Selection on codon usage bias is well documented in a number of microorganisms. Whether codon usage is also generally shaped by natural selection in large organisms, despite their relatively small effective population size (Ne), is unclear. In animals, the population genetics of codon usage bias has only been studied in a handful of model organisms so far, and can be affected by confounding, nonadaptive processes such as GC-biased gene conversion and experimental artefacts. Using population transcriptomics data, we analyzed the relationship between codon usage, gene expression, allele frequency distribution, and recombination rate in 30 nonmodel species of animals, each from a different family, covering a wide range of effective population sizes. We disentangled the effects of translational selection and GC-biased gene conversion on codon usage by separately analyzing GC-conservative and GC-changing mutations. We report evidence for effective translational selection on codon usage in large-Ne species of animals, but not in small-Ne ones, in agreement with the nearly neutral theory of molecular evolution. C- and T-ending codons tend to be preferred over synonymous G- and A-ending ones, for reasons that remain to be determined. In contrast, we uncovered a conspicuous effect of GC-biased gene conversion, which is widespread in animals and the main force determining the fate of AT↔GC mutations. Intriguingly, the strength of its effect was uncorrelated with Ne.

According to current assemblies, avian genomes differ from those of the other lineages of amniotes in 1) containing a lower number of genes; 2) displaying a high stability of karyotype and recombination map; and 3) lacking any correlation between evolutionary rates (dN/dS) and life-history traits, unlike mammals and nonavian reptiles. We question the reality of the bird missing genes and investigate whether insufficient representation of bird gene content might have biased previous evolutionary analyses. Mining RNAseq data, we show that the vast majority of the genes missing from avian genome assemblies are actually present in most species of birds. These mainly correspond to the GC-rich fraction of the bird genome, which is the most difficult to sequence, assemble and annotate. With the inclusion of these genes in a phylogenomic analysis of high-quality alignments, we uncover a positive and significant correlation between the ratio of nonsynonymous to synonymous substitution rate (dN/dS) and life-history traits in Neoaves. We report a strong effect of GC-biased gene conversion on the dN/dS ratio in birds and a peculiar behavior of Palaeognathae (ostrich and allies) and Galloanserae (chickens, ducks and allies). Avian genomes do not contain fewer genes than mammals or nonavian reptiles. Previous analyses have overlooked ∼15% of the bird gene complement. GC-rich regions, which are the most difficult to access, are a key component of amniote genomes. They experience peculiar molecular processes and must be included for unbiased functional and comparative genomic analyses in birds.