; R. marina, [START_REF] Overgaard | Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina[END_REF]) exhibit an increase in lactic acid production with temperatures above PBT in both resting and active animals. Increased lactic acid production indicates that http://mc.manuscriptcentral.com/icbiol high temperatures induce anaerobic respiration, even as aerobic scope is maintained, and therefore incur an oxygen debt for recovery. Further data are needed to assess whether the potential mismatch between maximal aerobic scope and T OPT in anurans can be explained by animals balancing the increased costs of repaying oxygen debt resulting from increased anaerobic respiration at high temperatures with the benefits of concurrent increases in aerobic scope.

Assuming maximal aerobic scope, T OPT , and PBT are linked as predicted by HMTL, they are not uniformly affected by experimental hypoxia as might initially be predicted. Only extreme hypoxia affects PBT (Fig. 2) whereas the temperature that maximizes aerobic scope and presumably whole-organism T OPT is predicted to drop continuously with hypoxia (Fig. 1). This discrepancy might indicate that adult reptiles and amphibians cannot sense and respond to hypoxia-induced changes in aerobic scope in real time, which might be expected given that these animals evolved in terrestrial environments where oxygen availability is relatively stable within a lifetime. Thus, we propose that individuals choose the same body temperature regardless of the oxygen environment so long as basic metabolic demands are met, and thus predict that the "breakpoint" in Fig. 2A occurs when hypoxia causes resting-aerobic T CRIT to fall below evolved PBT. Data comparing resting aerobic T CRIT and PBT when oxygen environment or demand is manipulated are needed to test this prediction. Given the predicted relationships between aerobic scope, T OPT , and PBT, we also expect species adapted to low-oxygen environments to have relatively lower T OPT and PBT, or greater oxygen-handling capacity. However, covariation between temperature and oxygen with elevation make testing this prediction in terrestrial environments difficult.

http://mc.manuscriptcentral.com/icbiol

In addition to further exploring the potential importance of resting-and active-aerobic T CRIT , additional research is needed to identify the subcellular components that underlie subcellular T CRIT. A subset of evolutionarily conserved components might constrain thermal tolerance across a diversity of taxa, or the components that are most important could be taxonomically specific. Currently, the data needed to differentiate these possibilities are not available. We think that measures of ATPase and HSP provide useful candidates for further exploration, but caution that focusing on a single or few potential indicators in isolation will likely provide a contorted view of subcellular limitation. Advances in "-omics" technologies, particularly differential expression RNAseq, metabolomics, and proteomics could provide much useful information about subcellular physiological function, and provide additional candidate molecules for detailed analysis (e.g. [START_REF] Verberk | Anaerobic metabolism at thermal extremes: A metabolomic test of the oxygen limitation hypothesis in an aquatic insect[END_REF][START_REF] Williams | Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster[END_REF][START_REF] Campbell-Staton | Winter storms drive rapid phenotypic, regulatory, and genomic shifts in the green anole lizard[END_REF][START_REF] Telemeco | Physiology at near-critical temperatures, but not critical limits, varies between two lizard species that partition the thermal environment[END_REF]. We recommend that experiments manipulating the thermal and oxygen environment of organisms endeavor to collect subcellular data as well as wholeorganism performance data. Where possible, an integrative approach combining measurements of subcellular components and whole-organism performance will best illuminate the mechanisms that underlie tolerance and their interactions.

Finally, the HMTL framework makes predictions for how populations could be affected by global change. Numerous species are expanding or shifting their range to higher elevations in response to climate change-related temperature increases (e.g. [START_REF] Sinervo | Erosion of lizard diversity by climate change and altered thermal niches[END_REF][START_REF] Pincheira-Donoso | The evolution of viviparity opens opportunities for lizard radiation but drives it into a climate cul-de-sac[END_REF][START_REF] Pauchard | Non-native and native organisms moving into high elevation and high latitude ecosystems in an era of climate change: New challenges for ecology and conservation[END_REF]). However, HMTL predicts that reduced oxygen partial pressures at high elevation will lower both T OPT and maximal performance. Thus, species must seek cooler environments as they move to higher elevation to maintain optimal performance, and performance potential will go down regardless of thermal environment selected. An evolutionary http://mc.manuscriptcentral.com/icbiol Downloaded from https://academic.oup.com/icb/advance-article-abstract/doi/10.1093/icb/icy005/4985721 by guest on 26 April 2018 change appears necessary for animals to seek out reduced body temperatures because moderate hypoxia does not affect PBT (Fig. 2). The HMTL framework also predicts that species with greater oxygen capacity will be more buffered from lost performance when exposed to increased environmental temperatures. Thus, oxygen capacity might be a prime target of natural selection as climates warm, even if it does not underlie CT MAX or T LETHAL . Finally, given the great diversity in modes of gas exchange, metabolic demands, and shifts across life-history stages in reptiles and amphibians, we emphasize the need to explore these hypotheses in a greater number and variety of taxa.

Conclusions

Available data suggest that both subcellular-and organ-system-level mechanisms shape thermal performance and tolerance in amphibians and reptiles. The HMTL framework that we propose describes how both mechanisms co-affect animals, with their relative importance driven by their respective T CRIT . We think that the HMTL hypothesis improves upon current frameworks by explicitly removing the false dichotomy between subcellular mechanisms and oxygen limitation, identifying useful parameters for further research (subcellular T CRIT , resting-aerobic T CRIT , and active-aerobic T CRIT ), and describing how aerobic and subcellular limitations interact to affect TPCs. Moreover, HMTL appears to explain a wide range of initially perplexing observations in reptiles and amphibians such as reduced aerobic scope at high temperatures without aerobic failure at critical temperatures, extreme hypoxia affecting thermal tolerance and behavior in adults with no effect of moderate hypoxia, moderate hypoxia reducing thermal tolerance in embryos, and HSP and ATPase activity suggesting loss of subcellular function near critical temperatures. Still, the HMTL framework makes numerous predictions for which additional data http://mc.manuscriptcentral.com/icbiol are needed, including animals only reducing their body temperature when resting-aerobic T CRIT drops below PBT, CT MAX being lower when resting-aerobic T CRIT is responsible than when subcellular T CRIT is responsible, maximum aerobic capacity underlying whole-organism thermal optima, and reduced performance when species invade higher elevations without increased oxygen capacity. Moreover, virtually no data are available to address the potential importance of aerobic-or subcellular-T CRIT as evolutionary constraints shaping the adaptive landscape. Further data better representing the diversity of reptile and amphibian taxa are needed both to understand the potential relevance of the HMTL mechanism in extant reptiles and amphibians, and how such a mechanism could have shaped the evolution of these animals. We think that many of the ideas that make up our HMTL framework are already widely accepted within the scientific community, and hope that explicitly describing them within a single framework with clear, testable predictions will facilitate further research. Although inspired by reptiles and amphibians, this integrated framework could have broad applicability across ectothermic animals. We look forward to continued investigation further integrating, refining, and testing these ideas across mechanisms and taxa. (2014). These data were used to fit curves for resting and active metabolic rate, which were then used to estimate T OPT (maximal aerobic scope), resting-aerobic T CRIT , active-aerobic T CRIT , and thermal performance curve shape. The critical thermal maximum is 42˚C [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF].

Figure legends

Panel B) Moderate hypoxia (10-20 kPa) is predicted to have no effect on CT MAX or resting metabolism because resting aerobic T CRIT is above subcellular T CRIT . Even so, active-aerobic T CRIT will be reduced thereby lowering aerobic scope, maximal performance, and T OPT . Panel C), Exposure to extreme hypoxia (< 10 kPa) will reduce CT MAX because resting-aerobic T CRIT drops below subcellular T CRIT . Active-aerobic T CRIT will be strongly affected by such hypoxic environments as well, with large reductions in aerobic scope, maximal performance, and T OPT .

In Panels B) and C), gray lines illustrate values predicted for normoxia and are included to aid interpretation of changes predicted to result from reduced environmental oxygen availability.

Similarly, arrows in Panels B) and C) indicate predicted displacement of maximum oxygen capacity, resting-aerobic T CRIT , T OPT , and CT MAX values given the oxygen environment. Subcellular T CRIT is not predicted to be affected by oxygen environment and therefore is never displaced.

http://mc.manuscriptcentral.com/icbiol Fig. 2 Thermal preference and tolerance are unaffected by moderate hypoxia but reduced in extremely hypoxic environments (< 10 kPa). This "broken-stick" pattern is apparent for A) preferred body temperature (i.e. behavioral anapyrexia), B) critical thermal maximum, C) gaping temperature, and D) panting temperature across species. Data are mostly available for lizards (solid lines) but preferred body temperature data are also available for alligator (dashed line). derived from the literature. See Table S1 for data and citations. Panel A) displays these relationships in normoxic environments. Data are means ± s.e.m. for the snake, Python regius, from [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]. These data were used to fit curves for resting and active metabolic rate, which were then used to estimate TOPT (maximal aerobic scope), resting-aerobic TCRIT, active-aerobic TCRIT, and thermal performance curve shape. The critical thermal maximum is 42˚C [START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]). Panel B) Moderate hypoxia (10-20 kPa) is predicted to have no effect on CTMAX or resting metabolism because resting aerobic TCRIT is above subcellular TCRIT. Even so, active-aerobic TCRIT will be reduced thereby lowering aerobic scope, maximal performance, and TOPT. Panel C), Exposure to extreme hypoxia (< 10 kPa) will reduce CTMAX because resting-aerobic TCRIT drops below subcellular TCRIT. Active-aerobic TCRIT will be strongly affected by such hypoxic environments as well, with large reductions in aerobic scope, maximal performance, and TOPT. In Panels B) and C), gray lines illustrate values predicted for normoxia and are included to aid interpretation of changes predicted to result from reduced environmental oxygen availability. Similarly, arrows in Panels B) and C) indicate predicted displacement of maximum oxygen capacity, resting-aerobic TCRIT, TOPT, and CTMAX values given the oxygen environment. Subcellular TCRIT is not predicted to be affected by oxygen environment and therefore is never displaced.

176x88mm (300 x 300 DPI)

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Thermal preference and tolerance are unaffected by moderate hypoxia but reduced in extremely hypoxic environments (< 10 kPa). This "broken-stick" pattern is apparent for A) preferred body temperature (i.e. behavioral anapyrexia), B) critical thermal maximum, C) gaping temperature, and D) panting temperature across species. Data are mostly available for lizards (solid lines) but preferred body temperature data are also available for alligator (dashed line). Lines between points are included to aid visualization, but connections between values at normoxia and hypoxia are likely non-linear. See Table S1 for data and citations.

231x168mm (300 x 300 DPI)

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Fig. 1

 1 Fig. 1 Schematic of the Hierarchical Mechanisms of Thermal Limitation (HMTL) framework

  Lines between points are included to aid visualization, but connections between values at normoxia and hypoxia are likely non-linear. Colors (online only) and initials denote species: A.m. = Alligator mississippiensis, B.v. = Basiliscus vittatus, C.p. = Ctenosaura pectinata, I.i. = Iguana iguana, P.v. = Pogona vitticeps, S.g. = Sceloporus graciosus, S.o. = Sceloporus occidentalis, V.e. = Varanus exanthematicus, V.v. = Varanus varius. Data are means ± s.e.m.

Fig. 3

 3 Fig.3As the environment becomes increasingly hypoxic, A) arterial P O 2 decreases, whereas B)

Fig. 1

 1 Fig.1Schematic of the Hierarchical Mechanisms of Thermal Limitation (HMTL) framework illustrating proposed relationships between body temperature, oxygen environment, metabolism, thermal limits, and performance. The top row of plots displays resting and active metabolic rates as a function of temperature within thermal and physiological limits, and the bottom row displays predicted thermal performance curves.Panel A) displays these relationships in normoxic environments. Data are means ± s.e.m. for the snake, Python regius, from[START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]. These data were used to fit curves for resting and active metabolic rate, which were then used to estimate TOPT (maximal aerobic scope), resting-aerobic TCRIT, active-aerobic TCRIT, and thermal performance curve shape. The critical thermal maximum is 42˚C[START_REF] Fobian | Oxygen transport is not compromised at high temperature in pythons[END_REF]). Panel B) Moderate hypoxia (10-20 kPa) is predicted to have no effect on CTMAX or resting metabolism because resting aerobic TCRIT is above subcellular TCRIT. Even so, active-aerobic TCRIT will be reduced thereby lowering aerobic scope, maximal performance, and TOPT. Panel C), Exposure to extreme hypoxia (< 10 kPa) will reduce CTMAX because resting-aerobic TCRIT drops below subcellular TCRIT. Active-aerobic TCRIT will be strongly affected by such hypoxic environments as well, with large reductions in aerobic scope, maximal performance, and TOPT. In Panels B) and C), gray lines illustrate values predicted for normoxia and are included to aid interpretation of changes predicted to result from reduced environmental oxygen availability. Similarly, arrows in Panels B) and C) indicate predicted displacement of maximum oxygen capacity, resting-aerobic TCRIT, TOPT, and CTMAX values given the oxygen environment. Subcellular TCRIT is not predicted to be affected by oxygen environment and therefore is never displaced.

  Colors (online only) and initials denote species: A.m. = Alligator mississippiensis, B.v. = Basiliscus vittatus, C.p. = Ctenosaura pectinata, I.i. = Iguana iguana, P.v. = Pogona vitticeps, S.g. = Sceloporus graciosus, S.o. = Sceloporus occidentalis, V.e. = Varanus exanthematicus, V.v. = Varanus varius. Data are means ± s.e.m. derived from the literature.

  Table S1 for data and citations. List of terms and abbreviations

	ATP	Adenosine triphosphate
	active-aerobic T CRIT Critical temperature where aerobic respiration is maximized for
		active individuals (i.e. ܸ ሶ O 2MAX is maximized, ˚C)
	resting-aerobic T CRIT Critical temperature where aerobic respiration would be maximized
		for resting individuals, assuming they survive to such high
		temperatures (i.e. ܸ ሶ O 2REST is maximized, ˚C)
	subcellular T CRIT	Critical temperature for subcellular function (˚C)
	CT MAX	Critical thermal maximum (˚C)
	HMTL	Hierarchical Mechanisms of Thermal Limitation hypothesis
	HSP	Heat shock protein
	MPMO	Multiple Performances -Multiple Optima hypothesis (sensu Clark et
		al. 2013)
	OCLTT	Oxygen-and Capacity-Limited Thermal Tolerance hypothesis
		(sensu Pörtner 2002 and Pörtner et al. 2017)
	T GAPE	Gaping temperature: Temperature at which animals gape to promote
		evaporative cooling (˚C)
	T LETHAL	Lethal temperature: Temperature at which an organism dies under
		acute exposure (˚C)
	T OPT	Optimal temperature for performance (˚C)
	T PANT	Panting temperature: Temperature at which animals pant to promote
		evaporative cooling (˚C)
	T PEJUS	Pejus (i.e. getting worse) temperature (˚C, sensu Frederich and
		Pörtner 2000)
	TPC	Thermal performance curve (sensu Huey and Stevenson 1979)
	PBT	Preferred body temperature (˚C)
	P CTMAX	Oxygen partial pressure below which CT MAX is reduced (kPa) (sensu
		Ern et al. 2016)
	P O 2	Partial pressure of oxygen (kPa)
	P CO 2 ܸ ሶ O 2	Partial pressure of carbon dioxide (kPa) Oxygen consumption rate (generally mL O 2 min -1 )

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Table 1 .

 1 Experimental designs for testing aspects of the Hierarchical Mechanisms of Thermal Limitation hypothesis in reptiles and amphibians with example studies.

	Experiment
	type: Factor	Manipulation	Dependent variable	What it demonstrates	Examples
	manipulated
		Temperature treatments or ramp in	Oxygen capacity parameters	Maintenance of aerobic scope at high	Carey 1979
		lab	(ܸ ሶ O 2 , Active ܸ ሶ O 2 , alveolar/arterial P O 2 , heart rate), lactate production	temperatures; No evidence of transition to anaerobic respiration	Seebacher and Franklin 2011 Overgaard et al. 2012 Fobian et al. 2014 Gangloff et al. 2016
		Temperature treatments in lab	Skeletal muscle	No transition from aerobic to	Telemeco et al. 2017
			metabolome, mitochondrial	anaerobic metabolism or subcellular
	Temperature	Ex vivo temperature treatments	function Enzyme activity	damage at high temperatures Temperature where subcellular components lose function	Licht 1964 Paulson and Hutchinson 1987 Irie et al. 1998 Al-Jassabi 2002
		Temperature treatments in lab	Heat shock protein	Temperature that induces a	Ulmasov et al. 1992
			induction	subcellular-protection response	Fernando and Heikkila 2000
					Zatespina et al. 2000
					McMillan et al. 2011
					Gao et al. 2014
					Simoniello et al. 2016
					Tedeschi et al. 2016
		Oxygen treatments in lab	Oxygen capacity parameters	Aerobic capacity is maintained under	Boyer 1963
	Ambient Oxygen	Oxygen treatments in lab	(ܸ ሶ O 2 , heartrate, alveolar/arterial O 2 , ventilation rate) PBT	conditions of mild hypoxia, but is limited under extreme hypoxia PBT is unaffected by mild hypoxia, but is reduced under extreme hypoxia	Boyer 1966 Withers and Hillman 1983 Pörtner et al. 1991 Branco et al. 1993 Wang et al. 1994 Hicks and Wood 1985 Branco et al. 1993
					Cadena and Tattersall 2009

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Acknowledgements

We are grateful to M. Angilletta, B. Bodensteiner, and J. VandenBrooks for comments on earlier drafts of this manuscript, and for the support of A. Bronikowski, T. Schwartz, B. Slater, M.

Telemeco, and A. Toth.

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Funding R.S.T. received support from Auburn University and California State University, Fresno. E.J.G. was supported by the U.S. National Science Foundation (IOS-1558071 to A. Bronikowski), the "Laboratoire d'Excellence (LABEX)" TULIP (ANR-10-LABX-41), and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 752299. http://mc.manuscriptcentral.com/icbiol

Supplementary data

Summary data compiled from the literature for Figs 2-3 are available in Table S1