whenever males tend to compete with males to whom they are more genetically related than the population average, for example when population viscosity limits dispersal and competition is not exclusively local [14,15]. In this context, a male may indirectly reduce his own inclusive fitness by harming females that could also reproduce with his male relatives, and this is expected to relax male–male competition and selection for male traits that harm females [6,11–14].
While the expectation that, under some circumstances, within-group male relatedness reduces the intensity of intra sexual competition has received empirical support (e.g. [16–19]), the notion that within-group male relatedness might also reduce female harm is only beginning to be investigated. Consistent with this idea, female least killifish, Heterandria formosa, died younger and produced progressively smaller offspring when experimentally mated to males that are unrelated to each other, compared with females mated to males highly related to each other (but always unrelated to the female; [20]). Similarly, female bulb mites, Rhizoglyphus robini, laid more eggs over a 2-day period when paired for 5 days with males that had experimentally evolved in populations comprising their full siblings than when paired with stock males [21].
The influence of male relatedness on female harm has also been explored in the fruit fly, Drosophila melanogaster. Carazo et al. [22] found that females had higher lifetime reproductive success and slower reproductive ageing (a more gradual decline in fecundity and fertility with age) when exposed to a triplet of brothers that were unrelated to the female but had been raised together as larvae than when exposed to a triplet of males that were unrelated to each other and had been raised separately as larvae. These patterns have now been explored by different research groups and in different D. melanogaster populations [23–26] resulting in some studies reporting results consistent with Carazo et al.’s findings and others reporting no effects (summarized in electronic supplementary material, table S1), suggesting that these effects are not entirely consistent and that they might be modulated by other mechanisms.
One such mechanism might be familiarity. Hollis et al. [24] identified larval familiarity among males as a requirement for reduced harm to females. By introducing a new treatment in which females were exposed to males that were related to each other but raised apart as larvae, this study showed that males were only benign to females when they were related and raised together as larvae. These results are consistent with larval familiarity acting as a kin recognition mechanism, as demonstrated in other taxa [27–32]. In principle, these results may also indicate that male flies might have evolved to reduce female harm strategically in response to male familiarity per se, independently of relatedness, through direct (rather than indirect) fitness effects [24]. For example, mechanisms such as reciprocity might reduce competition among familiar males, and this may in turn reduce female harm.
A scenario in which variation in male harm is entirely predicted by relatedness, not familiarity, would suggest that flies use genetic cues to recognize kin and reduce harm in the presence of relatives to gain indirect fitness benefits. A scenario in which variation in male harm is entirely predicted by male familiarity, not relatedness, would be consistent both with the idea that direct benefits associated with familiarity drive changes in female harm, and the idea that female harm is driven by indirect effects, whereby flies may rely entirely on familiarity cues to recognize kin. Finally, variation in male harm may be predicted by the interaction between relatedness and familiarity cues. For example, indirect fitness effects may reduce male harm to females when males are related, but male flies may only be able to recognize relatives under familiarity [33]. However, because no study has tested the fully factorial combination of relatedness and familiarity [22–26], the relative roles of these factors remain unresolved.
In this study, we conducted an experiment using a novel, fully factorial design to isolate the separate effects of relatedness, familiarity (shared larval environment) and their interaction on male sexual behaviour (as measured through assays of male–male aggression, courtship and mating rates) and female harm (as measured through female lifetime reproductive success, reproductive ageing, lifespan and reproductive lifespan) in D. melanogaster. We used four different social environments in which males were: (i) related and familiar, (ii) related and unfamiliar, (iii) unrelated and familiar and (iv) unrelated and unfamiliar. While we found no effect on male behaviours, we did observe an interaction between male relatedness and larval familiarity, thereby showing that larval familiarity alone is insufficient to reduce harm to females. Male relatedness increased female reproductive success, lifespan and reproductive lifespan, and slowed reproductive ageing, but only when males were familiar.
Methods
Stock cultures
We used a laboratory-adapted, wild-type Dahomey stock of Drosophila melanogaster, maintained in large, outbred populations since 1970 [34,35] at 25°C in a non-humidified room and a 12:12 h light:dark cycle. This is the same stock used by Carazo et al. [22,23]. All flies were maintained in cages containing bottles of Lewis medium [36] with overlapping generations.
Male treatments
We produced triplets of males belonging to one of four treatments generated from a fully factorial cross of relatedness and familiarity in the larval environment: related and familiar, related and unfamiliar, unrelated and familiar, and unrelated and unfamiliar (figure 1).
To generate each experimental male triplet, we created families using parents that were 2 days post-eclosion, and had been collected as eggs from the stock population and reared at standard larval density at 25°C [34]. We paired a single virgin male and female for 12 h in individual larval collection chambers containing a Petri-dish filled with hard grape agar (550 ml water, 25 g agar, 300 ml grape juice concentrate and 21.25 ml 10%w/v Nipagin) with a smear of live yeast paste, before discarding the males. Twenty-four to thirty-six hours after egg laying, we picked larvae with a mounted needle into 36 ml vials containing 8 ml of Lewis medium, collecting 60 larvae in total per family over a period of 3 days. Any families that failed to produce 60 larvae were excluded.
From each of 135 families, 45 larvae were divided equally among three ‘single family’ vials and 15 larvae were distributed individually among each of 15 ‘mixed family’ vials. Thus, each ‘single family’ vial contained 15 larvae from a single family, and each ‘mixed family’ vial contained 15 larvae from 15 randomly allocated families (figure 1). These vials were kept at 18°C and adult virgin males were collected within 16 h of eclosion.
