Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10–20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8–1.2 µM−1), folding stabilities of cetacean Mbs are ∼2–4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.
In this work, we identify positive selection in cetacean myoglobins and an early, significant divergence event. While O2-binding is nearly unchanged, positive selection acts to introduce and later maintain stability. Stability correlates with abundance across the species, supporting that selection for increased stability concurred with the known 10–20 fold increase in myoglobin abundance of cetaceans relative to terrestrial mammals, which itself resulted from speciation towards longer dive lengths of the animals. We suggest that this selection acted to keep constant the otherwise increasing number of unfolded Mb. Altogether, this work for the first time links protein phenotype (stability and abundance) in a specific, real protein to organism-level evolution and fitness of mammals.