AbstractUnderstanding functional adaptation demands an integrative framework that captures the complex interactions between form, function, ecology, and evolutionary processes. In this review, we discuss how to integrate the following two distinct approaches to better understand functional evolution: (1) the adaptive landscape approach (ALA), aimed at finding adaptive peaks for different ecologies, and (2) the performance landscape approach (PLA), aimed at finding performance peaks for different ecologies. We focus on the Ornstein-Uhlenbeck process as the evolutionary model for the ALA and on biomechanical modeling to estimate performance for the PLA. Whereas both the ALA and the PLA have each given insight into functional adaptation, separately they cannot address how much performance contributes to fitness or whether evolutionary constraints have played a role in form-function evolution. We show that merging these approaches leads to a deeper understanding of these issues. By comparing the locations of performance and adaptive peaks, we can infer how much performance contributes to fitness in species' current environments. By testing for the relevance of history on phenotypic variation, we can infer the influence of past selection and constraints on functional adaptation. We apply this merged framework in a case study of turtle shell evolution and explain how to interpret different possible outcomes. Even though such outcomes can be quite complex, they represent the multifaceted relations among function, fitness, and constraints.

AbstractParasites can affect host behavior, cognition, locomotion, body condition, and many other physiological traits. Changes to host aerobic metabolism may be responsible for these parasite-induced performance alterations. Whole-organism metabolic rate is underpinned by cellular energy metabolism driven most prominently by mitochondria. However, few studies have explored how mitochondrial enzymatic activity relates to body condition and parasite infection, despite it being a putative site for metabolic disruptions related to health status. We studied correlations among natural parasite infection, host body condition, and activity of key mitochondrial enzymes in target organs from wild-caught pumpkinseed sunfish () to better understand the cellular responses of fish hosts to endoparasite infection. Enzymatic activities in the gills, spleen, and brain of infected fish were not significantly related to parasite infection or host body condition. However, the activity of cytochrome c oxidase, an enzyme involved in oxidative phosphorylation, in fish hearts was higher in individuals with a lower body condition. Activities of citrate synthase, electron transport system (complexes I and III), and carnitine palmitoyltransferase were also significantly different among organ types. These results provide preliminary information regarding the likely mitochondrial pathways affecting host body condition, the maintenance energetic requirements of different organs, and the organs' specific dependency on particular mitochondrial pathways. These results help pave the way for future studies on the effects of parasite infection on mitochondrial metabolism.

AbstractThrough artificial selection and inbreeding, strains of laboratory mice have been developed that vary in the expression of a single or suite of desired traits valuable to biomedical research. In addition to the selected trait(s), these strains also display variation in pelage color, body size, physiology, and life history. This article exploits the broad phenotypic variation across lab mouse strains to evaluate the relationships between life history and metabolism. Life history variation tends to exist along a fast-slow continuum. There has been considerable interest in understanding the ecological and evolutionary factors underlying life history variation and the physiological and metabolic processes that support them. Yet it remains unclear how these key traits scale across hierarchical levels, as ambiguous empirical support has been garnered at the intraspecific level. Within-species investigations have been thwarted by methodological constraints and environmental factors that obscure the genetic architecture underlying the hypothesized functional integration of life history and metabolic traits. In this analysis, we used the publicly available Mouse Phenome Database by the Jackson Laboratory to investigate the relationships among life history traits (e.g., body size, reproduction, and life span) and metabolic traits (e.g., daily energy expenditure and insulin-like growth factor 1 concentration). Our findings revealed significant variation in reproductive characteristics across strains of mice as well as relationships among life history and metabolic traits. We found evidence of variation along the fast-slow life history continuum, though the direction of some relationships among these traits deviated from interspecific predictions laid out in previous literature. Furthermore, our results suggest that the strength of these relationships are strongest earlier in life.

AbstractMigrating birds perform extreme endurance exercise when flying. This shifts the balance between the production of reactive oxygen species and the antioxidant defense system toward the former, potentially generating oxidative damages. In between migratory flights, birds make stopovers, where besides accumulating fuel (mainly fats), they are assumed to rest and recover from the strenuous flight. We performed a series of studies on both temporarily caged (northern wheatears) and free-flying (northern wheatears and European robins) migrants to investigate whether migrants recover during stopover by decreasing the amount of oxidative lipid damage (malondialdehyde [MDA]) and/or increasing the total nonenzymatic antioxidant capacity (AOX). In caged wheatears, MDA decreased within a single day. These birds were able to simultaneously accumulate considerable amounts of fuel. Also, in the free-flying wheatears, there was a decrease in MDA during stopover; however, this process seemed incompatible with refueling. The reason for this difference could relate to constraints in the wild that are absent in caged birds, such as food limitation/composition and locomotor activity. In the robins, there was a near significant decrease in MDA concentration in relation to how long the birds were already at stopover, suggesting that this species also physiologically recovers during stopover. AOX did not change during stopover in either of the wheatear studies. For the robins, however, uric acid-corrected AOX declined during stopover. Our results show that during stopover, migrating birds rapidly reduce oxidative lipid damage, thereby likely recovering their physiological state. In addition to the commonly accepted function of refueling, stopovers thus probably serve physiological recovery.

AbstractDuring the colonization of freshwater by marine fish, adaptation to hypoosmotic conditions may impact their ability to osmoregulate in seawater. The prickly sculpin () is a euryhaline fish with marine ancestors that postglacially colonized many freshwater habitats. Previous work on suggested that isolation in freshwater habitats has resulted in putative adaptations that improve ion regulation in freshwater populations compared with populations with current access to estuaries. To determine whether long-term colonization of freshwater is associated with a reduced ability to ion regulate in seawater, we acclimated populations from three habitat types that vary in the extent to which they are isolated from marine habitats and compared their seawater osmoregulation. Seawater acclimation revealed that lake populations exhibited a reduced capacity to osmoregulate in seawater compared with coastal river populations with ongoing access to estuaries. In particular, when acclimated to seawater for several weeks, lake populations had lower gill Na/K-ATPase activity and lower intestinal H-ATPase activity than coastal river populations. Lake populations also had a reduced ability to maintain plasma ion concentrations, and they produced lower quantities of intestinal carbonate precipitates in seawater than coastal river populations. Furthermore, there was a positive relationship between the anterior intestinal Na/K-ATPase activity and the amount of precipitate produced by the intestine, which suggests that the anterior intestine plays a role in seawater osmoregulation. Our results suggest that the extent of isolation from the sea could, in part, explain the reduced osmoregulation in seawater in postglacial freshwater populations of .

AbstractSeasonality in infectious disease prevalence is predominantly attributed to changes in exogenous risk factors. For vectored pathogens, high abundance, activity, and/or diversity of vectors can exacerbate disease risk for hosts. Conversely, many host defenses, particularly immune responses, are seasonally variable. Seasonality in host defenses has been attributed, in part, to the proximate (i.e., metabolic) and ultimate (i.e., reproductive fitness) costs of defense. In this study, our goal was to discern whether any seasonality is observable in how a common avian host, the house sparrow (), copes with a common zoonotic arbovirus, the West Nile virus (WNV), when hosts are studied under controlled conditions. We hypothesized that if host biorhythms play a role in vector-borne disease seasonality, birds would be most vulnerable to WNV when breeding and/or molting (i.e., when other costly physiological activities are underway) and thus most transmissive of WNV at these times of year (unless birds died from infection). Overall, the results only partly supported our hypothesis. Birds were most transmissive of WNV in fall (after their molt is complete and when WNV is most prevalent in the environment), but WNV resistance, WNV tolerance, and WNV-dependent mortality did not vary among seasons. These results collectively imply that natural arboviral cycles could be partially underpinned by endogenous physiological changes in hosts. However, other disease systems warrant study, as this result could be specific to the nonnative and highly commensal nature of the house sparrow or a consequence of the relative recency of the arrival of WNV to the United States.

AbstractEctothermic species are dependent on temperature, which drives many aspects of their physiology, including locomotion. The distribution of the native populations of is characterized by an exceptional range in latitude and altitude. Along altitudinal gradients, thermal environments change, and populations experience different temperatures. In this study, we compared critical thermal limits and thermal performance curves of populations from the native range across an altitudinal gradient to test whether optimal temperatures for exertion differ depending on altitude. Data on exertion capacity were collected at six different temperatures (8°C, 12°C, 16°C, 19°C, 23°C, and 27°C) for four populations spanning an altitudinal gradient (60, 1,016, 1,948, and 3,197 m asl). Results show that the thermal performance optimum differs among populations. Populations from cold environments at high altitudes exhibit a lower optimal performance temperature than populations from warmer environments at lower altitudes. The ability of this species to change its optimal temperature for locomotor exertion across extremely different climatic environments within the native range may help explain its exceptional invasive potential. These results suggest that ectothermic species capable of adapting to broad altitudinal ranges may be particularly good at invading novel climatic areas, given their ability to cope with a wide range of variation in environmental temperatures.

AbstractMorphological variation is sometimes used as an indicator of environmental stress in animals. Here, we assessed how multiple morphological traits covaried in exposed to five common forms of environmental stress (high temperature, presence of predator cues, high salinity, low food abundance, and low Ca). We measured animal body length, body width, head width, eyespot diameter, and tail spine length along with mass in animals of five different ages (3, 6, 9, 12, and 15 d). There were strong allometric relationships among all morphological traits in reference animals and strong univariate effects of environmental stress on body mass and body length. We found that environmental stressors altered bivariate relationships between select pairwise combinations of morphological traits, with effects being dependent on animal age. Multivariate analyses further revealed high connectivity among body size-related traits but that eyespot diameter and tail spine length were less tightly associated with body size. Animals exposed to natural lake water with and without supplemental food also varied in morphology, with body size differences being suggestive of starvation and other unknown nutritional deficiencies. Yet our results demonstrate that the scaling of body morphological traits of is largely invariant with possible context-dependent plasticity in eye size and tail spine lengths. The strong coordination of traits indicates tight molecular coordination of body size during development despite strong and varied environmental stress.

AbstractTo maximize energy savings, entry into torpor should involve a fast reduction of metabolic rate and body temperature (); that is, animals should thermoconform. However, animals often defend against the decrease in via a temporary increase in thermoregulatory heat production, slowing the cooling process. We investigated how thermoregulating or thermoconforming during torpor entry affects temporal and thermoenergetic aspects in relation to body mass and age in juvenile and adult fat-tailed dunnarts (; Marsupialia: Dasyuridae). During torpor entry, juvenile thermoconformers cooled twice as fast as and used less energy during cooling than juvenile thermoregulators. While both juvenile and adult thermoconformers had a lower minimum , a lower torpor metabolic rate, and longer torpor bouts than thermoregulators, these differences were more pronounced in the juveniles. Rewarming from torpor took approximately twice as long for juvenile thermoconformers, and the costs of rewarming were greater. To determine the difference in average daily metabolic rate between thermoconformers and thermoregulators independent of body mass, we compared juveniles of a similar size (∼13 g) and similarly sized adults (∼17 g). The average daily metabolic rate was 7% (juveniles) and 17% (adults) less in thermoconformers than in thermoregulators, even though thermoconformers were active for longer. Our data suggest that thermoconforming during torpor entry provides an energetic advantage for both juvenile and adult dunnarts and may aid growth for juveniles. While thermoregulation during torpor entry is more costly, it still saves energy, and the higher permits greater alertness and mobility and reduces the energetic cost of endogenous rewarming.

AbstractTrade-offs between life history traits are context dependent; they vary depending on environment and life stage. Negative associations between development and growth often characterize larval life stages. Both growth and development consume large parts of the energy budget of young animals. The metabolic rate of animals should reflect differences in growth and developmental rates. Growth and development can also have negative associations with immune function because of their costs. We investigated how intraspecific variation in growth and development affected the metabolism of larval amphibians and whether intraspecific variation in growth, development, and metabolic rate could predict mortality and viral load in larvae infected with ranavirus. We also compared the relationship between growth and development before and after infection with ranavirus. We hypothesized that growth and development would affect metabolism and predicted that each would have a positive correlation with metabolic rate. We further hypothesized that allocation toward growth and development would increase ranavirus susceptibility and therefore predicted that larvae with faster growth, faster development, and higher metabolic rates would be more likely to die from ranavirus and have higher viral loads. Finally, we predicted that growth rate and developmental rate would have a negative association. Intraspecific variation in growth rate and developmental rate did not affect metabolism. Growth rate, developmental rate, and metabolism did not predict mortality from ranavirus or viral load. Larvae infected with ranavirus exhibited a trade-off between developmental rate and growth rate that was absent in uninfected larvae. Our results indicate a cost of ranavirus infection that is potentially due to both the infection-induced anorexia and the cost of infection altering priority rules for resource allocation.

AbstractInsulin-like growth factor 1 (IGF-1) is positively linked with growth and reproduction but negatively linked with survival, so a potential role of IGF-1 in modulating life history trade-offs has been proposed. However, the underlying mechanisms of the negative link between IGF-1 and survival are not yet clear, and oxidative stress has been proposed as a candidate. Immune activation is one important source of oxidative stress, and both immune activation and oxidative stress are known to reduce survival. We experimentally administrated an immune or oxidative insult to Japanese quails to evaluate whether oxidative stress is a proximate cost of holding elevated IGF-1 levels during a life challenge (e.g., infection, intoxication). IGF-1 levels increased in the presence of the immune insult, but they were not affected by the oxidative insult. Hence, IGF-1 may be linked to the survival costs of activating an immune response, but oxidative stress might not be directly involved as an underlying mechanism.

AbstractBy allowing for increased absorption or reflectance of solar radiation, changes in pigmentation may assist ectotherms in responding to immune challenges by enabling a more precise regulation of behavioral fever or hypothermia. Variation in epigenetic characteristics may also assist in regulating immune-induced pigmentation changes and managing the body's energetic reserves following infection. Here, we explore how dorsal pigmentation, metabolic rate, and DNA methylation in the Florida scrub lizard () respond to two levels of immune challenge across two habitat types. We found changes in pigmentation that are suggestive of efforts to assist in behavioral fever and hypothermia depending on the intensity of immune challenge. We also found correlations between DNA methylation in liver tissue and pigmentation change along the dorsum, indicating that color transitions may be part of a multifaceted immune response across tissue types. The relationship between immune response and metabolic rate supports the idea that energetic reserves may be conserved for the costs associated with behavioral fever when immune challenge is low and the immune functions when immune challenge is high. While immune response appeared to be unaffected by habitat type, we found differences in metabolic activity between habitats, suggesting differences in the energetic costs associated with each. To our knowledge, these results present the first potential evidence of pigmentation change in ectotherms in association with immune response. The relationship between immune response, DNA methylation, and pigmentation change also highlights the importance of epigenetic mechanisms in organism physiology.

AbstractHeat waves are becoming more frequent across the globe and may impose severe thermoregulatory challenges for endotherms. Heat stress can induce both behavioral and physiological responses, which may result in energy deficits with potential fitness consequences. We studied the responses of reindeer (), a cold-adapted ungulate, to a record-breaking heat wave in northern Finland. Activity, heart rate, subcutaneous body temperature, and body mass data were collected for 14 adult females. The post-heat wave autumn body masses were then analyzed against longitudinal body mass records for the herd from 1990 to 2021. With increasing air temperature during the day, reindeer became less active and had reduced heart rate and increased body temperature, reflecting both behavioral and physiological responses to heat stress. Although they increased activity in the late afternoon, they failed to compensate for lost foraging time on the hottest days (daily mean temperature ≥20°C), and total time active was reduced by 9%. After the heat wave, the mean September body mass of herd females ( kg, ) was on average 16.4% ± 4.8% lower than predicted ( kg). Among focal females, individuals with the lowest levels of activity during the heat wave had the greatest mass loss during summer. We show how heat waves impose a thermoregulatory challenge on endotherms, resulting in mass loss, potentially as a result of the loss of foraging time. While it is well known that environmental conditions affect large herbivore fitness indirectly through decreased forage quality and limited water supply, direct effects of heat may be increasingly common in a warming climate.

AbstractIn the face of increasing environmental temperatures, operative differences between mitochondrial function and whole-animal phenotypic response to the environment are underrepresented in research, especially in subtemperate ectothermic vertebrates. A novel approach to exploring this connection is to examine model species that are genetically similar but that have different whole-animal phenotypes, each of which inhabits different environments. The blind Mexican cavefish () has the following two morphotypes: a surface form found in aboveground rivers and an obligate cave-dwelling form. Each morphotype inhabits vastly different thermal and oxygen environments. Whole-animal and mitochondrial responses to thermal acclimation and oxidative stress, with respect to increasing temperatures, have not been previously determined in either morphotype of this species. Here, we chronically acclimated both morphotypes to three temperatures (14°C, 25°C, and 31°C) to establish potential for acclimation and critical thermal maxima (CT) for each morphotype of this species. After measuring CT in six cohorts, we additionally measured enzymatic antioxidant capacity (catalase, superoxide dismutase, and glutathione peroxidase activities), peroxyl scavenging capacity, and lipid peroxidation damage in white epaxial muscle for each individual. We found a significant effect of acclimation temperature on CT (, ) but no effect of morphotype on CT (, ). Additionally, we found that morphotype had a significant effect on glutathione peroxidase activity, with the surface morphotype having increased glutathione peroxidase activity compared with the cave morphotype (, ). No other oxidative stress variable demonstrated significant differences. Increases in CT with chronic thermal acclimation to higher temperatures suggests that there is some degree of phenotypic plasticity in this species that nominally occupies thermally stable environments. The decreased glutathione peroxidase activity in the cave morphotype may be related to decreased environmental oxygen concentration and decreased metabolic rate in this environmentally constrained morphotype compared to in its surface-living counterparts.

AbstractAntioxidants have important physiological roles in limiting the amount of oxidative damage that an organism experiences. One putative antioxidant is biliverdin, a pigment that is most commonly associated with the blue or green colors of avian eggshells. However, despite claims that biliverdin functions as an antioxidant, neither the typical physiological concentrations of biliverdin in most species nor the ability of biliverdin to oppose oxidative damage at these concentrations has been examined. Therefore, we quantified biliverdin in the plasma of six bird species and found that they circulated levels of biliverdin between 0.02 and 0.5 μM. We then used a pool of plasma from northern bobwhite quail () and spiked it with one of seven different concentrations of biliverdin, creating plasma-based solutions ranging from 0.09 to 231 μM biliverdin. We then compared each solution's ability to oppose oxidative damage in response to hydrogen peroxide relative to a control addition of water. We found that hydrogen peroxide consistently induced moderate amounts of oxidative damage (quantified as reactive oxygen metabolites) but that no concentration of biliverdin ameliorated this damage. However, biliverdin and hydrogen peroxide interacted, as the amount of biliverdin in hydrogen peroxide-treated samples was reduced to approximately zero, unless the initial concentration was over 100 μM biliverdin. These preliminary findings based on in vitro work indicate that while biliverdin may have important links to metabolism and immune function, at physiologically relevant concentrations it does not detectably oppose hydrogen peroxide-induced oxidative damage in plasma.

AbstractThermal physiology helps us understand how ectotherms respond to novel environments and how they persist when introduced to new locations. Researchers generally measure thermal physiology traits immediately after animal collection or after a short acclimation period. Because many of these traits are plastic, the conclusions drawn from such research can vary depending on the duration of the acclimation period. In this study, we measured the rate of change and extent to which cold tolerance (critical thermal minimum [CT]) of nonnative Italian wall lizards () from Hempstead, New York, changed during a cold acclimation treatment. We also examined how cold acclimation affected heat tolerance (critical thermal maximum [CT]), thermal preference (), evaporative water loss (EWL), resting metabolic rate (RMR), and respiratory exchange ratio (RER). We predicted that CT, CT, and would decrease with cold acclimation but that EWL and RMR would increase with cold acclimation. We found that CT decreased within 2 wk and that it remained low during the cold acclimation treatment; we suspect that this cold tolerance plasticity reduces risk of exposure to lethal temperatures during winter for lizards that have not yet found suitable refugia. CT and also decreased after cold acclimation, while EWL, RMR, and RER increased after cold acclimation, suggesting trade-offs with cold acclimation in the form of decreased heat tolerance and increased energy demands. Taken together, our findings suggest that cold tolerance plasticity aids the persistence of an established population of invasive lizards. More generally, our findings highlight the importance of accounting for the plasticity of physiological traits when investigating how invasive species respond to novel environments.

AbstractEarly developmental environments can shape how organisms respond to later environments, but despite the potential for this phenomenon to alter the evolution of phenotypes and their underlying mechanisms in variable environments, details of this process are not understood. For example, both temperature and parental age can alter offspring metabolic plasticity and growth within species, yet the extent of such effects is unknown. We measured the reaction norms of embryonic heart rate in response to egg temperature and the change in egg mass over the incubation period in wild house sparrows. Using Bayesian linear mixed models, we estimated covariation in the intercepts and slopes of these reaction norms among clutches and eggs. We found that heart rate intercepts, not slopes, varied among clutches and that neither intercepts nor slopes varied among eggs within clutches. In contrast, egg mass intercepts and slopes varied among clutches and eggs. Ambient temperature did not explain variance in reaction norms. Instead, individuals from older mothers were more metabolically sensitive to egg temperature and lost less mass over the incubation period than individuals from younger mothers. Nevertheless, heart rate reaction norms and egg mass reaction norms did not covary. Our results suggest that early environments influenced by parents may contribute to variation in embryonic reaction norms. The structure of variation in embryonic reaction norms that exists both among clutches and among eggs demonstrates a complexity in plastic phenotypes that should be explored in future work. Furthermore, the potential for the embryonic environment to shape the reaction norms of other traits has implications for the evolution of plasticity more broadly.

AbstractReduced energy intake can compromise the ability of a mammal to maintain body temperature within a narrow 24-h range, leading to heterothermy. To investigate the main drivers of heterothermy in a bulk grazer, we compared abdominal temperature, body mass, body condition index, and serum leptin levels in 11 subadult Cape buffalo () during a drought year and a nondrought year. Low food availability during the drought year (as indexed by grass biomass, satellite imagery of vegetation greenness, and fecal chlorophyll) resulted in lower body condition index, lower body mass relative to that expected for an equivalent-aged buffalo, and lower leptin levels. The range of 24-h body temperature rhythm was 2°C during the nondrought year and more than double that during the drought year, and this was caused primarily by a lower minimum 24-h body temperature rhythm during the cool dry winter months. After rain fell and vegetation greenness increased, the minimum 24-h body temperature rhythm increased, and the range of 24-h body temperature rhythm was smaller than 2°C. In order of importance, poor body condition, low minimum 24-h air temperature, and low serum leptin levels were the best predictors of the increase in the range of 24-h body temperature rhythm. While the thermoregulatory role of leptin is not fully understood, the association between range of 24-h body temperature rhythm and serum leptin levels provides clues about the underlying mechanism behind the increased heterothermy in large mammals facing food restriction.

AbstractMany attempts at providing a single-scale exponent and mechanism to explain metabolic rate assert a monolithic selective mechanism for allometries, characterized by a universal allometric scale power (usually chosen to be 0.75). To test for the deviations from universal allometric scaling, we gathered data from previously published metabolic measurements on 903 bird species and performed regressions of log(basal metabolic rate) and log(body mass) for (1) all birds and (2) 20 monophyletic clades within birds. We constructed two Bayesian linear mixed models-one included ecological variables and the other included data for mammals from Sieg et al. (2009). Overall allometric patterns differed significantly among clades of birds, and some clades were not consistent with the 0.75 scale power. We were unable to find apparent physiological, morphological, phylogenetic, or ecological characteristics among clades, predicting a difference in allometry or consistency with any previously proposed universal allometry. The Bayesian analysis illuminated novel bivariate, clade-specific differences in scaling slope-intercept space, separating large groups of birds and mammals. While significantly related to basal metabolic rate, feeding guild and migratory tendency had small effects compared to clade and body mass. We propose that allometric hypotheses, in general, must extend beyond simple overarching mechanisms to allow for conflicting and interacting influences that produce allometric patterns at narrower taxonomic scales-perhaps including other processes whose optimization may interfere with that of the system proposed by the metabolic theory of ecology.

AbstractHow do large and small reptiles defend against infections, given the consequences of body mass for physiology and disease transmission? Functionally equivalent mammalian and avian granulocytes increased disproportionately with body mass (i.e., scaled hypermetrically), such that large organisms had higher concentrations than expected by a prediction of proportional protection across sizes. However, as these scaling relationships were derived from endothermic animals, they do not necessarily inform the scaling of leukocyte concentration for ectothermic reptiles that have a different physiology and evolutionary history. Here, we asked whether and how lymphocyte and heterophil concentrations relate to body mass among more than 120 reptile species. We compared these relationships to those found in birds and mammals and to existing scaling frameworks (i.e., protecton, complexity, rate of metabolism, or safety factor hypotheses). Both lymphocyte and heterophil concentrations scaled almost isometrically among reptiles. In contrast, functionally equivalent granulocytes scaled hypermetrically and lymphocytes scaled isometrically in birds and mammals. Life history traits were also poor predictors of variation in reptilian heterophil and lymphocyte concentrations. Our results provide insight into differences in immune protection in birds and mammals relative to that in reptiles through a comparative lens. The shape of scaling relationships differs, which should be considered when modeling disease dynamics among these groups.