Positive and purifying selection influence the evolution of doublesex in the Anastrepha fraterculus species group

The gene doublesex (dsx) is considered to be under strong selective constraint along its evolutionary history because of its central role in somatic sex differentiation in insects. However, previous studies of dsx used global estimates of evolutionary rates to investigate its molecular evolution, which potentially miss signals of adaptive changes in generally conserved genes. In this work, we investigated the molecular evolution of dsx in the Anastrepha fraterculus species group (Diptera, Tephritidae), and test the hypothesis that this gene evolved solely by purifying selection using divergence-based and population-based methods. In the first approach, we compared sequences from Anastrepha and other Tephritidae with other Muscomorpha species, analyzed variation in nonsynonymous to synonymous rate ratios (dN/dS) in the Tephritidae, and investigated radical and conservative changes in amino acid physicochemical properties. We show a general selective constraint on dsx, but with signs of positive selection mainly in the common region. Such changes were localized in alpha-helices previously reported to be involved in dimer formation in the OD2 domain and near the C-terminal of the OD1 domain. In the population-based approach, we amplified a region of 540 bp that spanned almost all of the region common to both sexes from 32 different sites in Brazil. We investigated patterns of selection using neutrality tests based on the frequency spectrum and locations of synonymous and nonsynonymous mutations in a haplotype network. As in the divergence-based approach, these analyses showed that dsx has evolved under an overall selective constraint, but with some events of positive selection. In contrast to previous studies, our analyses indicate that even though dsx has indeed evolved as a conserved gene, the common region of dsx has also experienced bouts of positive selection, perhaps driven by sexual selection, during its evolution.