Our understanding of postcopulatory sexual selection has been constrained by an inability to discriminate competing sperm of different males, coupled with challenges of directly observing live sperm inside the female reproductive tract. Real-time and spatiotemporal analyses of sperm movement, storage, and use within female Drosophila melanogaster inseminated by two transgenic males with, respectively, green and red sperm heads allowed us to unambiguously discriminate among hypothesized mechanisms underlying sperm precedence, including physical displacement and incapacitation of "resident" sperm by second males, female ejection of sperm, and biased use of competing sperm for fertilization. We find that competitive male fertilization success derives from a multivariate process involving ejaculate-female and ejaculate-ejaculate interactions, as well as complex sperm behavior in vivo.

Cryptic female choice (CFC) represents postmating intersexual selection arising from female-driven mechanisms at or after mating that bias sperm use and impact male paternity share. Although biologists began to study CFC relatively late, largely spurred by Eberhard’s book published 20 years ago, the field has grown rapidly since then. Here, we review empirical progress to show that numerous female processes offer potential for CFC, from mating through to fertilization, although seldom has CFC been clearly demonstrated. We then evaluate functional implications, and argue that, under some conditions, CFC might have repercussions for female fitness, sexual conflict, and intersexual coevolution, with ramifications for related evolutionary phenomena, such as speciation. We conclude by identifying directions for future research in this rapidly growing field.

Success in sperm competition, occurring whenever females mate with multiple males, is predicted to be influenced by variation in ejaculate quality and interactions among competing sperm. Yet, apart from sperm number, relevant ejaculate characteristics and sperm-sperm interactions are poorly understood, particularly within a multivariate framework and the natural selective environment of the female reproductive tract. Here, we used isogenic lines of Drosophila melanogaster with distinguishable sperm to demonstrate and partition genetic variation in multiple sperm quality and performance traits. Next, by competing males from different lines, we show how rival sperm significantly influence each other's velocity and reveal that males with relatively slow and/or long sperm better displace rival sperm and resist displacement, thus avoiding ejection by the female from her reproductive tract. Finally, we establish fitness consequences of genetic variation in sperm quality and its role in securing a numerical advantage in storage by showing that offspring paternity is determined strictly by the representation of stored, competing sperm. These results provide novel insight into complex postcopulatory processes, illustrate that different ejaculate traits are critical at different biologically relevant time-points, and provide a critical foundation for elucidating the role of postcopulatory sexual selection in trait diversification and speciation.

How females store and use sperm after remating can generate postcopulatory sexual selection on male ejaculate traits. Variation in ejaculate performance traits generally is thought to be intrinsic to males but is likely to interact with the environment in which sperm compete (e.g., the female reproductive tract). Our understanding of female contributions to competitive fertilization success is limited, however, in part because of the challenges involved in observing events within the reproductive tract of internally fertilizing species while discriminating among sperm from competing males. Here, we used females from crosses among isogenic lines of Drosophila melanogaster, each mated to two genetically standardized males (the first with green-and the second with red-tagged sperm heads) to demonstrate heritable variation in female remating interval, progeny production rate, sperm-storage organ morphology, and a number of sperm performance, storage, and handling traits. We then used multivariate analyses to examine relationships between this female-mediated variation and competitive paternity. In particular, the timing of female ejection of excess second-male and displaced first-male sperm was genetically variable and, by terminating the process of sperm displacement, significantly influenced the relative numbers of sperm from each male competing for fertilization, and consequently biased paternity. Our results demonstrate that females do not simply provide a static arena for sperm competition but rather play an active and pivotal role in postcopulatory processes. Resolving the adaptive significance of genetic variation in female-mediated mechanisms of sperm handling is critical for understanding sexual selection, sexual conflict, and the coevolution of male and female reproductive traits.cryptic female choice | heritability | sperm ejection

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male-male competition and female choice as discrete processes.