A number of statistical tests have been proposed to detect positive Darwinian selection affecting a few amino acid sites in a protein, exemplified by an excess of nonsynonymous nucleotide substitutions. These tests are often more powerful than pairwise sequence comparison, which averages synonymous (d S ) and nonsynonymous (d N ) rates over the whole gene. In a recent study, however, Hughes AL and Friedman R (2005. Variation in the pattern of synonymous and nonsynonymous difference between two

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... ungal genomes. Mol Bio Evol. 22: 1320-1324) argue that d S and d N are expected to fluctuate along the sequence by chance and that an excess of nonsynonymous differences in individual codons is no evidence for positive selection. The authors compared codons in protein-coding genes from the genomes of 2 yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus. They calculated the proportions of synonymous and nonsynonymous differences per site (p S and p N ) in every codon and discovered that p N is often greater than p S and that among some codons p S and p N are negatively correlated. The authors argued that these results invalidate previous tests of codons under positive selection. Here I discuss several errors of statistics in the analysis of Hughes and Friedman, including confusion of statistics with parameters, arbitrary data filtering, and derivation of hypotheses from data. I also apply likelihood ratio tests of positive selection to the yeast data and illustrate empirically that Hughes and Friedman's criticisms on such tests are not valid.