Divergent and Parallel Routes of Biochemical Adaptation in High-altitude Passerine Birds from the Qinghai-Tibet Plateau

When different species experience similar selection pressures, the probability of evolving similar adaptive solutions may be influenced by legacies of evolutionary history, such as lineage-specific changes in genetic background. Here we test for adaptive convergence in hemoglobin (Hb) function among high-altitude passerine birds that are native to the Qinghai-Tibet Plateau, and we examine whether convergent increases in Hb-O affinity have a similar molecular basis in different species. We documented that high-altitude parid and aegithalid species from the Qinghai-Tibet Plateau have evolved derived increases in Hb-O affinity in comparison with their closest lowland relatives in East Asia. However, convergent increases in Hb-O affinity and convergence in underlying functional mechanisms were seldom attributable to the same amino acid substitutions in different species. Using ancestral protein resurrection and site-directed mutagenesis, we experimentally confirmed two cases in which parallel substitutions contributed to convergent increases in Hb-O affinity in codistributed high-altitude species. In one case involving the ground tit () and gray-crested tit (), parallel amino acid replacements with affinity-enhancing effects were attributable to nonsynonymous substitutions at a CpG dinucleotide, suggesting a possible role for mutation bias in promoting recurrent changes at the same site. Overall, most altitude-related changes in Hb function were caused by divergent amino acid substitutions, and a select few were caused by parallel substitutions that produced similar phenotypic effects on the divergent genetic backgrounds of different species.