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.

Foliar flavonoids from Tanacetum vulgare var. boreale and their geographical variation.

Foliar flavonoids of Tanacetum vulgare var. boreale were isolated. Eight flavonoid glycosides, 7-O-glucosides of apigenin, luteolin, scutellarein and 6- hydroxyluteolin, and 7-O-glucuronides of apigenin, luteolin, chrysoeriol and eriodictyol were identified. Moreover, eight flavonoid aglycones, apigenin, luteolin, hispidulin, nepetin, eupatilin, jaceosidin, pectolinarigenin and axillarin were also isolated and identified. The flavonoid composition of two varieties of T. vulgare, i.e. var. boreale and var. vulgare, were compared. All samples of var. boreale and one sample of var. vulgare had the same flavonoid pattern, and could be distinguished from almost all the samples of var. vulgare. Thus, the occurrence of chemotypes, which are characterized by either the presence or absence of scutellarein 7-O-glucoside, eriodictyol 7-O-glucuronide and pectolinarigenin was shown in T. vulgare sensu lato.

Further characterization of foliar flavonoids in Crossostephium chinense and their geographic variation.

Foliar flavonoids of Crossostephium chinense in Japan and Taiwan were isolated and further characterized. Eighteen flavonoid aglycones, luteolin, apigenin, hispidulin, chrysoeriol, 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone, jaceosidin, cilsimaritin, quercetin 3-methyl ether, axillarin, chrysosplenol-D, cirsiliol, apometzgerin, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, luteolin 3',4'-dimethyl ether, cirsilineol, eupatilin, nepetin and 5,7,3',4'-tetrahydroxy-6,5'-dimethoxyflavone, were identified by UV, 1H and 13C NMR spectroscopic, LC-MS and HPLC comparisons w ith authentic samples. The compounds existed on the leaf surface. Four flavonoid glycosides, quercetin 3,7-di-O-glucoside, quercetin 3-O-rutinoside, luteolin 7-O-glucoside and apigenin 7-O-rutinoside, were also isolated as the intracellular flavonoids. It was shown by HPLC survey that variation of the species' flavonoids occurs among the collection sites.

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.

Flavonoids from the Japanese monotypic genus, Nipponanthemum.

Foliar flavonoids of Nipponanthemum, which is a monotypic genus and consists of only one Japanese endemic species, N. nipponicum, were isolated for the first time. Fifteen flavonoids, i.e. six glycosides, quercetin 3,7-di-O-glucoside, vicenin-2, quercetin 3-O-glucoside, luteolin 7-O-glucoside, kaempferol 3-O-glucoside and apigenin 7-O-glucoside, and nine aglycones, luteolin, nepetin, quercetin 3-methyl ether, axillarin, apigenin, hispidulin, chrysoeriol, jaceosidin and sudachitin were identified. Of these compounds, the flavonoid aglycones existed on the leaf surface.