The role of female reproductive fluid in mediating reproductive isolation between ecotypes of the three-spined stickleback

Abstract Female reproductive fluid (FRF) is increasingly recognised as a potent mediator of gametic-level cryptic female mating bias in external fertilisers, yet its role in reproductive isolation during early ecological divergence remains largely untested. We examined whether FRF from three-spined stickleback ( Gasterosteus aculeatus ) females on North Uist differentially attracts and sustains sperm from armourless (acidic loch) versus armoured (basic loch) ecotypes. Using a fully crossed diallel design across four allopatric populations and a sympatric contact zone, we quantified spermatozoa accumulation in a sperm-selection chamber and spermatozoa swimming longevity in response to conspecific and heterospecific FRF versus loch-water controls. More spermatozoa accumulated in selection chambers in response to FRF than in paired controls. Both sperm attraction and swimming longevity were enhanced by FRF. Attraction was stronger in same-ecotype pairings, whereas longevity showed clear male-environment-by-medium interactions, with acid- and basic-adapted males performing best in water chemistry matching their natal habitat. FRF therefore has the potential to function as a previously unrecognised gametic barrier, reinforcing pre-mating isolation and reducing gene flow between recently diverged ecotypes in this adaptive radiation.