Larger sperm size may contribute to reproductive isolation between Etheostoma species.

Etheostoma is a genus of North American darter fish whose species have similar habitats and breeding seasons, yet hybridization is rare. Behavioral barriers have been demonstrated to play a key role in maintaining species boundaries. Further, conspecific (same species) sperm precedence has also been observed when the gametes of two different species come into contact. In this study, we investigated if physical characteristics of sperm could be a mechanism for the lower fertilization success of heterospecific (different species) males when eggs are simultaneously exposed to conspecific and heterospecific sperm. We chose to examine the sperm of two closely related species, E. zonale and E. barrenense. Using toluidine blue and immunofluorescent labeling methods, we compared head diameter and tail length of sperm cells between the two species. We found that head diameter was significantly larger for E. barrenense sperm compared to E. zonale. This difference in cell morphology may point to a physical mechanism underlying conspecific sperm precedence in Etheostoma. Our results are the first to describe a morphological difference in sperm between species in this genus and provide initial evidence for the role of sperm morphology in prezygotic reproductive isolation.