The structure of an ancient genotype-phenotype map shaped the functional evolution of a protein family

Mutation is more likely to produce some phenotypes than others, but the causal role of these production propensities in the evolution of phenotypic diversity remains unclear. There are two major challenges: it is difficult to separate the effect of the genotype-phenotype (GP) map from that of natural selection when analyzing natural diversity, and most extant phenotypes evolved long ago in species whose GP maps cannot be recovered. Using reconstructed ancestral transcription factors, we created libraries containing all possible amino acid combinations at historically variable sites in the proteins’ DNA binding interface (the genotypes) and measured their capacity to specifically bind DNA elements containing all possible combinations of nucleotides at historically variable sites (the phenotypes). The two ancestral GP maps were strongly anisotropic (the distribution of phenotypes encoded by genotypes is highly nonuniform) and heterogeneous (the phenotypes accessible around each genotype vary dramatically among genotypes), but the extent and direction of these properties differed dramatically between the maps. In both cases, these properties steered evolution toward the lineage-specific phenotypes that evolved during history. Our findings establish that ancient properties of the GP relationship were causal factors in the evolutionary process that produced present-day patterns of functional conservation and diversity.