Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose

Abstract

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of function-altering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function.

First de novo whole genome sequencing and assembly of the bar-headed goose

“…Bar-headed goose, a species occupying a wide variety of habitats on the Qinghai-Tibetan Plateau, is considered a good model for studying highaltitude adaptation in birds. Much of what is known about the mechanisms of highaltitude adaptation in bar-headed geese comes from studies that have taken physiological, biochemical, and morphological methods (Snyder, Byers & Kayar, 1984;Scott & Milsom, 2007;Scott et al, 2009;Butler, 2010;McCracken, Barger & Sorenson, 2010;Scott et al, 2015;Natarajan et al, 2018). An understanding of the genetic basis of this adaptation, however, has lagged behind due to the unavailability of the bar-headed goose genome.…”

“…Many studies have sought to determine the physiological, molecular and behavioral basis for the successful adaptation of bar-headed geese to high-altitude flying and living (Butler, 2010;Scott et al, 2015). For example, bar-headed geese have evolved multiple mechanisms that enhance the uptake, circulation and peripheral diffusion of oxygen during hypoxia, including the increased lung mass and total ventilation (Scott & Milsom, 2007), hemoglobin with an increased oxygen affinity because of a single amino acid point mutation in the alpha polypeptide chain (McCracken, Barger & Sorenson, 2010;Natarajan et al, 2018), greater capillary density in flight and cardiac muscles increasing oxygen supply (Scott et al, 2009), and a higher proportion of mitochondria in a subsarcolemmal location reducing oxygen diffusion distances (Snyder, Byers & Kayar, 1984). In addition, bar-headed geese were found to take a roller coaster strategy, rising and falling with the underlying terrain, to conserve energy during Himalayan migrations (Bishop et al, 2015).…”

UNVEILing connections between genotype, phenotype, and fitness in natural populations

“…The genotype-phenotype map is fundamentally a description of the actions and interactions of biomolecules. Perhaps nowhere is this more evident than in the study of amino acid polymorphisms and substitutions in proteins (Bridgham, Ortlund, & Thornton, 2009;Harms & Thornton, 2013;Natarajan et al, 2018;Storz, Natarajan, Cheviron, Hoffmann, & Kelly, 2012). Changes to protein sequence impact structure and function in complex and often unpredictable ways, and epistasis among substitutions has become a central theme of protein evolution.…”

“…Speakers at the UNVEIL Symposium applied these tools to studies of adaptation in the wild, demonstrating that the functional effects of mutation depends upon the genetic background and thus the evolutionary context under which they arose ( Figure 1). Jay Storz (University of Nebraska, Lincoln) used ancestral protein resurrection to understand the mutational pathways of biochemical adaptation in haemoglobin of bar-headed geese that routinely fly over the Qinghai-Tibetan plateau at over 5,000 m above sea level (Natarajan et al, 2018). First, Storz found that bar-headed geese have a higher haemoglobin-O 2 affinity than their strictly lowland relative, the greylag goose, and identified five amino acid substitutions between the two taxa, three in the α-globin chain and two in the β-globin chain.…”

“…This is most likely because the functional consequences of a mutation are dependent on the genetic background in which the mutation arises (see Lesson 2 above; Figure 1). Work from Jay Storz's laboratory, for example, has eloquently demonstrated this point: convergence in haemoglobin-oxygen affinity across high-altitude avian taxa proceeds via unpredictable changes at key amino acid residues, and the functional effect of each mutation depends on the genetic context in which it is expressed (Natarajan et al, 2018). Thus, although the genes that underlie adaptive evolution may be predictable in that they are repeated targets of selection, the precise mechanisms by which any single mutation leads to adaptive trait variation is highly species-and context-dependent.…”