The effects of experimental design on mating preferences and reproductive isolation in killifish

Determining the direction and magnitude of mating preference is fundamental to many questions in evolutionary biology. Unlike the measurement of traits such as body size, the measurement of mating preferences is likely affected by experimental design. Scientists must choose both the behavioral assay in which to measure preference and the metrics that serve as a proxy for preference. The accuracy of these assays and metrics, however, is often unknown and seldom tested. Here, we compared the accuracy of 3 assays (dichotomous choice, audience assay, and no-choice assay) and 3 metrics (association time, courtship bouts, and number of eggs produced) in the bluefin killifish, which possesses strong, conspecific mating preferences when in sympatry with rainwater killifish. We consistently detected preferences in both males and females when using metrics associated with mating (i.e., courting bouts and number of eggs spawned). However, we failed to consistently detect preference when using time...