Male alternative reproductive tactics reflect complex spatial and temporal dynamics of fertilization in the squid

Abstract

Abstract Males adopting alternative reproductive tactics (ARTs) often face uneven sperm competition, leading to asymmetrical pressures on ejaculate expenditure. These asymmetries are further influenced by spatio-temporal variations in fertilization context, such as where and when males release sperm relative to female oocytes. In loliginid squids, females have two spatially separated fertilization sites (internal oviduct opening and external buccal membrane) associated with consort and sneaker tactics, respectively. Males with alternative phenotypes, therefore, face asymmetrical pressures both in sperm competition and fertilization context. Consort males have long been predicted to dominate paternity due to their access to the internal site, making it difficult to understand the role of sneakers, whose sperm are stored at the buccal membrane. Using the loliginid Doryteuthis pleii , we empirically obtained evidence that fertilization sites dictate male success. Sperm at the oviduct monopolizes fertilization when spawning females have sperm at both sites. This clarifies long-standing enigmas regarding squid ARTs. For example, previous studies showed that sneakers have lower sperm investment per mating than consorts – our results indicate that this strategy could be associated with the lower availability of unfertilized oocytes at the buccal membrane. Conversely, when females lack sperm at the oviduct, the last sneaker to mate sires most offspring. This indicates that while the buccal membrane provides long-term sperm storage for several sneaker males, last-male sperm precedence ultimately dictates fertilization success at the external site, revealing complex postcopulatory sexual selection at the buccal membrane. Overall, these results corroborate the critical importance of fertilization context in the evolution of ARTs and highlight loliginid squids as excellent models to test how fertilization environment and sperm competition asymmetries shape diverging adaptations in male ARTs.