RESUMEN
Reinforcement occurs when behavioral isolation is strengthened between species due to selection against hybridization in sympatry. Mate preferences and their target traits may change in sympatry as a consequence of reinforcement. This can potentially generate further behavioral isolation within species if sympatric populations evolve extreme preferences or traits that cause them to reject individuals from foreign populations as mates or be rejected as mates. This process is known as cascade reinforcement. We measured behavioral isolation between sympatric and allopatric populations of Lucania killifish to determine whether isolation evolves due to reinforcement between species and whether reinforcement affects preferences within species, consistent with the cascade reinforcement hypothesis. We measured mate preferences in both sexes between species (Lucania parva vs. Lucania goodei) and within species (among populations of L. parva). Between species, both male and female preferences for conspecifics were highest in sympatric populations. Within species, L. parva females from sympatric populations preferred their own native males over foreign males. Allopatric L. parva females and all L. parva males showed no preferences within species. Within species, behavioral isolation showed no association with ecological variables, such as salinity. Thus, reinforcement is a primary factor generating behavioral isolation in Lucania killifish, creating strong preferences in both sexes among species and leading to cascade reinforcement of female mate preference within species.