Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in Drosophila melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated.

Codon usage bias (CUB), where certain codons are used more frequently than expected by chance, is a ubiquitous phenomenon and occurs across the tree of life. The dominant paradigm is that the proportion of preferred codons is set by weak selection. While experimental changes in codon usage have at times shown large phenotypic effects in contrast to this paradigm, genome-wide population genetic estimates have supported the weak selection model. Here we use deep genomic population sequencing of two Drosophila melanogaster populations to measure selection on synonymous sites in a way that allowed us to estimate the prevalence of both weak and strong purifying selection. We find that selection in favor of preferred codons ranges from weak (|Nes| ∼ 1) to strong (|Nes| > 10), with strong selection acting on 10–20% of synonymous sites in preferred codons. While previous studies indicated that selection at synonymous sites could be strong, this is the first study to detect and quantify strong selection specifically at the level of CUB. Further, we find that CUB-associated polymorphism accounts for the majority of strong selection on synonymous sites, with secondary contributions of splicing (selection on alternatively spliced genes, splice junctions, and spliceosome-bound sites) and transcription factor binding. Our findings support a new model of CUB and indicate that the functional importance of CUB, as well as synonymous sites in general, have been underestimated.

The availability of sequenced genomes from multiple related organisms allows the detection and localization of functional genomic elements based on the idea that such elements evolve more slowly than neutral sequences. Although such comparative genomics methods have proven useful in discovering functional elements and ascertaining levels of functional constraint in the genome as a whole, here we outline limitations intrinsic to this approach that cannot be overcome by sequencing more species. We argue that it is essential to supplement comparative genomics with ultra-deep sampling of populations from closely related species to enable substantially more powerful genomic scans for functional elements. The convergence of sequencing technology and population genetics theory has made such projects feasible and has exciting implications for functional genomics.

Comparative genomics has become widely accepted as the major framework for the ascertainment of functionally important regions in genomes. The underlying paradigm of this approach is that most of the functional regions are assumed to be under selective constraint, which in turn reduces the rate of evolution relative to neutrality. This assumption allows detection of functional regions through sequence conservation. However, constraint does not always lead to sequence conservation. When purifying selection is weak and mutation is biased, constrained regions can even evolve faster than neutral sequences and thus can appear to be under positive selection. Moreover, conservation estimates depend also on the orientation of selection relative to mutational biases and can vary over time. In the light of recent data of the ubiquity of mutational biases and weak selective forces, these effects should reduce the power of conservation analyses to define functional regions using comparative genomics data. We argue that the estimation of true mutational biases and the use of explicit evolutionary models are essential to improve methods inferring the action of natural selection and functionality in genome sequences.

Codon usage bias (CUB), where certain codons are used more frequently than expected by chance, is a ubiquitous phenomenon and occurs across the tree of life. The dominant paradigm is that the proportion of preferred codons is set by weak selection. While experimental changes in codon usage have at times shown large phenotypic effects in contrast to this paradigm, genome-wide population genetic estimates have supported the weak selection model. Here we use deep genomic sequencing of two Drosophila melanogaster populations to measure selection on synonymous sites in a way that allowed us to estimate the prevalence of both weak and strong selection. We find that selection in favor of preferred codons ranges from weak (|N e s| ∼ 1) to strong (|N e s| > 10). While previous studies indicated that selection at synonymous sites could be strong, this is the first study to detect and quantify strong selection specifically at the level of CUB. We suggest that the level of CUB in the genome is determined by the proportion of synonymous sites under no, weak, and strong selection. This model challenges the standard Li-Bulmer model and explains some of the longest-standing puzzles in the field.