Ancient biases in phenotype production drove the functional evolution of a protein family

Biological systems may be biased in the phenotypes they can access by mutation^1–7, but the extent of these biases and their causal role in the evolution of extant phenotypic diversity remains unclear. There are three major challenges: it is difficult to isolate the effect of bias in the genotype-phenotype (GP) map from that of natural selection in producing natural diversity^6,8–11, the universe of possible genotypes and phenotypes is so vast and complex that a direct characterization has been impossible, and most extant phenotypes evolved long ago in species whose GP maps cannot be recovered. Here we develop exhaustive multi-phenotype deep mutational scanning to experimentally characterize the complete GP maps of two reconstructed ancestral steroid receptor proteins, which existed during an ancient phylogenetic interval when a new phenotype—specific binding of a new DNA response element—evolved¹². We measured all possible DNA specificity phenotypes encoded by all possible amino acid combinations at sites in the protein’s DNA binding interface. We found that the ancestral GP maps are structured by strong global bias—unequal propensity to encode the various phenotypes—and extreme heterogeneity in the phenotypes accessible around each genotype, which strongly affect evolution on both long and short timescales. Distinct biases in the two ancestral maps steered evolution toward the lineage-specific functional phenotypes that evolved during history. Our findings establish that ancient biases in the GP relationship were causal factors in the evolutionary process that produced the present-day patterns of phenotypic conservation and diversity in this protein family.