A Genetic Test for Whether Pairs of Hermaphrodites Can Cross-Fertilize in a Selfing Killifish

Kryptolebias marmoratus, a small killifish that lives in mangrove habitat from southern Florida to Brazil, is one of the planet's only known self-fertilizing hermaphroditic vertebrates. Generation after generation, hermaphroditic individuals simultaneously produce sperm and eggs and internally self-fertilize to produce what are, in effect, highly inbred clones of themselves. Although populations are composed primarily of hermaphrodites, they also contain some true males. The frequency of males in a population varies geographically, from < 2% in Florida to as high as 25% in Belize. Males are known to mate occasionally with hermaphrodites, thereby releasing genetic variation that has profound consequences for population genetic structure. However, it is unknown whether hermaphrodites can or do sporadically mate with each other also. Here, we test whether hermaphroditic individuals of the killifish Kryptolebias marmoratus are capable of crossing with one another, in addition to their much more common habits of self-fertilization and occasional outcrossing with pure males. We employ an experimental design in which replicate hermaphrodite pairs were housed together and allowed to reproduce naturally. Among 173 embryos screened at diagnostic microsatellite loci, all were found to result from selfing (i.e., no embryos were the product of a hermaphrodite cross). We thus conclude that hermaphrodite pairs are unlikely to cross, or do so exceedingly rarely.