Male sexual ornament size is positively associated with reproductive morphology and enhanced fertility in the stalk-eyed fly Teleopsis dalmanni

Background Exaggerated male ornaments and displays often evolve in species where males only provide females with ejaculates during reproduction. Although "good genes" arguments are typically invoked to explain this phenomenon, a simpler alternative is possible if variation in male reproductive quality (e.g. sperm number, ejaculate content, mating rate) is an important determinant of female reproductive success. The "phenotype-linked fertility hypothesis" states that female preference for male ornaments or displays has been selected to ensure higher levels of fertility and has driven the evolution of exaggerated male traits. Females of the stalk-eyed fly Teleopsis dalmanni must mate frequently to maintain high levels of fertility and prefer to mate with males exhibiting large eyespan, a condition-dependent sexual ornament. If eyespan indicates male reproductive quality, females could directly increase their reproductive success by mating with males with large eyespan. Here we investigate whether male eyespan indicates accessory gland and testis length, and then ask whether mating with large eyespan males affects female fertility. Results Male eyespan was a better predictor of two key male reproductive traits – accessory gland and testis length – than was body size alone. This positive relationship held true over three levels of increasing environmental stress during the maturation of the adult accessory glands and testes. Furthermore, females housed with a large eyespan male exhibited higher levels of fertility than those with small eyespan males. Conclusion Male eyespan in stalk-eyed flies is subject to strong directional mate preference and is a reliable indicator of male reproductive quality – both because males with larger eyespan have bigger accessory glands and testes, and also as they confer higher fertility on females. Fertility enhancement may have arisen because males with larger eyespan mated more often and/or because they transferred more sperm or other substances per ejaculate. The need to ensure high levels of fertility could thus have been an important selective force in the coevolution of female preference and male eyespan in stalk-eyed flies. Our results support the phenotype-linked fertility hypothesis and suggest that it might be of general importance in explaining the evolution of exaggerated male ornaments and displays in species where males only provide females with ejaculates during reproduction.

that larvae were reared under low nutritional stress (>2g of puréed corn per 13 eggs) to minimize variation in body size and eyespan and so minimize reproductive organ allometry.
To further reduce the effects of allometric scaling, body size was measured at eclosion and males with body length outside the range of one standard deviation above and below the mean were discarded. Remaining males were assigned to one of five adult diets: 0% corn, 25% corn, 50% corn, 75% corn, 100% corn. Reproductive organs were measured every 7 days for 49 days, with a total of 11 flies per diet dissected on each day. Data analysis was performed using a general linear model including body size, diet and age as predictors (both diet and age were treated as categorical variables, allowing comparison of differences between levels of each factor).
We were able to eliminate the positive allometry of both reproductive organs. Body size failed to predict either accessory gland length (F 1,371 = 0.23, p = 0.6307) or testis length (F 1,369 = 0.01, p = 0.9346). Age was a significant predictor of reproductive organ size; both accessory gland length (F 6,371 = 316.70, p < 0.0001) and testis length (F 6,369 = 16.91, p < 0.0001) increased with age. Multiple pairwise comparisons (Tukey HSD) revealed significant increases in accessory gland length over each of the first 4 weeks (day 8 < 15 < 22 < 36) but no significant difference between the last three weeks. Similarly, testis length increased over each of the first 2 weeks (day 8 < 15) and between day 15 and days 36-50. There was no 2 significant difference in testis length between the last three weeks.
Diet also had important effects on reproductive organ size; both accessory gland length (F 4, 371 = 89.72, p < 0.0001) and testis length (F 4,369 = 5.97, p = 0.0001) decreased with increasing levels of nutritional stress. Multiple pairwise comparisons (Tukey HSD) revealed significant differences in accessory gland length between 0% corn, 25% corn, and the three least stressful diets (50% corn, 75% corn, 100%) which were not significantly different from one another.
Similarly, testis length was significantly smaller in males raised on 0% corn than in the three least stressful diets (no significant difference was observed between the 25% corn group and any other diet).
As the three least stressful diets were equivalent, we chose to examine the effects of eyespan on reproductive organ growth across three different levels of adult nutritional stress: 0%, 25%, and 75% corn. We also chose to measure reproductive organs at three time points: 14 days, 28 days and 42 days post-eclosion to include periods of rapid growth and the attainment of final size for both the testes and the accessory glands.

Figure S1
The effect of adult nutritional stress on growth of (a) the accessory glands and (b) the testes, in male stalk-eyed flies raised under low larval stress. Organ growth trajectories for flies exposed to one of five different levels of nutritional stress. Data points represent the least squares means ± s.e. log reproductive organ length.