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

Abstract

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.

Figure 1. Map of sampling sites in Brazil.

The haplotypes from each site are shown in the table at right.

Figure 2. Unrooted phylogenetic trees estimated by maximum likelihood.

A) Male isoform. B) Female isoform. The foreground branch is marked by an asterisk (*). The branch lengths are as nucleotide substitutions per nucleotide site.

Figure 3. Bayes Empirical Bayes of male and female isoform.

A) Posterior probability for ω >1 in male isoform. B) Schematic representation of male and female transcripts. The dashed box stands for the amplified segment used in populational analyses. C) Posterior probability for ω>1 in female isoform. The dashed line represents the 0.95 limit of posterior probability for ω>1. Arrows indicate codon positions with posterior probability greater than 0.95 for ω >1. DM/OD1 – DNA-binding and dimerization domain 1. OD2 – dimerization domain 2.

Figure 4. Sliding window plots of the z-scores of radically changed properties showing regions under positive-destabilizing selection in (A) female and (B) male isoform.

DM/OD1 – DNA-binding and dimerization domain. OD2 – dimerization domain 2. α₁, α₂ and α₃ – alpha-helices that compose the predicted UBA-like domain. α* - disordered C-terminal tail, proposed to fold as an alpha-helix. Dashed horizontal line indicates the Bonferroni corrected significant limit (z-score = 3.07, p<0.05, male exon; z-score = 2.95, p<0.05, female exon).

Figure 5. Haplotype network estimated by statistical parsimony (TCS ver 1.21).

Thicker lines represent nonsynonymous substitutions and small circles stand for inferred haplotypes. Haplotypes were labeled sequentially and the subsequent letters represent the respective identified species. O – A. obliqua, F – A. fraterculus, S – A. sororcula, G – A. grandis, T – A. striata, B – A. bistrigata, E– A. serpentina. Letters near lines represent internal mutations labels.