Abstract
A major conclusion from comparative genomics is that many sequences that do not code for proteins are conserved beyond neutral expectations, indicating that they evolve under the influence of purifying selection and are likely to have functional roles. Due to the degeneracy of the genetic code, synonymous sites within protein-coding genes have previously been seen as "silent" with respect to function and thereby invisible to selection. However, there are indications that synonymous sites of vertebrate genomes are also subject to selection and this is not necessarily because of potential codon bias. We used divergence in ancestral repeats as a neutral reference to estimate the constraint on 4-fold degenerate sites of avian genes in a whole-genome approach. In the pairwise comparison of chicken and zebra finch, constraint was estimated at 24-32%. Based on three-species alignments of chicken, turkey, and zebra finch, lineage-specific estimates of constraint were 43%, 29%, and 24%, respectively. The finding of significant constraint at 4-fold degenerate sites from data on interspecific divergence was replicated in an analysis of intraspecific diversity in the chicken genome. These observations corroborate recent data from mammalian genomes and call for a reappraisal of the use of synonymous substitution rates as neutral standards in molecular evolutionary analysis, for example, in the use of the well-known d(N)/d(S) ratio and in inferences on positive selection. We show by simulations that the rate of false positives in the detection of positively selected genes and sites increases several-fold at the levels of constraint at 4-fold degenerate sites found in this study.