Sadamu Matsumoto

NEW AND RARE FLAVONOL GLYCOSIDES FROM ASPLENIUM TRICHOMANES-RAMOSUM AS STABLE CHEMOTAXONOMIC MARKERS

Abstract Sixteen flavonol glycosides, including new and rare ones, were isolated from Asplenium trichomanes-ramosum . Major glycosides were quercetin 3-methyl ether 5- O -glucoside, kaempferol 3- O -arabinoside-7- O -rhamnoside, kaempferol 3- O -glucoside-7- O -rhamnoside, kaempferol 3,7-di- O -rhamnoside, quercetin 3- O -rhamnoside-7- O -arabinoside and quercetin 3- O -glucoside-7-rhamnoside, which were accompanied with traces of two quercetin 3-methyl ether 5- O -glycosides and three kaempferol 3,5- O -glycosides. Flavonol 5- O -glycosides were found in the genus Asplenium for the first time. The new or rare glycosides were consistently detected in all samples, which were collected at representative places in the Northern Hemisphere (the oldest sample was collected in 1879) and show that the flavonoids are extremely stable as chemotaxonomic markers for defining the species.

Reconstructing Hybrid Speciation Events in the Pteris cretica Group (Pteridaceae) in Japan and Adjacent Regions

Abstract Polyploidy, hybridization, and agamospory have been considered important mechanisms in fern speciation. By integrating the methods of cytology, molecular phylogeny, and morphology, we examined the origins of polyploid species in the Pteris cretica group, which comprises five agamosporous taxa and six sexual species. Phylogenetic analysis was conducted using both cpDNA (rbcL and trnV-trnM) and a low-copy nuclear gene (gapCp). The combined results of cytology and the phylogenetic trees suggested that the sexual diploid P. kidoi had played a central role in the diversification of polyploid species in the P. cretica group. Some triploid clones of agamosporous P. cretica var. cretica originated through hybridization between the diploid cytotype of P. cretica var. cretica and the sexual diploid P. kidoi. The sexual polyploid species, P. henryi, P. multifida, P. ryukyuensis, and P. yamatensis, and the hexaploid race of P. deltodon have arisen through allopolyploidization between their respective ancestral parent species and P. kidoi. Additionally, the agamosporous triploid P. nipponica and P. cretica var. albolineata might have originated through hybridization between P. cretica var. cretica and an unknown ancestral diploid parent of P. ryukyuensis. Agamosporous P. cretica var. cretica harbored considerable genetic variation within both the diploid and triploid cytotypes. Because we could not find clear genetic differences between the diploid and triploid P. cretica, both cytotypes might be autopolyploids, or alternatively have originated through hybridization among the same members of ancestral sexual species.

Flavonoid variation and evolution inAsplenium normale and related species (Aspleniaceae)

Flavonoid profiles of 132 populations (472 individuals) ofAsplenium normale, and related species,A. boreale, A. shimurae, andA. oligophlebium var.oligophlebium and var.iezimaense in Japan were surveyed by HPLC and 2D-PC. Of the five taxa, each ofAsplenium boreale, A. shimurae andA. oligophlebium including var.iezimaense had consistent flavonoid composition: apigenin 7, 4′-di-O-rhamnoside (9) inAsplenium boreale, 7-O-glucosyirhamnosides of apigenin and luteolin (6 and 7) inA. shimurae and genkwanin 4′-O-glucosyl-rhamnoside (5) in twoA. oligophlebium varieties. On the other hand,Asplenium normale was divided into seven chemotypes A-G: A-type has 7-O-dirhamnosides of apigenin and luteolin (1 and 2) and genkwanin 4′-O-glucosylrhamnoside (5); B-type, 5 alone; C-type, apigenin 7-O-rhamnoside-4′-O-glucosylrhamnoside (8); D-type, 1 and 2; E-type, 1,2 and 8; F-type, 1, 2, 5 and 8; and G-type, 5 and 8. Among them, the most frequent types were A, B and C, and A-type was mainly distributed in inland of Honshu, Shikoku and Kyushu, while B- and C-types extended their distribution areas southwards in general and occur along the Pacific coast with several exception. Chemical and evolutionary relationships amongAsplenium boreale, A. shimurae, A. oligophlebium, and the chemotypes ofA. normale were discussed on the basis of general biosynthetic pathway.

Apigenin 7-rhamnoside-4′-glucosylrhamnoside from Asplenium normale

Abstract A new flavone glycoside, apigenin 7- O -α- l -rhamnoside-4′- O -α- l -glucosylrhamnoside was identified in fronds of Asplenium normale .

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.×stenosiphon) 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.

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.

Evidence by flavonoid markers of four natural hybrids among Asplenium normale and related species (Aspleniaceae) in Japan

Abstract The flavonoid patterns of sterile hybrids, Asplenium normale × A. boreale , A. normale × A. shimurae , A. normale × A. oligophlebium and A. boreale × A. oligophlebium , were surveyed by 2D-PC and HPLC, and compared with those of their parental species. Apigenin-7- O -rhamnoside (1), luteolin-7- O -dirhamnoside (2), genkwanin-4′- O -glucosylrhamnoside (3), apigenin-7- O -rhamnoside-4′- O -glucosyl-rhamnoside and its derivatives (4) and apigenin 7,4′-di- O -rhamnoside (5) were found in ten populations, and 3+4+5, 4 and 3+5 in one population each of A. normale × A. boreale ; apigenin-7- O -glucosylrhamnoside (6), luteolin-7- O -glucosylrhamnoside (7), 1 and 2 in two populations, and 3+6+7 and 6+7 in one population each of A. normale × A. shimurae ; 1+2+3 or 1+2+3+4 in ten populations, and 4 in 2 populations of A. normale × A. oligophlebium ; and 3+4+5 in A. boreale × A. oligophlebium. Two C -glycosylflavones, apigenin-6,8-di- C -glucoside (vicenin-2) (8) and luteolin-6,8-di- C -glycoside (9) were detected from all the individuals of the four natural hybrids. Most flavonoids in MeOH frond extracts of the hybrids show total addition of parental attributes.

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.

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.