Chiaki Kuroda

Natural hybridization and introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae, Senecioneae) in four different locations.

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.

Natural hybridization and introgression in sympatric Ligularia species (Asteraceae, Senecioneae)

Abstractu2002 The difficulty in clarifying species of genus Ligularia Cass. has been attributed to rapid and continuous allopatric speciation in small and isolated populations, combined with interspecific diploid hybridization in the Qinghai‐Tibetan Plateau and adjacent areas. However, no concrete example has been reported to prove this hypothesis. We studied a natural mixed population of six species of Ligularia in which some individuals were morphologically intermediate between L. subspicata and L. nelumbifolia. Based on DNA sequences (trnC‐F, trnL‐rpL32, trnQ‐5′rps16, trnK‐rps16, and internal transcribed spacer) and inter‐simple sequence repeat data, we concluded that putative hybrids are primarily products of hybridization between L. nelumbifolia and L. subspicata. The other four species or additional, unknown species may also be involved in hybridization. This hybridization is bidirectional but asymmetrical. Hybrid individuals were mostly the first generation, but F2 and later‐generation hybrids were also present. Moreover, the backcrossed individuals detected indicate that natural gene flow occurs among at least three Ligularia species. Hybrids may become stabilized to form new species or may function as intermediates in evolutionary diversification.

Chemical and genetic similarity and diversity of Ligularia anoleuca and L. fischeri collected in the Hengduan Mountains of China.

The sesquiterpenoid composition in the root and the DNA sequences of evolutionarily neutral regions were studied in Ligularia anoleuca and Ligularia fischeri (Asteraceae) collected in the Sichuan Province of China. LC-MS analysis showed that L. anoleuca populations from different localities had different chemical compositions. However, the isolated compounds were similar to each other, indicating that the differences in chemical composition were not large. The DNA analysis suggested that the two species were indistinguishable. Seventeen furanoeremophilanes and an eremophilane acetal were isolated.

Chemical and genetic diversity of Cremanthodium lineare

Chemical constituents and evolutionally neutral DNA sequences of six samples of Cremanthodium lineare Maxim., collected in the Sichuan Province of China, were studied. Three samples produced furanoeremophilanes and the other three, eremophilan-8-ones. The chemotypes were found to be correlated with DNA sequence types, suggesting that the chemical diversity observed has a genetic origin. Production of furanoeremophilanes by a Cremanthodium species suggests an evolutionary relationship between Cremanthodium and Ligularia species, and possibly to related genera.

Direct MS measurement of the extract of Ligularia virgaurea collected in Yunnan and Sichuan provinces of China

INTRODUCTIONnThere are numerous Ligularia species in the eastern Qinghai-Tibet Plateau and adjacent areas. L. virgaurea has been used as a traditional folk medicine for the treatment of stomachache and nausea. Objective - To analyse chemical constituents of L. virgaurea, grown in Yunnan and Sichuan provinces of China.nnnMETHODOLOGYnThe direct MS measurement of the crude extract of plant samples was used for grouping of this species. As the main compounds were available in pure form, the main peaks were analysed by LCMS.nnnRESULTSnAn easy and speedy method of analysis using MS spectra was developed. On the basis of the findings, L. virgaurea could be divided into two groups. The genetic studies also supported this grouping. Type 1 mainly includes virgaurenones and virgaurenolides. The MS of type 2 is quite different because it includes mainly ligularol and its congeners. Both MS were easily distinguished.nnnCONCLUSIONnThe crude extracts of 11 L. virgaurea samples already collected in recent years were analysed and it was possible to identify them as type 1 or 2. This method was applied to three samples collected in 2009 to successfully classify them as either type 1 or 2.

Chemical Constituents in Hybrids of Ligularia tongolensis and L. cymbulifera: Chemical Introgression in L. tongolensis

Two samples with morphologies intermediate between Ligularia tongolensis and L. cymbulifera were collected in Desha, Sichuan Province, and one, in Pachahai, Yunnan Province, P. R. China. The DNA sequencing confirmed that the samples were hybrids of the two species. Tetradymol (1), the major compound of L. cymbulifera not found in L. tongolensis, was isolated from the hybrid samples collected at both locations, while furanoeremophilan‐15‐oic acid derivative 4, a compound characteristic to L. tongolensis, was found in the Pachahai hybrid but not in the Desha hybrids. Thus, the chemical consequence of hybridization can be variable. In addition, analysis of L. tongolensis samples at Pachahai indicated that introgression has been a mechanism of generating chemical diversity in the plant. Eleven compounds including three new ones were isolated.

Terpenoids and Phenylpropanoids in Ligularia duciformis, L. kongkalingensis, L. nelumbifolia, and L. limprichtii

The diversity in root chemicals and evolutionally neutral DNA regions in the complex of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia (the d/k/n complex) was studied using eight samples collected in central and northern Sichuan Province of China. Cacalol (14) and epicacalone (15), rearranged eremophilanes, were isolated from the complex for the first time. Two new phenylpropanoids were also obtained. Seven of the eight samples produced phenylpropanoids and the other produced lupeol alone. Two of the seven samples also produced furanoeremophilanes or their derivatives and one produced oplopanes. The geographical distribution of the sesquiterpene-producing populations suggests that the production of sesquiterpenes evolved independently in separate regions. L. limprichtii collected in northern Sichuan was also analyzed and its chemical composition and the sequence of internal transcribed spacers (ITSs) in the ribosomal RNA gene cluster were found to be similar to that in the d/k/n complex and L. yunnanensis, which are morphologically similar.