Abstract
In detecting natural selection operating at the amino acid sequence level by comparing the rates of synonymous (rS) and nonsynonymous (rN) substitutions, the rates of synonymous and nonsynonymous mutations are assumed to be the same, which may not be the case in reality. Here, we develop the evolutionary pathway methods for comparing rS and rN at multiple codon sites (all-sites analysis) and at single codon sites (single-site analysis) that take into account the hypermutability at CpG dinucleotides in estimating the numbers of synonymous (dS) and nonsynonymous (dN) substitutions. Computer simulations show that the bias in estimation of dN/dS caused by the hypermutability is greatly reduced when using the methods we propose to account for the hypermutability. In an all-sites analysis of protamine P1 genes from primates, dN/dS>1 for many pairs if the hypermutability was ignored. However, dN/dS≦1 for most of these pairs when the CpG sites are assumed to be hypermutable. Therefore, statistical indications of positive selection in some sequences or codons may be caused by mutation rate differences in synonymous and nonsynonymous sites.