Equivocal support for the climate variability hypothesis within a Neotropical bird assemblage.

The climate variability hypothesis posits that an organism's exposure to temperature variability determines the breadth of its thermal tolerance and has become an important framework for understanding variation in species' susceptibilities to climate change. For example, ectotherms from more thermally stable environments tend to have narrower thermal tolerances and greater sensitivity to projected climate warming. Among endotherms, however, the relationship between climate variability and thermal physiology is less clear, particularly with regard to microclimate variation-small-scale differences within or between habitats. To address this gap, we explored associations between two sources of temperature variation (habitat type and vertical forest stratum) and (1) thermal physiological traits and (2) temperature sensitivity metrics within a diverse assemblage of Neotropical birds (n = 89 species). We used long-term temperature data to establish that daily temperature regimes in open habitats and forest canopy were both hotter and more variable than those in the forest interior and forest understory, respectively. Despite these differences in temperature regime, however, we found little evidence that species' thermal physiological traits or temperature sensitivity varied in association with either habitat type or vertical stratum. Our findings provide two novel and important insights. First, and in contrast to the supporting empirical evidence from ectotherms, the thermal physiology of birds at our study site appears to be largely decoupled from local temperature variation, providing equivocal support for the climate variability hypothesis in endotherms. Second, we found no evidence that the thermal physiology of understory forest birds differed from that of canopy or open-habitat species-an oft-invoked, yet previously untested, mechanism for why these species are so vulnerable to environmental change.

Thermal acclimation in a non-migratory songbird occurs via changes to thermogenic capacity, but not conductance.

Thermoregulatory performance can be modified through changes in various subordinate traits, but the rate and magnitude of change in these traits is poorly understood. We investigated flexibility in traits that affect thermal balance between black-capped chickadees (Poecile atricapillus) acclimated for 6 weeks to cold (-5°C) or control (23°C) environments (n=7 per treatment). We made repeated measurements of basal and summit metabolic rates via flow-through respirometry and of body composition using quantitative magnetic resonance of live birds. At the end of the acclimation period, we measured thermal conductance of the combined feathers and skins. Cold-acclimated birds had a higher summit metabolic rate, reflecting a greater capacity for endogenous heat generation, and an increased lean mass. However, birds did not alter their thermal conductance. These results suggest that chickadees respond to cold stress by increasing their capacity for heat production rather than increasing heat retention, an energetically expensive strategy.

Adaptive structural and functional evolution of the placenta protects fetal growth in high-elevation deer mice.

Environmental hypoxia challenges female reproductive physiology in placental mammals, increasing rates of gestational complications. Adaptation to high elevation has limited many of these effects in humans and other mammals, offering potential insight into the developmental processes that lead to and protect against hypoxia-related gestational complications. However, our understanding of these adaptations has been hampered by a lack of experimental work linking the functional, regulatory, and genetic underpinnings of gestational development in locally adapted populations. Here, we dissect high-elevation adaptation in the reproductive physiology of deer mice (Peromyscus maniculatus), a rodent species with an exceptionally broad elevational distribution that has emerged as a model for hypoxia adaptation. Using experimental acclimations, we show that lowland mice experience pronounced fetal growth restriction when challenged with gestational hypoxia, while highland mice maintain normal growth by expanding the compartment of the placenta that facilitates nutrient and gas exchange between gestational parent and fetus. We then use compartment-specific transcriptome analyses to show that adaptive structural remodeling of the placenta is coincident with widespread changes in gene expression within this same compartment. Genes associated with fetal growth in deer mice significantly overlap with genes involved in human placental development, pointing to conserved or convergent pathways underlying these processes. Finally, we overlay our results with genetic data from natural populations to identify candidate genes and genomic features that contribute to these placental adaptations. Collectively, these experiments advance our understanding of adaptation to hypoxic environments by revealing physiological and genetic mechanisms that shape fetal growth trajectories under maternal hypoxia.

Gene regulatory changes underlie developmental plasticity in respiration and aerobic performance in highland deer mice.

Phenotypic plasticity can play an important role in the ability of animals to tolerate environmental stress, but the nature and magnitude of plastic responses are often specific to the developmental timing of exposure. Here, we examine changes in gene expression in the diaphragm of highland deer mice (Peromyscus maniculatus) in response to hypoxia exposure at different stages of development. In highland deer mice, developmental plasticity in diaphragm function may mediate changes in several respiratory traits that influence aerobic metabolism and performance under hypoxia. We generated RNAseq data from diaphragm tissue of adult deer mice exposed to (1) life-long hypoxia (before conception to adulthood), (2) post-natal hypoxia (birth to adulthood), (3) adult hypoxia (6-8 weeks only during adulthood) or (4) normoxia. We found five suites of co-regulated genes that are differentially expressed in response to hypoxia, but the patterns of differential expression depend on the developmental timing of exposure. We also identified four transcriptional modules that are associated with important respiratory traits. Many of the genes in these transcriptional modules bear signatures of altitude-related selection, providing an indirect line of evidence that observed changes in gene expression may be adaptive in hypoxic environments. Our results demonstrate the importance of developmental stage in determining the phenotypic response to environmental stressors.

Widespread Torpor Use in Hummingbirds from the Thermally Stable Lowland Tropics.

AbstractTorpor, the temporary reduction of metabolic rate and body temperature, is a common energy-saving strategy in endotherms. Because of their small body size and energetically demanding life histories, hummingbirds have proven useful for understanding when and why endotherms use torpor. Previous studies of torpor in hummingbirds have been largely limited to tropical montane species or long-distance migrants that regularly experience challenging thermal conditions. Comparatively little is known, however, about the use of torpor in hummingbirds of the lowland tropics, where relatively high and stable year-round temperatures may at least partially negate the need for torpor. To fill this knowledge gap, we tested for the occurrence of torpor in tropical lowland hummingbirds (n=37 individuals of six species) from central Panama. In controlled experimental conditions simulating the local temperature regime, all six species used torpor to varying degrees and entered torpor at high ambient temperatures (i.e., ≥28°C), indicating that hummingbirds from the thermally stable lowland tropics regularly use torpor. Torpor reduced overnight mass loss, with individuals that spent more time in torpor losing less body mass during temperature experiments. Body mass was the best predictor of torpor depth and duration among and within species-smaller species and individuals tended to use torpor more frequently and enter deeper torpor. Average mass loss in our experiments (∼8%-10%) was greater than that reported in studies of hummingbirds from higher elevation sites (∼4%). We therefore posit that the energetic benefits accrued from torpor may be limited by relatively high nighttime temperatures in the lowland tropics, although further studies are needed to test this hypothesis.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

Genetic variation in HIF-2α attenuates ventilatory sensitivity and carotid body growth in chronic hypoxia in high-altitude deer mice.

The gene encoding HIF-2α, Epas1, has experienced a history of natural selection in many high-altitude taxa, but the functional role of mutations in this gene is still poorly understood. We investigated the influence of the high-altitude variant of Epas1 in North American deer mice (Peromyscus maniculatus) on the control of breathing and carotid body growth during chronic hypoxia. We created hybrids between high- and low-altitude populations of deer mice to disrupt linkages between genetic loci so that the physiological effects of Epas1 alleles (Epas1H and Epas1L , respectively) could be examined on an admixed genomic background. In general, chronic hypoxia (4 weeks at 12 kPa O2 ) enhanced ventilatory chemosensitivity (assessed as the acute ventilatory response to hypoxia), increased total ventilation and arterial O2 saturation during progressive poikilocapnic hypoxia, and increased haematocrit and blood haemoglobin content across genotypes. However, the effects of chronic hypoxia on ventilatory chemosensitivity were attenuated in mice that were homozygous for the high-altitude Epas1 allele (Epas1H/H ). Carotid body growth and glomus cell hyperplasia, which was strongly induced in Epas1L/L mice in chronic hypoxia, was not observed in Epas1H/H mice. Epas1 genotype also modulated the effects of chronic hypoxia on metabolism and body temperature depression in hypoxia, but had no effects on haematological traits. These findings confirm the important role of HIF-2α in modulating ventilatory sensitivity and carotid body growth in chronic hypoxia, and show that genetic variation in Epas1 is responsible for evolved changes in the control of breathing and metabolism in high-altitude deer mice. KEY POINTS: High-altitude natives of many species have experienced natural selection on the gene encoding HIF-2α, Epas1, including high-altitude populations of deer mice. HIF-2α regulates ventilation and carotid body growth in hypoxia, and so the genetic variants in Epas1 in high-altitude natives may underlie evolved changes in control of breathing. Deer mice from controlled crosses between high- and low-altitude populations were used to examine the effects of Epas1 genotype on an admixed genomic background. The high-altitude variant was associated with reduced ventilatory chemosensitivity and carotid body growth in chronic hypoxia, but had no effects on haematology. The results help us better understand the genetic basis for the unique physiological phenotype of high-altitude natives.

Genetic variation in haemoglobin is associated with evolved changes in breathing in high-altitude deer mice.

Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated whether genetic variants in haemoglobin (Hb) that contribute to high-altitude adaptation in deer mice (Peromyscus maniculatus) are associated with evolved changes in the control of breathing. We created F2 inter-population hybrids of highland and lowland deer mice to test for phenotypic associations of α- and ß-globin variants on a mixed genetic background. Hb genotype had expected effects on Hb-O2 affinity that were associated with differences in arterial O2 saturation in hypoxia. However, high-altitude genotypes were also associated with breathing phenotypes that should contribute to enhancing O2 uptake in hypoxia. Mice with highland α-globin exhibited a more effective breathing pattern, with highland homozygotes breathing deeper but less frequently across a range of inspired O2, and this difference was comparable to the evolved changes in breathing pattern in deer mouse populations native to high altitude. The ventilatory response to hypoxia was augmented in mice that were homozygous for highland ß-globin. The association of globin variants with variation in breathing phenotypes could not be recapitulated by acute manipulation of Hb-O2 affinity, because treatment with efaproxiral (a synthetic drug that acutely reduces Hb-O2 affinity) had no effect on breathing in normoxia or hypoxia. Therefore, adaptive variation in Hb may have unexpected effects on physiology in addition to the canonical function of this protein in circulatory O2 transport.

Temperature heterogeneity correlates with intraspecific variation in physiological flexibility in a small endotherm.

Phenotypic flexibility allows individuals to reversibly modify trait values and theory predicts an individual's relative degree of flexibility positively correlates with the environmental heterogeneity it experiences. We test this prediction by integrating surveys of population genetic and physiological variation with thermal acclimation experiments and indices of environmental heterogeneity in the Dark-eyed Junco (Junco hyemalis) and its congeners. We combine field measures of thermogenic capacity for 335 individuals, 22,006 single nucleotide polymorphisms genotyped in 181 individuals, and laboratory acclimations replicated on five populations. We show that Junco populations: (1) differ in their thermogenic responses to temperature variation in the field; (2) harbor allelic variation that also correlates with temperature heterogeneity; and (3) exhibit intra-specific variation in thermogenic flexibility in the laboratory that correlates with the heterogeneity of their native thermal environment. These results provide comprehensive support that phenotypic flexibility corresponds with environmental heterogeneity and highlight its importance for coping with environmental change.

Fetal growth, high altitude, and evolutionary adaptation: a new perspective.

Residence at high altitude is consistently associated with low birthweight among placental mammals. This reduction in birthweight influences long-term health trajectories for both the offspring and mother. However, the physiological processes that contribute to fetal growth restriction at altitude are still poorly understood, and thus our ability to safely intervene remains limited. One approach to identify the factors that mitigate altitude-dependent fetal growth restriction is to study populations that are protected from fetal growth restriction through evolutionary adaptations (e.g., high altitude-adapted populations). Here, we examine human gestational physiology at high altitude from a novel evolutionary perspective that focuses on patterns of physiological plasticity, allowing us to identify 1) the contribution of specific physiological systems to fetal growth restriction and 2) the mechanisms that confer protection in highland-adapted populations. Using this perspective, our review highlights two general findings: first, that the beneficial value of plasticity in maternal physiology is often dependent on factors more proximate to the fetus; and second, that our ability to understand the contributions of these proximate factors is currently limited by thin data from altitude-adapted populations. Expanding the comparative scope of studies on gestational physiology at high altitude and integrating studies of both maternal and fetal physiology are needed to clarify the mechanisms by which physiological responses to altitude contribute to fetal growth outcomes. The relevance of these questions to clinical, agricultural, and basic research combined with the breadth of the unknown highlight gestational physiology at high altitude as an exciting niche for continued work.

The adaptive benefit of evolved increases in hemoglobin-O2 affinity is contingent on tissue O2 diffusing capacity in high-altitude deer mice.

BACKGROUND: Complex organismal traits are often the result of multiple interacting genes and sub-organismal phenotypes, but how these interactions shape the evolutionary trajectories of adaptive traits is poorly understood. We examined how functional interactions between cardiorespiratory traits contribute to adaptive increases in the capacity for aerobic thermogenesis (maximal O2 consumption, V̇O2max, during acute cold exposure) in high-altitude deer mice (Peromyscus maniculatus). We crossed highland and lowland deer mice to produce F2 inter-population hybrids, which expressed genetically based variation in hemoglobin (Hb) O2 affinity on a mixed genetic background. We then combined physiological experiments and mathematical modeling of the O2 transport pathway to examine the links between cardiorespiratory traits and V̇O2max. RESULTS: Physiological experiments revealed that increases in Hb-O2 affinity of red blood cells improved blood oxygenation in hypoxia but were not associated with an enhancement in V̇O2max. Sensitivity analyses performed using mathematical modeling showed that the influence of Hb-O2 affinity on V̇O2max in hypoxia was contingent on the capacity for O2 diffusion in active tissues. CONCLUSIONS: These results suggest that increases in Hb-O2 affinity would only have adaptive value in hypoxic conditions if concurrent with or preceded by increases in tissue O2 diffusing capacity. In high-altitude deer mice, the adaptive benefit of increasing Hb-O2 affinity is contingent on the capacity to extract O2 from the blood, which helps resolve controversies about the general role of hemoglobin function in hypoxia tolerance.

Broad Concordance in the Spatial Distribution of Adaptive and Neutral Genetic Variation across an Elevational Gradient in Deer Mice.

When species are continuously distributed across environmental gradients, the relative strength of selection and gene flow shape spatial patterns of genetic variation, potentially leading to variable levels of differentiation across loci. Determining whether adaptive genetic variation tends to be structured differently than neutral variation along environmental gradients is an open and important question in evolutionary genetics. We performed exome-wide population genomic analysis on deer mice sampled along an elevational gradient of nearly 4,000 m of vertical relief. Using a combination of selection scans, genotype-environment associations, and geographic cline analyses, we found that a large proportion of the exome has experienced a history of altitude-related selection. Elevational clines for nearly 30% of these putatively adaptive loci were shifted significantly up- or downslope of clines for loci that did not bear similar signatures of selection. Many of these selection targets can be plausibly linked to known phenotypic differences between highland and lowland deer mice, although the vast majority of these candidates have not been reported in other studies of highland taxa. Together, these results suggest new hypotheses about the genetic basis of physiological adaptation to high altitude, and the spatial distribution of adaptive genetic variation along environmental gradients.

X chromosome-dependent disruption of placental regulatory networks in hybrid dwarf hamsters.

Embryonic development in mammals is highly sensitive to changes in gene expression within the placenta. The placenta is also highly enriched for genes showing parent-of-origin or imprinted expression, which is predicted to evolve rapidly in response to parental conflict. However, little is known about the evolution of placental gene expression, or if divergence of placental gene expression plays an important role in mammalian speciation. We used crosses between two species of dwarf hamsters (Phodopus sungorus and Phodopus campbelli) to examine the genetic and regulatory underpinnings of severe placental overgrowth in their hybrids. Using quantitative genetic mapping and mitochondrial substitution lines, we show that overgrowth of hybrid placentas was primarily caused by genetic differences on the maternally inherited P. sungorus X chromosome. Mitochondrial interactions did not contribute to abnormal hybrid placental development, and there was only weak correspondence between placental disruption and embryonic growth. Genome-wide analyses of placental transcriptomes from the parental species and first- and second-generation hybrids revealed a central group of co-expressed X-linked and autosomal genes that were highly enriched for maternally biased expression. Expression of this gene network was strongly correlated with placental size and showed widespread misexpression dependent on epistatic interactions with X-linked hybrid incompatibilities. Collectively, our results indicate that the X chromosome is likely to play a prominent role in the evolution of placental gene expression and the accumulation of hybrid developmental barriers between mammalian species.

Physiological Genomics of Adaptation to High-Altitude Hypoxia.

Population genomic studies of humans and other animals at high altitude have generated many hypotheses about the genes and pathways that may have contributed to hypoxia adaptation. Future advances require experimental tests of such hypotheses to identify causal mechanisms. Studies to date illustrate the challenge of moving from lists of candidate genes to the identification of phenotypic targets of selection, as it can be difficult to determine whether observed genotype-phenotype associations reflect causal effects or secondary consequences of changes in other traits that are linked via homeostatic regulation. Recent work on high-altitude models such as deer mice has revealed both plastic and evolved changes in respiratory, cardiovascular, and metabolic traits that contribute to aerobic performance capacity in hypoxia, and analyses of tissue-specific transcriptomes have identified changes in regulatory networks that mediate adaptive changes in physiological phenotype. Here we synthesize recent results and discuss lessons learned from studies of high-altitude adaptation that lie at the intersection of genomics and physiology.

High-altitude rodents have abundant collaterals that protect against tissue injury after cerebral, coronary and peripheral artery occlusion.

Collateral number/density varies widely in brain and other tissues among strains of Mus musculus mice due to differences in genetic background. Recent studies have shown that prolonged exposure to reduced atmospheric oxygen induces additional collaterals to form, suggesting that natural selection may favor increased collaterals in populations native to high-altitude. High-altitude guinea pigs (Cavia) and deer mice (Peromyscus) were compared with lowland species of Peromyscus, Mus and Rattus (9 species/strains examined). Collateral density, diameter and other morphometrics were measured in brain where, importantly, collateral abundance reflects that in other tissues of the same individual. Guinea pigs and high-altitude deer mice had a greater density of pial collaterals than lowlanders. Consistent with this, guinea pigs and highlander mice evidenced complete and 80% protection against stroke, respectively. They also sustained significantly less ischemia in heart and lower extremities after arterial occlusion. Vessels of the circle of Willis, including the communicating collateral arteries, also exhibited unique features in the highland species. Our findings support the hypothesis that species native to high-altitude have undergone genetic selection for abundant collaterals, suggesting that besides providing protection in obstructive disease, collaterals serve a physiological function to optimize oxygen delivery to meet oxygen demand when oxygen is limiting.

Chronic cold exposure induces mitochondrial plasticity in deer mice native to high altitudes.

KEY POINTS: Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in cold hypoxic environments remains unresolved. We examined high-altitude deer mice, which have evolved a high capacity for aerobic thermogenesis, to determine the mechanisms of mitochondrial plasticity during chronic exposure to cold and hypoxia, alone and in combination. Cold exposure in normoxia or hypoxia increased mitochondrial leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency, which may serve to augment non-shivering thermogenesis. Cold also increased muscle oxidative capacity, but reduced the capacity for mitochondrial respiration via complex II relative to complexes I and II combined. High-altitude mice had a more oxidative muscle phenotype than low-altitude mice. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitude. ABSTRACT: Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold and hypoxic environments. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in small mammals at high altitude remains unresolved. High-altitude deer mice (Peromyscus maniculatus) and low-altitude white-footed mice (P. leucopus) were born and raised in captivity, and chronically exposed as adults to warm (25°C) normoxia, warm hypoxia (12 kPa O2 ), cold (5°C) normoxia, or cold hypoxia. We then measured oxidative enzyme activities, oxidative fibre density and capillarity in the gastrocnemius, and used a comprehensive substrate titration protocol to examine the function of muscle mitochondria by high-resolution respirometry. Exposure to cold in both normoxia or hypoxia increased the activities of citrate synthase and cytochrome oxidase. In lowlanders, this was associated with increases in capillary density and the proportional abundance of oxidative muscle fibres, but in highlanders, these traits were unchanged at high levels across environments. Environment had some distinct effects on mitochondrial OXPHOS capacity between species, but the capacity of complex II relative to the combined capacity of complexes I and II was consistently reduced in both cold environments. Both cold environments also increased leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency in both species, which may serve to augment non-shivering thermogenesis. These cold-induced changes in mitochondrial function were overlaid upon the generally more oxidative phenotype of highlanders. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitudes.

Is there a role for sarcolipin in avian facultative thermogenesis in extreme cold?

Endotherms defend their body temperature in the cold by employing shivering (ST) and/or non-shivering thermogenesis (NST). Although NST is well documented in mammals, its importance to avian heat generation is unclear. Recent work points to a prominent role for the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in muscular NST. SERCA's involvement in both ST and NST, however, posits a tradeoff between these two heat-generating mechanisms. To explore this tradeoff, we assayed pectoralis gene expression of adult songbirds exposed to chronic temperature acclimations. Counter to mammal models, we found that cold-acclimated birds downregulated the expression of sarcolipin (SLN), a gene coding for a peptide that promotes heat generation by uncoupling SERCA Ca2+ transport from ATP hydrolysis, indicating a reduced potential for muscular NST. We also found differential expression of many genes involved in Ca2+ cycling and muscle contraction and propose that decreased SLN could promote increased pectoralis contractility for ST. Moreover, SLN transcript abundance negatively correlated with peak oxygen consumption under cold exposure (a proxy for ST) across individuals, and higher SLN transcript abundance escalated an individual's risk of hypothermia in acute cold. Our results therefore suggest that SLN-mediated NST may not be an important mechanism of-and could be a hindrance to-avian thermoregulation in extreme cold.

Coordinated changes across the O2 transport pathway underlie adaptive increases in thermogenic capacity in high-altitude deer mice.

Animals native to the hypoxic and cold environment at high altitude provide an excellent opportunity to elucidate the integrative mechanisms underlying the adaptive evolution and plasticity of complex traits. The capacity for aerobic thermogenesis can be a critical determinant of survival for small mammals at high altitude, but the physiological mechanisms underlying the evolution of this performance trait remain unresolved. We examined this issue by comparing high-altitude deer mice (Peromyscus maniculatus) with low-altitude deer mice and white-footed mice (P. leucopus). Mice were bred in captivity and adults were acclimated to each of four treatments: warm (25°C) normoxia, warm hypoxia (12 kPa O2), cold (5°C) normoxia or cold hypoxia. Acclimation to hypoxia and/or cold increased thermogenic capacity in deer mice, but hypoxia acclimation led to much greater increases in thermogenic capacity in highlanders than in lowlanders. The high thermogenic capacity of highlanders was associated with increases in pulmonary O2 extraction, arterial O2 saturation, cardiac output and arterial-venous O2 difference. Mechanisms underlying the evolution of enhanced thermogenic capacity in highlanders were partially distinct from those underlying the ancestral acclimation responses of lowlanders. Environmental adaptation has thus enhanced phenotypic plasticity and expanded the physiological toolkit for coping with the challenges at high altitude.

Body temperature maintenance acclimates in a winter-tenacious songbird.

Flexibility in heat generation and dissipation mechanisms provides endotherms the ability to match their thermoregulatory strategy with external demands. However, the degree to which these two mechanisms account for seasonal changes in body temperature regulation is little explored. Here, we present novel data on the regulation of avian body temperature to investigate how birds alter mechanisms of heat production and heat conservation to deal with variation in ambient conditions. We subjected dark-eyed juncos (Junco hyemalis) to chronic cold acclimations of varying duration and subsequently quantified their metabolic rates, thermal conductance and ability to maintain normothermia. Cold-acclimated birds adjusted traits related to both heat generation (increased summit metabolic rate) and heat conservation (decreased conductance) to improve their body temperature regulation. Increases in summit metabolic rate occurred rapidly, but plateaued after 1 week of cold exposure. In contrast, changes to conductance occurred only after 9 weeks of cold exposure. Thus, the ability to maintain body temperature continued to improve throughout the experiment, but the mechanisms underlying this improvement changed through time. Our results demonstrate the ability of birds to adjust thermoregulatory strategies in response to thermal cues and reveal that birds may combine multiple responses to meet the specific demands of their environments.