English: Codons are conserved from D. sechellia-D. grimshawi (excluding D. willistoni). The conserved codons were separated into those that were ancestrally preferred (P) and those that were ancestrally unpreferred (U) using polarization with the D. sechellia-D. grimshawi (excluding D. willistoni) outgroup. 10 bootstraps were done within each class. Error bars represent the s.e. of the estimates. The dark bars represent the counts of all sites that fall into each class while the light bars represent the number of sites estimated to be under strong constraint via the bootstrap procedure. The dashed line indicates what the count of total unpreferred conserved codons would have been had unpreferred 4D sites been conserved to the same extent as preferred 4D sites in otherwise conserved amino acids, i.e. the dashed line represents the proportion of U:P in all conserved amino acids. More than half (53%) of those unpreferred codons that are conserved across the ten Drosophila species are under strong purifying selection in D. melanogaster; 38% of preferred conserved codons are under strong selection.