Testes-restricted, Recently evolved, Clustered (TRC) canonical miRNAs in Drosophila. (A) An example of a novel TRC miRNA cluster (dan_86 → dan_373) in D. ananassae. The majority of miRNAs show high expression in the male-body libraries. (B) An example of a TRC miRNA cluster (dps_3416 → dps-mir-2536) in the obscura subgroup species. The D. pseudoobscura cluster contains 36 miRNAs, whereas its sister species, D. persimilis, contains 26 miRNAs. MiRNAs within the 3′ end region of these orthologous clusters (orange highlight) have preserved their order, whereas miRNAs within the 5′ region show high gene duplication. Colored circles and numbers represent miRNAs of the same family. (C) Expression heatmap for all D. pseudoobscura copies reveals a predominant testes-restricted profile. (D) Comparison of expression difference between TRC and solo canonical miRNAs present in D. virilis alone or within the virilis/mojavensis clade alone. TRC miRNAs of the virilis-subgroup show significantly higher expression than their age-matched solo canonical cohorts (Mann-Whitney U test, P < 10⁻⁸). All Drosophilid subclades have their own distinct TRC loci, and details of all the novel TRC loci cloned in this study are provided in Supplemental Figs. S12–S15.