Multiple mechanisms of cryptic female choice act on intraspecific male variation in Drosophila simulans

Postcopulatory sexual selection can arise when females mate with multiple males and is usually mediated by an interaction between the sexes. Cryptic female choice (CFC) is one form of postcopulatory sexual selection that occurs when female morphology, physiology, or behavior generates a bias in fertilization success. However, its importance in nonrandom reproductive success is poorly resolved due to challenges distinguishing the roles of females and males in generating patterns of fertilization bias. Nevertheless, two CFC mechanisms have recently been documented and characterized in Drosophila simulans within the context of gametic isolation in competitive hybrid matings with Drosophila mauritiana: sperm ejection and nonrandom use of sperm storage organs for fertilization. Here, we explore if and how female D. simulans employ these two mechanisms of CFC in response to intraspecific male size variation. We used transgenic males expressing green (GFP) or red fluorescent protein (RFP) in sperm heads to document postcopulatory processes, in conjunction with a probabilistic analytical model. We unexpectedly found that differential reproductive success was also a function of male population (GFP or RFP), suggesting that females use different CFC mechanisms to select for different male traits. Moreover, concordance of selection at the precopulatory (as measured by mating latency) and postcopulatory stages depends on both the male trait and the CFC mechanism examined. Larger males were more successful both before and after mating, but we unexpectedly found that females also mated more quickly with males with GFP-labeled sperm, while fertilization bias favored RFP-labeled sperm.