Relationship between the functionality of neo-sex-linked genes and constraint, estimated as the ratio of nonsynonymous to synonymous nucleotide substitutions per site (d_N/d_S) in Drosophila miranda (A), D. albomicans (B), and D. americana (C) lineages. Branches c (left), a (center), and b (right) correspond to those in Figure 4. Violin plots were used to show the distribution of d_N/d_S. Orthologs regarded to be functional in the outgroup species (D. obscura, D. kohkoa, and D. novamexicana, respectively) and located on the same Muller element without any inparalogs were used for this analysis. In total, 681 genes were analyzed for X_F-Y_F, 474 genes for X_F-Y_P, 70 genes for X_P-Y_F, and 92 genes for X_P-Y_P in the D. miranda lineage; 2551 genes were analyzed for X_F-Y_F, 277 genes for X_F-Y_P, 166 genes for X_P-Y_F, and 189 genes for X_P-Y_P in the D. albomicans lineage; and 1267 genes were analyzed for X_F-Y_F, 119 genes for X_F-Y_P, 85 genes for X_P-Y_F, and 57 genes for X_P-Y_P in the D. americana lineage. Differences of d_N/d_S ratios between groups were evaluated by a Mann–Whitney U test with correction for multiple testing under the null hypothesis of an equal d_N/d_S ratio. The same roman numerals indicate Q ≥ 0.05, whereas different numerals indicate Q < 0.05 between categories.