Crossing fitness valleys via double substitutions within codons

Single nucleotide substitutions in protein-coding genes can be divided into synonymous (S), with little fitness effect, and non-synonymous (N) ones that alter amino acids and have a greater effect. Most of the N substitutions are affected by purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can alleviate the deleterious effect of single substitutions, making them subject to positive selection. To elucidate the effects of selection on double substitutions in all codons, it is critical to differentiate selection from mutational biases. We addressed the evolutionary regimes of within-codon double substitutions in 37 groups of closely related prokaryotic genomes from diverse phyla by comparing within codons double substitutions to equivalent double S substitutions in adjacent codons. All within-codon double substitutions can be represented as “ancestral-intermediate-final” sequences and can be partitioned into 4 classes: 1) SS: S intermediate – S final, 2) SN: S intermediate – N final, 3) NS: N intermediate – S final, 4) NN: N intermediate – N final. We found that the selective pressure on the second substitution markedly differs among these classes. Analogous to single S substitutions, SS evolve neutrally whereas, analogous to single N substitutions, SN are subject to purifying selection. In contrast, NS show positive selection on the second step because the original amino acid is recovered. NN double substitutions are heterogeneous and depend on the amino acid similarity between the final, intermediate and the ancestral states. Positive selection compensating for deleterious substitutions can result in frequent crossing of valleys on the fitness landscape.