Reticulate Evolution in Japanese Sceptridium (Ophioglossaceae), including diploid, tetraploid, and hexaploid species

Homospory represents an intriguing reproductive strategy, including the potential for gametophytic selfing, considered the ultimate form of selfing. The plants of the fern genus Sceptridium (Ophioglossaceae) are generally considered to be predominantly selfing, making them suitable for analyzing speciation patterns and intraspecific variation characteristic of homosporous plants. The Sceptridium species in Japan not only exhibit variations in ploidy, including diploids, tetraploids, and hexaploids, but also pose taxonomic challenges due to morphologically distinctive forms that are difficult to be assigned to previously described species. We developed multiple single-copy nuclear markers and aimed to identify ancestral species of the polyploids and to elucidate the evolutionary entity of an undescribed species. Chromosome counts and analyses of individual gene trees confirmed that S. japonicum, S. atrovirens, and the undescribed species are allohexaploids originating through hybridization between a maternal tetraploid ancestor closely related to S. formosanum and a paternal diploid ancestor closely related to extant diploid species. By identifying homoeologs derived from the diploid ancestor, we successfully analyzed the phylogenetic relationship between the diploid ancestor of the hexaploid species and extant diploid species using the multispecies coalescent model. The examined undescribed species could not be distinguished from S. atrovirens by multiple nuclear markers or by SNP data obtained from multiplexed ISSR genotyping by sequencing. This species appears to represent one of the polymorphisms of S. atrovirens, which was fixed through gametophytic selfing.