Precise mechanisms underlying sperm storage and utilization are largely unknown, and data directly linking stored sperm to paternity remain scarce. We used competitive microsatellite PCR to study the effects of female morphology, copula duration and oviposition on the proportion of stored sperm provided by the second of two copulating males (S2) in Scathophaga stercoraria (Diptera: Scathophagidae), the classic model for sperm competition studies. We genotyped all offspring from potentially mixed-paternity clutches to establish the relationship between a second male’s stored sperm (S2) and paternity success (P2). We found consistent skew in sperm storage across the three female spermathecae, with relatively more second-male sperm stored in the singlet spermatheca than in the doublet spermathecae. S2 generally decreased with increasing spermathecal size, consistent with either heightened first-male storage in larger spermathecae, or less efficient sperm displacement in them. Additionally, copula duration and several two-way interactions influenced S2, highlighting the complexity of postcopulatory processes and sperm storage. Importantly, S2 and P2 were strongly correlated. Manipulation of the timing of oviposition strongly influenced observed sperm-storage patterns, with higher S2 when females laid no eggs before being sacrificed than when they oviposited between copulations, an observation consistent with adaptive plasticity in insemination. Our results identified multiple factors influencing sperm storage, nevertheless suggesting that the proportion of stored sperm is strongly linked to paternity (i.e. a fair raffle). Even more detailed data in this vein are needed to evaluate the general importance of sperm competition relative to cryptic female choice in postcopulatory sexual selection.

These data were collected in the course of a lab experiment that manipulated the possibility and timing of oviposition relative to the occurrence of two consecutive mating events for female yellow dung flies. Following this manipulation, females were sacrificed, their reproductive tracts dissected, and the sperm stored within analysed using molecular markers to quantify the relative contributions of two males to stored sperm.

Further details of data processing are described in the associated paper, and an R script is also included to allow the reproduction of results and figures.

Metadata including keys to column names are in the associated ReadMe file.