title: 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.
authors.affiliation: Department of Animal Biology, School of Integrative Biology, University of Illinois, Urbana-Champaign School of Biological Sciences, University of Nebraska, Lincoln cheviron@illinois.edu jstorz2@unl.edu.
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Cheviron
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Natarajan
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Joana
authors.initials: J
authors.lastname: Projecto-Garcia
authors.affiliation: Department of Animal Biology, School of Integrative Biology, University of Illinois, Urbana-Champaign.
authors.forename: Douglas K
authors.initials: DK
authors.lastname: Eddy
authors.affiliation: Department of Animal Biology, School of Integrative Biology, University of Illinois, Urbana-Champaign.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jones
authors.affiliation: Department of Zoology and Physiology, University of Wyoming.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Carling
authors.affiliation: Department of Biology, University of New Mexico Museum of Southwestern Biology, University of New Mexico.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Witt
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Moriyama
authors.affiliation: Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark.
authors.forename: Roy E
authors.initials: RE
authors.lastname: Weber
authors.affiliation: Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark.
authors.forename: Angela
authors.initials: A
authors.lastname: Fago
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln cheviron@illinois.edu jstorz2@unl.edu.
authors.forename: Jay F
authors.initials: JF
authors.lastname: Storz
mesh_terms: D000222:Adaptation, Physiological; D000483:Alleles; D000531:Altitude; D000818:Animals; D001692:Biological Transport; D015870:Gene Expression; D005787:Gene Frequency; D006454:Hemoglobins / Q000235:genetics* / Q000378:metabolism; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D046370:Sparrows / Q000502:physiology*; D055542:alpha-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D055542:alpha-Globins; D010100:Oxygen
keywords: adaptation; functional synthesis; high-altitude adaptation; hypoxia; protein evolution