Differential sperm usage from consecutive matings, or sperm precedence, is vital in determining male reproductive success and the outcome of sperm competition for many organisms. Sperm precedence also has significant consequences for mating system dynamics, including both male and female adaptations for increasing reproductive success and avoiding the costs of mating. Despite sexual selection being a strong driver of reproductive behaviour and morphology in cephalopods, surprisingly few studies have investigated sperm dynamics in this group. To redress this gap, we experimentally quantified sperm precedence patterns in the dumpling squid, Euprymna tasmanica, controlling for recent male mating history (first vs. second mating), mating position, and mating frequency. We found that the last male to mate gains an advantage in this system, with the second mating male siring up to 75% of offspring at the beginning of the laying period. The proportion of offspring attributable to the second mating male decreases to 54% by the end of the laying period, potentially as a result of changes in the velocity or number of sperm released from spermatangia over time. There is also significant variation among females in patterns of sperm precedence. This variation was not associated with whether it was the male's first or second mating, male mass, the duration of copulation or the number of pumps (sperm removal behaviour) by the second male. If widespread in cephalopods, last male sperm precedence could help to explain the evolution of mate guarding (or long copulation duration) and sperm removal behaviour in this group
