SUMMARY A major challenge in biology is to understand the genetic basis of adaptation. One compelling idea is that groups of tightly linked genes (i.e. ‘supergenes’ [1, 2]) facilitate adaptation in suites of traits that determine fitness. Despite their likely importance, little is known about how alternate supergene alleles arise and become differentiated, nor their ultimate fate within species. Herein we address these questions by investigating the evolutionary history of a supergene in white-throated sparrows, Zonotrichia albicollis. This species comprises two morphs, tan and white, that differ in pigmentation and components of social behavior [3–5]. Morph is determined by alternative alleles at a balanced >100Mb inversion-based supergene, providing a unique system for studying gene-behavior relationships. Using over two decades of field data we document near-perfect disassortative mating among morphs, as well as the fitness consequences of rare assortative mating. We use de novo whole genome sequencing coupled with population- and phylo-genomic data, to show that alternate supergene alleles are highly divergent at over 1,000 genes, that these alleles originated prior to the split of Z. albicollis from its sister species, and may be polymorphic in Z. albicollis due to a past hybridization event. We provide evidence that the ‘white' allele may be degrading, similar to neo-Y/Wsex chromosomes. We further show that the ‘tan’ allele has surprisingly low levels of genetic diversity, yet does not show several canonical signatures of recurrent positive selection. We discuss these results in the context of the origin, molecular evolution, and possible fate of this remarkable polymorphism.

SummaryHigh-altitude environments provide ideal testing grounds for investigations of mechanism and process in physiological adaptation. In vertebrates, much of our understanding of the acclimatization response to high-altitude hypoxia derives from studies of animal species that are native to lowland environments. Such studies can indicate whether phenotypic plasticity will generally facilitate or impede adaptation to high altitude. Here, we review general mechanisms of physiological acclimatization and genetic adaptation to high-altitude hypoxia in birds and mammals. We evaluate whether the acclimatization response to environmental hypoxia can be regarded generally as a mechanism of adaptive phenotypic plasticity, or whether it might sometimes represent a misdirected response that acts as a hindrance to genetic adaptation. In cases in which the acclimatization response to hypoxia is maladaptive, selection will favor an attenuation of the induced phenotypic change. This can result in a form of cryptic adaptive evolution in which phenotypic similarity between high-and low-altitude populations is attributable to directional selection on genetically based trait variation that offsets environmentally induced changes. The blunted erythropoietic and pulmonary vasoconstriction responses to hypoxia in Tibetan humans and numerous high-altitude birds and mammals provide possible examples of this phenomenon. When lowland animals colonize high-altitude environments, adaptive phenotypic plasticity can mitigate the costs of selection, thereby enhancing prospects for population establishment and persistence. By contrast, maladaptive plasticity has the opposite effect. Thus, insights into the acclimatization response of lowland animals to high-altitude hypoxia can provide a basis for predicting how altitudinal range limits might shift in response to climate change.

In response to hypoxic stress, many animals compensate for a reduced cellular O 2 supply by suppressing total metabolism, thereby reducing O 2 demand. For small endotherms that are native to high-altitude environments, this is not always a viable strategy, as the capacity for sustained aerobic thermogenesis is critical for survival during periods of prolonged cold stress. For example, survivorship studies of deer mice (Peromyscus maniculatus) have demonstrated that thermogenic capacity is under strong directional selection at high altitude. Here, we integrate measures of whole-organism thermogenic performance with measures of metabolic enzyme activities and genomic transcriptional profiles to examine the mechanistic underpinnings of adaptive variation in this complex trait in deer mice that are native to different elevations. We demonstrate that highland deer mice have an enhanced thermogenic capacity under hypoxia compared with lowland conspecifics and a closely related lowland species, Peromyscus leucopus. Our findings suggest that the enhanced thermogenic performance of highland deer mice is largely attributable to an increased capacity to oxidize lipids as a primary metabolic fuel source. This enhanced capacity for aerobic thermogenesis is associated with elevated activities of muscle metabolic enzymes that influence flux through fatty-acid oxidation and oxidative phosphorylation pathways in high-altitude deer mice and by concomitant changes in the expression of genes in these same pathways. Contrary to predictions derived from studies of humans at high altitude, our results suggest that selection to sustain prolonged thermogenesis under hypoxia promotes a shift in metabolic fuel use in favor of lipids over carbohydrates.functional genomics | RNA-seq | thermoregulation | transcriptomics D uring cold stress, homeothermic endotherms maintain a constant body temperature by increasing metabolic heat production. In small endotherms like mice that have high thermoregulatory demands, thermogenic capacity influences survival in cold environments and therefore has a clear connection to Darwinian fitness (1, 2). Indeed, thermogenic capacity in freeranging deer mice (Peromyscus maniculatus) is subject to strong directional selection at high altitude (3).Sustaining maximal thermogenic capacities during prolonged periods of cold stress requires a high rate of O 2 flux through oxidative pathways, and this requirement presents a unique challenge for endothermic animals that live under conditions of chronic O 2 deprivation at high altitude. The reduced partial pressure of O 2 (PO 2 ) at high altitude imposes well-documented constraints on aerobic metabolism (4-8), thereby exacerbating the increased thermoregulatory demands faced by endothermic animals that are native to cold, alpine environments.In rodents, aerobic thermogenesis is accomplished through both shivering and nonshivering mechanisms, and in deer mice, shivering accounts for roughly 35-50% of total thermogenic capacity (9). As with other forms of strenuous exerci...

The hypoxic and cold environment at high altitudes requires that small mammals sustain high rates of O2 transport for exercise and thermogenesis while facing a diminished O2 availability. We used laboratory-born and -raised deer mice (Peromyscus maniculatus) from highland and lowland populations to determine the interactive effects of ancestry and hypoxia acclimation on exercise performance. Maximal O₂consumption (V̇o(2max)) during exercise in hypoxia increased after hypoxia acclimation (equivalent to the hypoxia at ∼4,300 m elevation for 6-8 wk) and was consistently greater in highlanders than in lowlanders. V̇o(2max) during exercise in normoxia was not affected by ancestry or acclimation. Highlanders also had consistently greater capillarity, oxidative fiber density, and maximal activities of oxidative enzymes (cytochrome c oxidase and citrate synthase) in the gastrocnemius muscle, lower lactate dehydrogenase activity in the gastrocnemius, and greater cytochrome c oxidase activity in the diaphragm. Hypoxia acclimation did not affect any of these muscle traits. The unique gastrocnemius phenotype of highlanders was associated with higher mRNA and protein abundances of peroxisome proliferator-activated receptor γ (PPARγ). Vascular endothelial growth factor (VEGFA) transcript abundance was lower in highlanders, and hypoxia acclimation reduced the expression of numerous genes that regulate angiogenesis and energy metabolism, in contrast to the observed population differences in muscle phenotype. Lowlanders exhibited greater increases in blood hemoglobin content, hematocrit, and wet lung mass (but not dry lung mass) than highlanders after hypoxia acclimation. Genotypic adaptation to high altitude, therefore, improves exercise performance in hypoxia by mechanisms that are at least partially distinct from those underlying hypoxia acclimation.

Extreme environmental perturbations offer opportunities to observe the effects of natural selection in wild populations. During the winter of 2013-2014, the southeastern United States endured an extreme cold event. We used thermal performance, transcriptomics, and genome scans to measure responses of lizard populations to storm-induced selection. We found significant increases in cold tolerance at the species' southern limit. Gene expression in southern survivors shifted toward patterns characteristic of northern populations. Comparing samples before and after the extreme winter, 14 genomic regions were differentiated in the surviving southern population; four also exhibited signatures of local adaptation across the latitudinal gradient and implicate genes involved in nervous system function. Together, our results suggest that extreme winter events can rapidly produce strong selection on natural populations at multiple biological levels that recapitulate geographic patterns of local adaptation.