  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 D. 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.
