Embryo and adult viability determination

For embryo viability counts (Fig. 2), 3–4 day old adult virgin females were mated with males of the relevant genotypes for 2–3 days in glass vials supplemented with Drosophila medium and yeast paste. Following this period, the adults were transferred to an egg collection chamber containing a grape juice agar plate. Females were allowed to lay at 26 °C for 12 h, after which the adults were removed and the total number of embryos were scored. These embryos were kept on the agar surface at 26 °C for 24 h. The % survival was then determined by counting the number of unhatched embryos. One group of 100–300 embryos per cross was scored in each experiment, and each experiment was carried out in biological triplicate (total number of offspring scored is presented in Table ^S3). The results presented are averages from these three experiments. Embryo survival was normalized with respect to the % survival observed in parallel experiments carried out with the Oregon R wildtype strain, which was 91.66%. For adult fly counts (Fig. 2), the agar plates were transferred to 250 ml plastic bottles with Drosophila medium and kept at 26 °C for 7–10 days. Following this period, the number of adults that emerged was scored. The percentages of adult survival presented are averages from each cross normalized with respect to the % survival observed in Oregon R, which was 58.04% (total number of offspring scored is presented in Source Data file); all crosses were set in triplicate. Unpaired two-tailed t-test statistical analyses using GraphPad Prism version 8.2.1 for Windows (GraphPad Software, San Diego CA, www.graphpad.com) were carried out for both embryo and adult fly counts to compare expected and observed values. For species crossed to themselves, significant differences were found in embryo survival for species A2 and C1 compared to WT crossed to itself (p values = 0.0322 and 0.0325, respectively) but not for any of the remaining six species. For species outcrossed to WT, significant differences were found for all bidirectional crosses of each species to WT when compared to WT crossed to itself. This significance was seen in experiments for both embryo and adult fly survival (p < 0.05). Exact p values reported in Source Data file. Cohen’s d effect sizes were calculated using Stata (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC). Figure icons were created with Biorender.com