Allotetraploid cryptic species in Asplenium normale in the Japanese Archipelago, detected by chemotaxonomic and multi-locus genotype approaches.

PREMISE OF THE STUDY: Delimitation of cryptic species provides an understanding of biodiversity and opportunities to elucidate speciation processes. Extensive flavonoid variation has been reported in the tetraploid cytotype of the fern, Asplenium normale, although related species have no intraspecific variations in flavonoid composition. We hypothesized that Japanese A. normale still harbors multiple cryptic species with different flavonoid compositions, and tested this hypothesis using chemotaxonomic and multilocus genotyping approaches. METHODS: We determined the multilocus genotypes (MLGs) of 230 samples from 37 populations for one chloroplast DNA region and three nuclear genes. MLGs were used to delimit reproductively isolated lineages by population-genetic approaches. We also tested the correspondence between genetically recognized groups and flavonoid compositions. To identify the origins of putative cryptic species, we conducted phylogenetic analysis of the DNA markers used in genotyping. KEY RESULTS: The genetic clusters and flavonoid compositions showed clear correspondence. We recognized three putative cryptic species in tetraploid Asplenium normale in Japan. Phylogenetic analyses revealed that cryptic species I and III originated from allopolyploidization between a diploid A. normale and an unknown diploid of A. boreale, and cryptic species II originated from allopolyploidization between a diploid A. normale and A. oligophlebium. CONCLUSIONS: Our study demonstrated that intraspecific variation of secondary metabolites can be a good indicator of cryptic species in ferns. The presence of the two cryptic species having the same progenitor diploid pair suggests that speciation between allopolyploid lineages of independent origin may be more common than previously considered.

Enzymatic (13)C labeling and multidimensional NMR analysis of miltiradiene synthesized by bifunctional diterpene cyclase in Selaginella moellendorffii.

Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-(13)C(6)]mevalonate, all carbons were labeled with (13)C stable isotope (>99%). The fully (13)C-labeled product was subjected to (13)C-(13)C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and multidimensional (13)C NMR analyses of completely (13)C-labeled compound are powerful methods for biosynthetic studies.

Origin of Dryopteris shibipedis (Dryopteridaceae), a fern species extinct in the wild.

Dryopteris shibipedis was once treated as an extinct species in the Red List (2007) by the Japanese Ministry of the Environment, but 'rediscovered' in the Tsukuba Botanical Garden. To clarify its origin, using 'overlooked' cultivated stocks we analyzed nuclear PgiC intron sequences. As the PgiC genotype of D. shibipedis can be explained by a combination of alleles of D. kinkiensis and those of D. pacifica, the hypothesis of hybrid origin of the species is supported.

Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan.

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Hybridization involving independent gametophytes in the Vandenboschia radicans complex (Hymenophyllaceae): a new perspective on the distribution of fern hybrids.

To test our hypothesis of hybrid formation involving the 'independent gametophyte' phenomenon in ferns, we identified the genomic formulae and ploidy level of gametophytes of the Vandenboschia radicans complex at the periphery of a sporophyte population. We identified haploid gametophytes of V. kalamocarpa (one of the two putative parents of V.xstenosiphon) in a hybrid sporophyte population in Japan that lacks fertile non-hybrid individuals. Furthermore, diploid sporophytes of the species were not found within a 50-km radius. This finding supports a hypothesis of hybridization involving the 'independent gametophyte' phenomenon and provides a new perspective on the geographical distribution of fern hybrids.

The Relationship between Mating System and Genetic Diversity in Diploid Sexual Populations of Cyrtomium falcatum in Japan.

The impact of variation in mating system on genetic diversity is a well-debated topic in evolutionary biology. The diploid sexual race of Cyrtomium falcatum (Japanese holly fern) shows mating system variation, i.e., it displays two different types of sexual expression (gametangia formation) in gametophytes: mixed (M) type and separate (S) type. We examined whether there is variation in the selfing rate among populations of this species, and evaluated the relationship between mating system, genetic diversity and effective population size using microsatellites. In this study, we developed eight new microsatellite markers and evaluated genetic diversity and structure of seven populations (four M-type and three S-type). Past effective population sizes (Ne) were inferred using Approximate Bayesian computation (ABC). The values of fixation index (FIS), allelic richness (AR) and gene diversity (h) differed significantly between the M-type (FIS: 0.626, AR: 1.999, h: 0.152) and the S-type (FIS: 0.208, AR: 2.718, h: 0.367) populations (when admixed individuals were removed from two populations). Although evidence of past bottleneck events was detected in all populations by ABC, the current Ne of the M-type populations was about a third of that of the S-type populations. These results suggest that the M-type populations have experienced more frequent bottlenecks, which could be related to their higher colonization ability via gametophytic selfing. Although high population differentiation among populations was detected (FST = 0.581, F'ST = 0.739), there was no clear genetic differentiation between the M- and S-types. Instead, significant isolation by distance was detected among all populations. These results suggest that mating system variation in this species is generated by the selection for single spore colonization during local extinction and recolonization events and there is no genetic structure due to mating system.

Genkwanin 4'-O-glucosyl-(1 --> 2)-rhamnoside from new chemotype of Asplenium normale in Japan.

New flavone glycoside, genkwanin 4'-O-ß-glucopyranosyl-(1 --> 2)-O-α-rhamnopyranoside was isolated from the fronds of new chemotype of Asplenium normale D. Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structure of the isolated glycoside was established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Apigenin di- and trirhamnoside from Asplenium normale in Malaysia.

Two new flavone rhamnosides, apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside and apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside-4'-O-alpha-L-rhamnopyranoside were isolated from the fronds of Asplenium normale D. Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structures of the isolated glycosides were established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas.

Species complexes consisting of ill-defined "species" are widely known among ferns, and their involvement with reticulate evolution is expected. Nevertheless approaches to reticulation history with DNA markers are not yet commonly adopted. We have successfully elucidated the biological status of the Vandenboschia radicans complex in East Asian islands by combining analyses of ploidy level, a cpDNA marker (rbcL), and a nuclear DNA marker (GapCp). The results based on 266 individuals collected from 174 localities throughout Japan and Taiwan suggest that complicated hybridizations have occurred involving at least three parental diploid species from within the V. radicans complex and Vandenboschia liukiuensis, which was formerly considered to be distinct from this complex. Triploids are the most common cytotype, but they show no evidence of apogamous reproduction, while all nonhybrid diploids are rare and have very limited distribution. Possible accounts of this phenomenon will be briefly discussed including the possibility of relict distribution and occasional apogamous reproduction.

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants.

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.