Inferring natural selection operating on conservative and radical substitution at single amino acid sites

Natural selection operating on amino acid substitution at single amino acid sites can be detected by comparing the rates of synonymous (r(S)) and nonsynonymous (r(N)) nucleotide substitution at single codon sites. Amino acid substitutions can be classified as conservative or radical according to whether they retain the properties of the substituted amino acid. Here methods for comparing the rates of conservative (r(C)) and radical (r(R)) nonsynonymous substitution with r(S) at single codon sites were developed to detect natural selection operating on these substitutions at single amino acid sites. A method for comparing r(C) and r(R) at single codon sites was also developed to detect biases toward these substitutions at single amino acid sites. Charge was used as the property of the amino acids. In a computer simulation, false-positive rates of these methods were always < 5%, unless termination sites were included in the computation of the numbers of sites and estimates of transition/transversion rate ratio were highly biased. The frequency of detection of natural selection operating on conservative substitution was almost independent of the presence of natural selection operating on radical substitution, and vice versa. Natural selection operating specifically on conservative and radical substitution was detected more efficiently by comparing r(S) with r(C) and r(S) with r(R) than by comparing r(S) with r(N). These methods also appeared to be robust against the occurrence of recombination during evolution. In an analysis of class I human leukocyte antigen, negative selection operating on conservative substitution, but not positive selection operating on radical substitution, was observed at some of the codon sites with r(R) > r(C), suggesting that r(R) > r(C) may not necessarily be an indicator of positive selection operating on radical substitution.