title: Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas.
abstract: A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb-O2 affinity. These experiments revealed that the effects of mutations on Hb-O2 affinity are highly dependent on the temporal order in which they occur: Each of three β-chain substitutions produced a significant increase in Hb-O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb-O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Tufts
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Natarajan
authors.affiliation: Department of Bioscience, Zoophysiology, Aarhus University, Aarhus, Denmark.
authors.forename: Inge G
authors.initials: IG
authors.lastname: Revsbech
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Joana
authors.initials: J
authors.lastname: Projecto-Garcia
authors.affiliation: Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University.
authors.forename: Federico G
authors.initials: FG
authors.lastname: Hoffmann
authors.affiliation: Department of Bioscience, Zoophysiology, Aarhus University, Aarhus, Denmark.
authors.forename: Roy E
authors.initials: RE
authors.lastname: Weber
authors.affiliation: Department of Bioscience, Zoophysiology, Aarhus University, Aarhus, Denmark.
authors.forename: Angela
authors.initials: A
authors.lastname: Fago
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Moriyama
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln jstorz2@unl.edu.
authors.forename: Jay F
authors.initials: JF
authors.lastname: Storz
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology; D000531:Altitude; D000818:Animals; D004843:Epistasis, Genetic / Q000235:genetics*; D019143:Evolution, Molecular; D006454:Hemoglobins / Q000235:genetics*; D007793:Lagomorpha / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D012641:Selection, Genetic / Q000235:genetics
chemical_list: D006454:Hemoglobins; D010100:Oxygen
keywords: adaptation; epistasis; hemoglobin; high altitude; molecular evolution; protein evolution