Primers used in this study.

Unisexual hybrids that reproduce either clonally or hemiclonally are considered to be evolutionarily short-lived as they lack the ability to reduce deleterious mutations and increase genetic diversity. In the greenling (Teleostei: Hexagrammidae, genus Hexagrammos), unisexual hybrids that produce haploid eggs containing only the H. octogrammus (maternal species) genome generate hemiclonal offspring by fertilization with haploid sperm of H. agrammus (paternal species). When hemiclonal hybrids are backcrossed to a male of the maternal species, the offspring (BC-Hoc) are phenotypically similar to the maternal species and produce recombinant gametes through conventional meiosis. BC-Hoc (recombinant generation) individuals referred to as carriers harbor the genetic factor for hybridogenesis, thereby facilitating the production of new hemiclonal lineages through hybridization. Previous studies based on field research have suggested that the carriers produced by two-way backcrossing (mating pattern in which hemiclonal hybrids are backcrossed with both parental species) may overcome the evolutionary dead end imposed by the lack of recombination. The present study verified this hypothesis by regenerating a newly hemiclonal lineage through artificial hybridization. To clarify the genetic mode of hybrids produced by crosses between BC-Hoc and Hag, mature eggs were obtained from 16 individuals and fertilized with either Hag or Hoc sperm. Hybridogenesis was confirmed in one of the 16 individuals. Based on the low occurrence rate, these findings suggest that hemiclonal lineages can be regenerated, and that the hemiclonal factors are likely distributed across multiple genes on different chromosomes. The findings provide important evidence for the retention of a robust system for increasing genetic variability and maintaining evolutionary succession in unisexual hybrids that reproduce hemiclonally.