Predictable convergence in hemoglobin function has unpredictable molecular underpinnings

Abstract

To investigate the predictability of genetic adaptation, we examined the molecular basis of convergence in hemoglobin function in comparisons involving 56 avian taxa that have contrasting altitudinal range limits. Convergent increases in hemoglobin-oxygen affinity were pervasive among high-altitude taxa, but few such changes were attributable to parallel amino acid substitutions at key residues. Thus, predictable changes in biochemical phenotype do not have a predictable molecular basis. Experiments involving resurrected ancestral proteins revealed that historical substitutions have context-dependent effects, indicating that possible adaptive solutions are contingent on prior history. Mutations that produce an adaptive change in one species may represent precluded possibilities in other species because of differences in genetic background.

Fig. 1. Amino acid differences that distinguish the Hbs of each pair of high- and low-altitude taxa

Derived (nonancestral) amino acids are shown in red lettering, and rows corresponding to high-altitude taxa are shaded in blue. Subunits of the major HbA isoform are encoded by the α^A- and β^A-globin genes, whereas those of the minor HbD isoform are encoded by the α^D- and β^A-globin genes. Phylogenetically replicated β-chain replacements that contribute to convergent increases in Hb-O₂ affinity (N/G83S, A86S, D94E, and A116S) are outlined. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.

Fig. 2. Convergent increases in Hb-O₂ affinity in high-altitude Andean birds

(A) Plot of P_50(KCl+IHP) (± 1 SE) for HbA in 28 matched pairs of high- and low-altitude taxa. Data points that fall below the diagonal line (x = y) denote cases in which the high-altitude member of a given taxon pair possesses a higher Hb-O₂ affinity (lower P₅₀). Comparisons involve replicated pairs of taxa, so all data points are phylogenetically independent. (B) Plot of P_50(KCl+IHP) (± 1 SE) for the minor HbD isoform in a subset of the same taxon pairs in which both members of the pair express HbD. P_50(KCl+IHP), O₂ partial pressure at which Hb is 50% saturated in the presence of chloride and inositol hexaphosphate.

Fig. 3. Phenotypic effects of substitutions at β83 are conditional on genetic background

(A) The engineered G83S mutation produced a significant reduction in P_50(KCl+IHP) (increase in Hb-O₂ affinity) in the reconstructed Hb of the hummingbird ancestor. (B) The engineered A67V and N83S mutations produced additive reductions in P_50(KCl+IHP) in the reconstructed Hb of the flowerpiercer ancestor. Underlining indicates derived (nonancestral) amino acids. (C) Diagrammatic tree with time-scaled branch lengths showing internal nodes that we targeted for ancestral protein resurrection. Scale bar, 10 million years. (D) N/G83S mutations produced significant increases in Hb-O₂ affinity (expressed as reductions in P_50(KCl+IHP)) in the ancestors of hummingbirds and flowerpiercers. Substitutions at the same site produced no detectable effects in Anc Neoaves or Anc Neornithes.