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Site-specific evolutionary rates can be estimated from codon sequences or from amino-acid sequences. For codon sequences, the most popular methods use some variation of the  dN ∕ dS  ratio. For amino-acid sequences, one widely-used method is called Rate4Site, and it assigns a relative conservation score to each site in an alignment. How site-wise  dN ∕ dS  values relate to Rate4Site scores is not known. Here we elucidate the relationship between these two rate measurements. We simulate sequences with known  dN ∕ dS , using either  dN ∕ dS  models or mutation–selection models for simulation. We then infer Rate4Site scores on the simulated alignments, and we compare those scores to either true or inferred  dN ∕ dS  values on the same alignments. We find that Rate4Site scores generally correlate well with true  dN ∕ dS , and the correlation strengths increase in alignments with greater sequence divergence and more taxa. Moreover, Rate4Site scores correlate very well with inferred (as opposed to true)  dN ∕ dS  values, even for small alignments with little divergence. Finally, we verify this relationship between Rate4Site and  dN ∕ dS  in a variety of empirical datasets. We conclude that codon-level and amino-acid-level analysis frameworks are directly comparable and yield very similar inferences.

Calculating site-specific evolutionary rates at the amino-acid or codon level yields similar rate estimates