Speciation is poorly understood at the molecular level because of the difficulty in identifying hybrid incompatibility genes. Recently, we identified a new hybrid incompatibility gene, gfzf, that is required for hybrid F1 male inviability between Drosophila melanogaster and its sister species Drosophila simulans. Knockdown of only the D.simulans ortholog of gfzf in male hybrids is sufficient to rescue their viability. However, it remains unclear whether the incompatibility driven by gfzf is a general isolating mechanism between D.melanogaster and the D.simulans clade (D.simulans, D.mauritiana, and D.sechellia) or evolved along the D.simulans lineage specifically. To investigate the role of gfzf-meditated incompatibility in the D.simulans clade, we constructed D.melanogaster transgenic flies that express RNAi constructs to knock-down expression from the gfzf ortholog of each D.simulans clade species. We crossed D.melanogaster females that contain the RNAi construct to males of the D.simulans clade and counted male and female progeny. We find that gfzf knockdown produces variable hybrid male rescue across the D.simulans clade.
Further, it is unknown if the cell cycle defects observed in D.melanogaster / D.simulans hybrids remain consistent in hybrids between D.melanogaster and other species in the D.simulans clade. To investigate the defects in cell proliferation in developing hybrid larvae, we used a pair of fluorescent constructs that mark cells in a cell cycle specific pattern. We crossed D. melanogaster females that contain these transgenic constructs to males of the D.simulans clade. Our analyses using developing brains and imaginal discs via immunofluorescent confocal microscopy suggest that the severity of proliferation defects in hybrids of D.melanogaster and the D.simulans clade may not be uniform. The variation in both rescue of hybrid males by gfzf knockdown and hybrid cell cycle defects suggests either the presence of other genes that contribute to dominant male lethality, or a variable role for gfzf in this hybrid incompatibility.