Primers for 36 microsatellite loci were developed and employed to characterize genetic stocks and detect possible outcrossing between highly inbred laboratory strains of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. From attempted crosses involving hermaphrodites from particular geographic strains and gonochoristic males from others, 2 among a total of 32 surveyed progenies (6.2%) displayed multilocus heterozygosity clearly indicative of interstrain gametic syngamy. One of these outcross hybrids was allowed to resume self-fertilization, and microsatellite assays of progeny showed that heterozygosity decreased by approximately 50% after one generation, as expected. Although populations of K. marmoratus consist mostly of synchronous hermaphrodites with efficient mechanisms of internal self-fertilization, these laboratory findings experimentally confirm that conspecific males can mediate occasional outcross events and that this process can release extensive genic heterozygosity.

The hermaphroditic Mangrove Killifish, Kryptolebias marmoratus, is the world's only vertebrate that routinely self-fertilizes. As such, highly inbred and presumably isogenic “clonal” lineages of this androdioecious species have long been maintained in several laboratories and used in a wide variety of experiments that require genetically uniform vertebrate specimens. Here we conduct a genetic inventory of essentially all laboratory stocks of the Mangrove Killifish held worldwide. At 32 microsatellite loci, these stocks proved to show extensive interline differentiation as well as some intraline variation, much of which can be attributed to post-origin de novo mutations and/or to the segregation of polymorphisms from wild progenitors. Our genetic findings also document that many of the surveyed laboratory strains are not what they have been labeled, apparently due to the rather frequent mishandling or unintended mixing of various laboratory stocks over the years. Our genetic inventory should help to clarify much of this confusion about the clonal identities and genetic relationships of laboratory lines, and thereby help to rejuvenate interest in K. marmoratus as a reliable vertebrate model for experimental research that requires or can capitalize upon “clonal” replicate specimens.