Integrating Evolutionary and Functional Tests of Adaptive Hypotheses: A Case Study of Altitudinal Differentiation in Hemoglobin Function in an Andean Sparrow, Zonotrichia capensis

Abstract

In air-breathing vertebrates, the physiologically optimal blood-O2 affinity is jointly determined by the prevailing partial pressure of atmospheric O2, the efficacy of pulmonary O2 transfer, and internal metabolic demands. Consequently, genetic variation in the oxygenation properties of hemoglobin (Hb) may be subject to spatially varying selection in species with broad elevational distributions. Here we report the results of a combined functional and evolutionary analysis of Hb polymorphism in the rufous-collared sparrow (Zonotrichia capensis), a species that is continuously distributed across a steep elevational gradient on the Pacific slope of the Peruvian Andes. We integrated a population genomic analysis that included all postnatally expressed Hb genes with functional studies of naturally occurring Hb variants, as well as recombinant Hb (rHb) mutants that were engineered through site-directed mutagenesis. We identified three clinally varying amino acid polymorphisms: Two in the α(A)-globin gene, which encodes the α-chain subunits of the major HbA isoform, and one in the α(D)-globin gene, which encodes the α-chain subunits of the minor HbD isoform. We then constructed and experimentally tested single- and double-mutant rHbs representing each of the alternative α(A)-globin genotypes that predominate at different elevations. Although the locus-specific patterns of altitudinal differentiation suggested a history of spatially varying selection acting on Hb polymorphism, the experimental tests demonstrated that the observed amino acid mutations have no discernible effect on respiratory properties of the HbA or HbD isoforms. These results highlight the importance of experimentally validating the hypothesized effects of genetic changes in protein function to avoid the pitfalls of adaptive storytelling.

Hemoglobin–oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

“…For example, birds typically express two main Hb isoforms in definitive red blood cells -a major isoform, HbA, which incorporates α-type products of the α A -globin gene, and a minor isoform, HbD, which incorporates α-type products of the α D -globin gene; the two isoforms share the same β-type subunits Grispo et al, 2012;Opazo et al, 2015). In all bird species that have been examined to date, the minor HbD isoform has an appreciably higher O 2 affinity than the major HbA isoform in the presence of allosteric effectors (Grispo et al, 2012;Projecto-Garcia et al, 2013;Cheviron et al, 2014;Galen et al, 2015;Natarajan et al, 2015bNatarajan et al, , 2016. Other sauropsid taxa also tend to co-express multiple, structurally distinct Hb isoforms in definitive erythrocytes (Weber and Jensen, 1988;Storz et al, 2011bStorz et al, , 2015bDamsgaard et al, 2013).…”

“…As in the case with the mammals, the 29 pairwise comparisons are phylogenetically independent. For information regarding elevational ranges, see Projecto-Garcia et al (2013), Cheviron et al (2014), Galen et al (2015) and Natarajan et al (2015bNatarajan et al ( , 2016.…”

“…5B). These taxa include ground doves, nightjars, hummingbirds, passerines and waterfowl Cheviron et al, 2014;Galen et al, 2015;Natarajan et al, 2015bNatarajan et al, , 2016. In the case of the passerines and waterfowl, we can make comparisons between closely related species as well as conspecific populations.…”

Neutral and functionally important genes shed light on phylogeography and the history of high‐altitude colonization in a widespread New World duck

“…This result, together with functional analyses (Natarajan et al., 2015) suggest that a Thr/Ser‐69 substitution arising de novo did not facilitate the colonization of high‐elevation environments by Ruddy Ducks, implying that the previously proposed evolutionary scenario requires reevaluation. Other studies have revealed that patterns of elevational differentiation in hemoglobin sequences consistent with spatially variable selection need not imply adaptive evolution because amino acid replacements may have no effect on respiratory properties of hemoglobin isoforms (Cheviron et al., 2014). However, we note that recent analyses indicate that Ruddy Ducks throughout their range, including North America, possess hemoglobin with relatively high O 2 affinity, similar to other Andean species (Natarajan et al., 2015).…”

Selection on the morphology–physiology‐performance nexus: Lessons from freshwater stickleback morphs

“…Indeed results from genome scans increasingly reveal signals of selection acting on physiological processes (Akey et al., 2010; Gautier et al., 2009; Shimada, Shikano, & Merilä, 2011; Simonson et al., 2010), and on metabolism in particular (reviewed in Marden, 2013). While these types of studies can provide initial evidence in support of adaptive divergence, the case of the Andean Sparrow ( Zonotrichia capensis ) highlights their potential limitations: A genome scan suggested selection acting on metabolic pathways, but this result was contradicted by experimental tests of associated enzyme affinity (Cheviron et al., 2014). Thus, direct measures of physiological processes are integral in the context of studying putative adaptive divergence.…”