Figure 1.

Protocol for selection on sperm competitive ability. Only the high-P2 selection experiment is illustrated; differences in protocol between this and the other lines are described below. Matings are indicated in boxes. Seventy-five recessive, brown-eye (bw) males and females were collected as larvae from the LH_M-bw base population cage and were used to establish the selection line population. These individuals are referred herein as ‘selection line’ males and females and are indicated with bold and underlined text. All other flies (the competitor males and the females used in the sperm competition matings) are referred herein as ‘stock’ males and females. They were collected as larvae for each generation of the experiment from the LH_M (denoted as ‘+/+’) or LH_M-bw (bw) base population cages. Selection line and stock flies were collected on day 1 of the initial generation. Individual stock females (bw) were paired first with a single stock male (+/+) on the morning of day 5 and then with a single selection line male (bw) on the morning of day 8. (This mating order was reversed for the high-P1 and control-P1 lines.) All copulations were observed, and females and males were separated following copulation to prevent double matings. Stock females were transferred to new vials each day until the second mating on day 8. These vials (from days 5 to 7) were saved to count progeny prior to female re-mating. Stock males were discarded following copulation on day 8, and selection line males were transferred individually to fresh vials. Females from the sperm competition experiment were discarded on day 9, one day after re-mating. On day 15, all selection line males were paired with a single female from their own line; sib matings were avoided. Progeny prior to female re-mating were counted first. Next, progeny from the day 8 vials were counted and their eye colours were scored. The day 15 selection line mating vials with progeny from the 15 males with the highest P2 scores were retained. (15 random selection line mating vials were retained for the control lines.) When the progeny from these vials emerged, five males and five females (75 virgins of each sex in all) were collected from each vial. For the control-P1 and control-P2 lines, two males and two females (30 of each sex) were collected. These flies were used to begin the next generation of selection; stock flies were again collected from the base population cages.