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Osmunda regalis and O. japonica are geographically isolated independent species. Contrary to this morphologically based taxonomy, previous plastid DNA data have shown that O. regalis is paraphyletic, with the American O. regalis sister to O. japonica and O. lancea. It remains to be confirmed whether this paraphyletic topology results from hybridization, budding speciation, or classification issues. To clarify the evolutionary history of O. regalis and O. japonica, we performed multiple molecular phylogenetic analyses using 38 worldwide samples and sequences from 15 nuclear and six plastid DNA regions. The phylogenetic trees of the nuclear and plastid single-region analyses were incongruent. Phylogenetic networks were also discordant between the nuclear and plastid regions. Statistical analysis for detecting the introgressed sequences found hybridization signals between the American O. regalis and O. japonica lineage, particularly in plastid regions. Along with the paleobotanical data, the results suggest that the ancient introgression occurred at an estimated time of 12.8–4.1 Ma in East Asia, when and where both species coexisted. Post-hybridization geographical fluctuation led to the present amphi-Pacific distribution pattern. The phylogenetic network suggests that the New World O. regalis spread across continents in a relatively short time, while the Old World O. regalis experienced long-term geographical isolation between (sub)continents.

Premise of research. Osmunda regalis has a worldwide distribution, except for in eastern Asia, where Osmunda japonica and Osmunda lancea can be found instead. However, the present geographic separation does not necessarily mean geographical speciation. Molecular data indicated that American O. regalis is sister to O. japonica and O. lancea rather than to European O. regalis, but the relationship between the distribution and phylogeny remained unclear. An analysis of vegetative pinnule fossils, which were abundant in the Cenozoic time, would help elucidate the historical biogeography and evolution of these species if the taxonomy of the fossils would be clarified.Methodology. We measured interveinlet intervals in mature and juvenile plants of the extant species and fossils from Japan and the rest of the world, as well as the size of their pinnules, and compared them through geological time and during ontogeny.Pivotal results. The interveinlet interval differs between O. regalis and O. japonica, and part of...

The chromosome number and phylogenetic position of the monotypic eusporangiate genus Mankyua (Ophioglossaceae), which shares peculiar morphological characteristics with Ophioglossum s.l. and Helminthostachys, were determined. Meiotic and mitotic chromosome observations of Mankyua show n = 130 and 2n = ca. 260, suggesting that the base number of this genus is x = 130, which is a new finding and the highest base number reported so far for Ophioglossaceae. The molecular phylogenetic trees derived from nucleotide sequence variation of rbcL and matK genes differ in terms of the phylogenetic position of Mankyua. The molecular phylogenetic trees based on combined data sets of rbcL and matK suggest that Mankyua might be the earliest-diverging lineage of Ophioglossaceae.

The southern Chinese Osmunda ×mildei has been suggested to be an intersubgeneric hybrid, i.e., O. japonica (subgenus Osmunda) × O. angustifolia (subgenus Plenasium) or O. japonica × O. vachellii (subgenus Plenasium). These interpretations were based on morphological, cytological, and/or chloroplast DNA data, yet the parents of the hybrid remained unclear. Molecular phylogenetic relationships inferred here from chloroplast rbcL sequences and three nuclear DNA markers show that O. ×mildei is most likely a hybrid between the paternal O. japonica and the maternal O. vachellii.

Osmunda subgenus Osmunda is variable across its worldwide range. In addition to the three species assigned to the subgenus, there is an enigmatic group of plants in Laos and Myanmar. To reveal the taxonomic and evolutionary status of these plants, we performed taxonomic, cytological, and flow-cytometric analyses, and molecular phylogenetic analyses for 32 samples of subgenus Osmunda for three nuclear DNA markers and the chloroplast rbcL gene. Results show that the enigmatic plants are a tetraploid (n = 44, 2n = 88) of hybrid origin from Osmunda regalis and O. japonica, which is described as Osmunda hybrida sp. nov. This is the first record of a naturally occurring polyploid species in the most primitive leptosporangiate family Osmundaceae, previously thought to be diploid throughout. Molecular data suggest that Indian O. regalis is a putative maternal ancestor and O. japonica is a paternal ancestor. Osmunda hybrida shares the fully dimorphic or partially dimorphic leaves with its parental species...

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.