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.

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

Our results confirm that reproductive isolation can quickly arise from diversifying (allopatric) postcopulatory sexual selection. This experimental approach to "speciation phenotypes" illustrates how knowledge of sperm precedence mechanisms can be used to predict the mechanisms and extent of reproductive isolation between populations and species.

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