Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas

Danielle M. Tufts, Chandrasekhar Natarajan, Inge G. Revsbech, Joana Projecto-Garcia, Federico G. Hoffmann, Roy E. Weber, Angela Fago, Hideaki Moriyama, Jay F. Storz

Research output: Contribution to journalArticle

53 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)287-298
Number of pages12
JournalMolecular biology and evolution
Issue number2
StatePublished - Feb 1 2015


  • adaptation
  • epistasis
  • hemoglobin
  • high altitude
  • molecular evolution
  • protein evolution

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Molecular Biology
  • Genetics

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    Tufts, D. M., Natarajan, C., Revsbech, I. G., Projecto-Garcia, J., Hoffmann, F. G., Weber, R. E., Fago, A., Moriyama, H., & Storz, J. F. (2015). Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas. Molecular biology and evolution, 32(2), 287-298. https://doi.org/10.1093/molbev/msu311