3D printed models are an accurate, cost-effective, and reproducible tool for quantifying terrestrial thermal environments.

Predicting ecological responses to rapid environmental change has become one of the greatest challenges of modern biology. One of the major hurdles in forecasting these responses is accurately quantifying the thermal environments that organisms experience. The distribution of temperatures available within an organism's habitat is typically measured using data loggers called operative temperature models (OTMs) that are designed to mimic certain properties of heat exchange in the focal organism. The gold standard for OTM construction in studies of terrestrial ectotherms has been the use of copper electroforming which creates anatomically accurate models that equilibrate quickly to ambient thermal conditions. However, electroformed models require the use of caustic chemicals, are often brittle, and their production is expensive and time intensive. This has resulted in many researchers resorting to the use of simplified OTMs that can yield substantial measurement errors. 3D printing offers the prospect of robust, easily replicated, morphologically accurate, and cost-effective OTMs that capture the benefits but alleviate the problems associated with electroforming. Here, we validate the use of OTMs that were 3D printed using several materials across eight lizard species of different body sizes and living in habitats ranging from deserts to tropical forests. We show that 3D printed OTMs have low thermal inertia and predict the live animal's equilibration temperature with high accuracy across a wide range of body sizes and microhabitats. Finally, we developed a free online repository and database of 3D scans (https://www.3dotm.org/) to increase the accessibility of this tool to researchers around the world and facilitate ease of production of 3D printed models. 3D printing of OTMs is generalizable to taxa beyond lizards. If widely adopted, this approach promises greater accuracy and reproducibility in studies of terrestrial thermal ecology and should lead to improved forecasts of the biological impacts of climate change.

Transgenerational effects of maternal corticosterone across early life in a viviparous snake.

Glucocorticoids (GCs) are central mediators of vertebrate responses to intrinsic and extrinsic stimuli. Among the sources of variation in circulating GCs are transgenerational effects mediated by mothers. Here we studied potential maternal effects mediated by GCs on offspring phenotype in a live-bearing reptile, the western terrestrial garter snake (Thamnophis elegans). We evaluated the association between baseline corticosterone (CORT) levels during gestation (i.e., preparturition) in field-captured mothers and 1) reproductive success and offspring sex ratios, 2) birth phenotypic traits of offspring born under common-garden laboratory conditions, and 3) neonate (age 3 months) and juvenile (age 12 months) traits of offspring raised under two thermal regimes ('warm' and 'cool') during their first year of life. Reproductive success and offspring sex ratios were not associated with preparturition maternal CORT, but pregnant snakes with higher CORT levels gave birth to smaller, lighter offspring, which tended to grow faster to age three months. Neonate baseline CORT varied with preparturition maternal CORT in a sex-specific manner (positive trend for females, negative for males). Maternal CORT effects on offspring phenotype were no longer detectable in juveniles at age one year. Instead, juvenile phenotypes were most influenced by rearing environment, with offspring raised under the cool regime showing higher baseline CORT and slower growth than those raised under warmer conditions. Our findings support the notion that offspring phenotype might be continuously adjusted in response to environmental cues -both pre- and post-natal- and that the strength of maternal CORT effects declines as offspring develop and experience unique environmental challenges. Our results contribute to a growing literature on transgenerational effects of hormones and help to fill a gap in our knowledge of these effects in ectothermic amniotes.

Over a decade of field physiology reveals life-history specific strategies to drought in garter snakes (Thamnophis legans).

Changing climates and severe weather events can affect population viability. Individuals need to buffer such negative fitness consequences through physiological plasticity. Whether certain life-history strategies are more conducive to surviving changing climates is unknown, but theory predicts that strategies prioritizing maintenance and survival over current reproduction should be better able to withstand such change. We tested this hypothesis in a meta-population of garter snakes having naturally occurring variation in life-history strategies. We tested whether slow pace-of-life (POL) animals, that prioritize survival over reproduction, are more resilient than fast POL animals as measured by several physiological biomarkers. From 2006 to 2019, which included two multi-year droughts, baseline and stress-induced reactivity of plasma corticosterone and glucose varied annually with directionalities consistent with life-history theory. Slow POL animals exhibited higher baseline corticosterone and lower baseline glucose, relative to fast POL animals. These patterns were also observed in stress-induced measures; thus, reactivity was equivalent between ecotypes. However, in drought years, measures of corticosterone did not differ between different life histories. Immune cell distribution showed annual variation independent of drought or life history. These persistent physiological patterns form a backdrop to several extirpations of fast POL populations, suggesting a limited physiological toolkit to surviving periods of extreme drought.

Impacts of temperature on O2 consumption of the Pyrenean brook newt (Calotriton asper) from populations along an elevational gradient.

Global warming impacts biodiversity worldwide, leading to species' adaptation, migration, or extinction. The population's persistence depends on the maintenance of essential activities, which is notably driven by phenotypic adaptation to local environments. Metabolic rate - that increases with temperature in ectotherms - is a key physiological proxy for the energy available to fuel individuals' activities. Cold-adapted ectotherms can exhibit a higher resting metabolism than warm-adapted ones to maintain functionality at higher elevations or latitudes, known as the metabolic cold-adaptation hypothesis. How climate change will affect metabolism in species inhabiting contrasting climates (cold or warm) is still a debate. Therefore, it is of high interest to assess the pace of metabolic responses to global warming among populations adapted to highly different baseline climatic conditions. Here, we conducted a physiological experiment in the endemic Pyrenean brook newt (Calotriton asper). We measured a proxy of standard metabolic rate (SMR) along a temperature gradient in individuals sampled among 6 populations located from 550 to 2189 m a.s.l. We demonstrated that SMR increased with temperature, but significantly diverged depending on populations' origins. The baseline and the slope of the relationship between SMR and temperature were both higher for high-elevation populations than for low-elevation populations. We discussed the stronger metabolic response observed in high-elevation populations suggesting a drop of performance in essential life activities for these individuals under current climate change. With the increase of metabolism as the climate warms, the metabolic-cold adaptation strategy selected in the past could compromise the sustainability of cold-adapted populations if short-term evolutionary responses do not allow to offset this evolutionary legacy.

Effects of reliance on stored sperm on reproduction in the sailfin molly Poecilia latipinna.

The impacts of relying on stored sperm were evaluated in the sailfin molly, Poecilia latipinna. Females reliant on stored sperm had fewer offspring compared to remated females, but offspring size and short-term growth rate did not differ. Thus, females may use stored sperm in cases such as previous mating with a preferred male, lack of access to mating opportunities during a reproductive cycle, or to maximize egg fertilization. Females do not compensate for producing fewer offspring however, by allocating more resources to offspring relative to their size or initial growth.

Thermal-metabolic phenotypes of the lizard Podarcis muralis differ across elevation, but converge in high-elevation hypoxia.

In response to a warming climate, many montane species are shifting upslope to track the emergence of preferred temperatures. Characterizing patterns of variation in metabolic, physiological and thermal traits along an elevational gradient, and the plastic potential of these traits, is necessary to understand current and future responses to abiotic constraints at high elevations, including limited oxygen availability. We performed a transplant experiment with the upslope-colonizing common wall lizard (Podarcis muralis) in which we measured nine aspects of thermal physiology and aerobic capacity in lizards from replicate low- (400 m above sea level, ASL) and high-elevation (1700 m ASL) populations. We first measured traits at their elevation of origin and then transplanted half of each group to extreme high elevation (2900 m ASL; above the current elevational range limit of this species), where oxygen availability is reduced by ∼25% relative to sea level. After 3 weeks of acclimation, we again measured these traits in both the transplanted and control groups. The multivariate thermal-metabolic phenotypes of lizards originating from different elevations differed clearly when measured at the elevation of origin. For example, high-elevation lizards are more heat tolerant than their low-elevation counterparts (counter-gradient variation). Yet, these phenotypes converged after exposure to reduced oxygen availability at extreme high elevation, suggesting limited plastic responses under this novel constraint. Our results suggest that high-elevation populations are well suited to their oxygen environments, but that plasticity in the thermal-metabolic phenotype does not pre-adapt these populations to colonize more hypoxic environments at higher elevations.

Multiple Paternity in Garter Snakes With Evolutionarily Divergent Life Histories.

Many animal species exhibit multiple paternity, defined as multiple males genetically contributing to a single female reproductive event, such as a clutch or litter. Although this phenomenon is well documented across a broad range of taxa, the underlying causes and consequences remain poorly understood. For example, it is unclear how multiple paternity correlates with life-history strategies. Furthermore, males and females may differ in mating strategies and these patterns may shift with ecological context and life-history variation. Here, we take advantage of natural life-history variation in garter snakes (Thamnophis elegans) to address these questions in a robust field setting where populations have diverged along a slow-to-fast life-history continuum. We determine both female (observed) and male (using molecular markers) reproductive success in replicate populations of 2 life-history strategies. We find that despite dramatic differences in annual female reproductive output: 1) females of both life-history ecotypes average 1.5 sires per litter and equivalent proportions of multiply-sired litters, whereas 2) males from the slow-living ecotype experience greater reproductive skew and greater variance in reproductive success relative to males from the fast-living ecotype males despite having equivalent average reproductive success. Together, these results indicate strong intrasexual competition among males, particularly in the fast-paced life-history ecotype. We discuss these results in the context of competing hypotheses for multiple paternity related to population density, resource variability, and life-history strategy.

Surviving winter: Physiological regulation of energy balance in a temperate ectotherm entering and exiting brumation.

Characterizing the physiological response to prolonged cold exposure is essential for understanding the maintenance of long-term energy balance. As part of their natural life cycle, temperate ectotherms are often exposed to seasonal variation in temperatures, including extended periods of cold well below their activity range. Relatively little is known about variation in physiological responses as vertebrate ectotherms enter and exit brumation in response to sustained cold temperatures. We tested the influence of temperature on physiology before, during, and after a simulated brumation in the checkered garter snake (Thamnophis marcianus), a widespread ectothermic vertebrate. We tested for the relative effect of immediate temperature and physiological context (entering or exiting brumation) on hormones regulating energy balance, indicators of energy availability, and resting metabolic rate (V̇O2). Plasma corticosterone, glucose, and insulin, as well as immune cell heterophil: lymphocyte ratios responded to temperature, though they did so with different thermal response curves. Thermal sensitivity varied both among and within physiological measures depending on whether animals were going into or coming out of brumation. Additionally, V̇O2 was regulated beyond simple temperature-dependence, whereby post-brumation measures were depressed relative to pre-brumation measures at the same temperature. This pattern was characterized by a change in the temperature coefficient (Q10), with a larger pre-brumation Q10, suggesting reduced thermal sensitivity of metabolic rate following a period of extended cold exposure. The integrated physiological response presented here demonstrates not only temperature dependence across physiological axes, but seasonal variation in thermal responsiveness. Our results suggest that energy allocation decisions and hormonal regulation of underlying processes promote differing levels of thermal sensitivity when entering or exiting brumation.

Genetic background and thermal environment differentially influence the ontogeny of immune components during early life in an ectothermic vertebrate.

An understudied aspect of vertebrate ecoimmunology has been the relative contributions of environmental factors (E), genetic background (G) and their interaction (G × E) in shaping immune development and function. Environmental temperature is known to affect many aspects of immune function and alterations in temperature regimes have been implicated in emergent disease outbreaks, making it a critical environmental factor to study in the context of immune phenotype determinants of wild animals. We assessed the relative influences of environmental temperature, genetic background and their interaction on first-year development of innate and adaptive immune defences of captive-born garter snakes Thamnophis elegans using a reciprocal transplant laboratory experiment. We used a full-factorial design with snakes from two divergent life-history ecotypes, which are known to differ in immune function in their native habitats, raised under conditions mimicking the natural thermal regime-that is, warmer and cooler-of each habitat. Genetic background (ecotype) and thermal regime influenced innate and adaptive immune parameters of snakes, but in an immune-component specific manner. We found some evidence of G × E interactions but no indication of adaptive plasticity with respect to thermal environment. At the individual level, the effects of thermal environment on resource allocation decisions varied between the fast- and the slow-paced life-history ecotypes. Under warmer conditions, which increased food consumption of individuals in both ecotypes, the former invested mostly in growth, whereas the latter invested more evenly between growth and immune development. Overall, immune parameters were highly flexible, but results suggest that other environmental factors are likely more important than temperature per se in driving the ecotype differences in immunity previously documented in the snakes under field conditions. Our results also add to the understanding of investment in immune development and growth during early postnatal life under different thermal environments. Our finding of immune-component specific patterns strongly cautions against oversimplification of the highly complex immune system in ecoimmunological studies. In conjunction, these results deepen our understanding of the degree of immunological flexibility wild animals present, information that is ever more vital in the context of rapid global environmental change.

Transplanting gravid lizards to high elevation alters maternal and embryonic oxygen physiology, but not reproductive success or hatchling phenotype.

Increased global temperatures have opened previously inhospitable habitats, such as at higher elevations. However, the reduction of oxygen partial pressure with increase in elevation represents an important physiological constraint that may limit colonization of such habitats, even if the thermal niche is appropriate. To test the mechanisms underlying the response to ecologically relevant levels of hypoxia, we performed a translocation experiment with the common wall lizard (Podarcis muralis), a widespread European lizard amenable to establishing populations outside its natural range. We investigated the impacts of hypoxia on the oxygen physiology and reproductive output of gravid common wall lizards and the subsequent development and morphology of their offspring. Lowland females transplanted to high elevations increased their haematocrit and haemoglobin concentration within days and maintained routine metabolism compared with lizards kept at native elevations. However, transplanted lizards suffered from increased reactive oxygen metabolite production near the oviposition date, suggesting a cost of reproduction at high elevation. Transplanted females and females native to different elevations did not differ in reproductive output (clutch size, egg mass, relative clutch mass or embryonic stage at oviposition) or in post-oviposition body condition. Developing embryos reduced heart rates and prolonged incubation times at high elevations within the native range and at extreme high elevations beyond the current range, but this reduced oxygen availability did not affect metabolic rate, hatching success or hatchling size. These results suggest that this opportunistic colonizer is capable of successfully responding to novel environmental constraints in these important life-history stages.

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.

Geographic variation and within-individual correlations of physiological stress markers in a widespread reptile, the common garter snake (Thamnophis sirtalis).

Characterizing the baseline and stress-induced hormonal, metabolite, and immune profiles of wild animals is important to assess the impacts of variable environments, including human-induced landscape changes, on organismal health. Additionally, the extent to which these profiles are coordinated across physiological systems within individuals remains an important question in understanding how stressors can differentially affect aspects of an individual's physiology. Here, we present data from wild populations of the common garter snake (Thamnophis sirtalis) on both baseline and stress-induced biomarkers: plasma corticosterone (CORT) concentration, plasma glucose concentration, and whole blood heterophil:lymphocyte ratio. Using a standardized restraint protocol with individuals from populations in disparate portions of this species' range - the Sierra Nevada Mountains of California and the plains of Iowa - we collected blood plasma samples at nine time points over three days. Both CORT and glucose response curves differed between georegions, with Iowa snakes attaining higher glucose concentration and maintaining elevated CORT and glucose levels for a longer duration. Additionally, both the total amount and proportional increases of CORT and glucose were lower in larger and therefore older snakes, suggesting ontogenetic shifts in stress perception or response. Within-individual correlation among the three physiological indicators was significant at the time of capture, absent after 3h in captivity, and partially restored after 3days in captivity, demonstrating the effect of stress on the relationships among these physiological systems. Together, these results provide further evidence for the great physiological flexibility of ectothermic tetrapods in maintaining homeostasis across a range of factors.

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.

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.

Embryonic and juvenile snakes (Natrix maura, Linnaeus 1758) compensate for high elevation hypoxia via shifts in cardiovascular physiology and metabolism.

The colonization of novel environments requires a favorable response to conditions never, or rarely, encountered in recent evolutionary history. For example, populations colonizing upslope habitats must cope with lower atmospheric pressure at elevation, and thus reduced oxygen availability. The embryo stage in oviparous organisms is particularly susceptible, given its lack of mobility and limited gas exchange via diffusion through the eggshell and membranes. Especially little is known about responses of Lepidosaurian reptiles to reduced oxygen availability. To test the role of physiological plasticity during early development in response to high elevation hypoxia, we performed a transplant experiment with the viperine snake (Natrix maura, Linnaeus 1758). We maintained gravid females originating from low elevation populations (432 m above sea level [ASL]-normoxia) at both the elevation of origin and high elevation (2877 m ASL-extreme high elevation hypoxia; approximately 72% oxygen availability relative to sea level), then incubated egg clutches at both low and high elevation. Regardless of maternal exposure to hypoxia during gestation, embryos incubated at extreme high elevation exhibited altered developmental trajectories of cardiovascular function and metabolism across the incubation period, including a reduction in late-development egg mass. This physiological response may have contributed to the maintenance of similar incubation duration, hatching success, and hatchling body size compared to embryos incubated at low elevation. Nevertheless, after being maintained in hypoxia, juveniles exhibit reduced carbon dioxide production relative to oxygen consumption, suggesting altered energy pathways compared to juveniles maintained in normoxia. These findings highlight the role of physiological plasticity in maintaining rates of survival and fitness-relevant phenotypes in novel environments.

Physiological phenotypes differ among color morphs in introduced common wall lizards (Podarcis muralis).

Many species exhibit color polymorphisms which have distinct physiological and behavioral characteristics. However, the consistency of morph trait covariation patterns across species, time, and ecological contexts remains unclear. This trait covariation is especially relevant in the context of invasion biology and urban adaptation. Specifically, physiological traits pertaining to energy maintenance are crucial to fitness, given their immediate ties to individual reproduction, growth, and population establishment. We investigated the physiological traits of Podarcis muralis, a versatile color polymorphic species that thrives in urban environments (including invasive populations in Ohio, USA). We measured five physiological traits (plasma corticosterone and triglycerides, hematocrit, body condition, and field body temperature), which compose an integrated multivariate phenotype. We then tested variation among co-occurring color morphs in the context of establishment in an urban environment. We found that the traits describing physiological status and strategy shifted across the active season in a morph-dependent manner-the white and yellow morphs exhibited clearly different multivariate physiological phenotypes, characterized primarily by differences in plasma corticosterone. This suggests that morphs have different strategies in physiological regulation, the flexibility of which is crucial to urban adaptation. The white-yellow morph exhibited an intermediate phenotype, suggesting an intermediary energy maintenance strategy. Orange morphs also exhibited distinct phenotypes, but the low prevalence of this morph in our study populations precludes clear interpretation. Our work provides insight into how differences among stable polymorphisms exist across axes of the phenotype and how this variation may aid in establishment within novel environments.

Temperature-dependence of metabolism and fuel selection from cells to whole organisms.

Temperature affects nearly every aspect of how organisms interact with and are constrained by their environment. Measures of organismal energetics, such as metabolic rate, are highly temperature-dependent and governed through temperature effects on rates of biochemical reactions. Characterizing the relationships among levels of biological organization can lend insight into how temperature affects whole-organism function. We tested the temperature dependence of cellular oxygen consumption and its relationship to whole-animal metabolic rate in garter snakes (Thamnophis elegans). Additionally, we tested whether thermal responses were linked to shifts in the fuel source oxidized to support metabolism with the use of carbon stable isotopes. Our results demonstrate temperature dependence of metabolic rates across levels of biological organization. Cellular (basal, adenosine triphosphate-linked) and whole-animal rates of respiration increased with temperature but were not correlated within or among individuals, suggesting that variation in whole-animal metabolic rates is not due simply to variation at the cellular level, but rather other interacting factors across scales of biological organization. Counter to trends observed during fasting, elevated temperature did not alter fuel selection (i.e., natural-abundance stable carbon isotope composition in breath, δ13 Cbreath ). This consistency suggests the maintenance and oxidation of a single fuel source supporting metabolism across a broad range of metabolic demands.