The effectiveness of selection in a species affects the direction of amino acid frequency evolution

Nearly neutral theory predicts that species with higher effective population size (N _e ) are better able to purge slightly deleterious mutations. We compare evolution in high-N _e vs. low-N _e vertebrates to reveal which amino acid frequencies are subject to subtle selective preferences. We take three complementary approaches, two measuring flux and one measuring outcomes. First, we fit non-stationary substitution models of amino acid flux using maximum likelihood, comparing the high-N _e clade of rodents and lagomorphs to its low-N _e sister clade of primates and colugos. Second, we compare evolutionary outcomes across a wider range of vertebrates, via correlations between amino acid frequencies and N _e . Third, we dissect the details of flux in human, chimpanzee, mouse, and rat, as scored by parsimony - this also enables comparison to a historical paper. All three methods agree on which amino acids are preferred under more effective selection. Preferred amino acids tend to be smaller, less costly to synthesize, and to promote intrinsic structural disorder. Parsimony-induced bias in the historical study produces an apparent reduction in structural disorder, perhaps driven by slightly deleterious substitutions. Within highly exchangeable pairs of amino acids, arginine is strongly preferred over lysine, and valine over isoleucine, consistent with more effective selection preferring a marginally larger free energy of folding. These two preferences match differences between thermophiles and mesophilic relatives. These results reveal the biophysical consequences of mutation-selection-drift balance, and demonstrate the utility of nearly neutral theory for understanding protein evolution.