206
Ligularia tangutica (Maximowicz) Bergmans obtained from Berlin, Germany
(sample 29), afforded 3α-angeloyloxyeremophila-9,11-dien-8-one (55), (−)-germacrene D (758), and cis-β-ocimene (875) [13].
Sample 30, L. trichocephala (Maximowicz) Matsumura et Koidzumi (from
Moscow, Russia), produced seven 1,10-epoxyfuranoeremophilanes (288, 290, 291,
293 (major constituent), 296, 300, and 303) [13]. The chemical constituents of
L. trichocephala were found to be similar to those of L. sagitta (Sect. 3.21).
Ligularia vorobierii Worosh (sample 31 from Moscow, Russia) produced nine
1β,10β,-epoxyfuranoeremophilanes (288, 290 (major constituent), 291, 293, 296,
299, 300, 302, 303) and (−)-germacrene D (758) [164]. All of the above specimens
except for samples 30 and 31 produced cis-β-ocimene (875).
4 Genetic Analyses
In our study on the chemical diversity in Ligularia species growing in the Hengduan
Mountains area of China, also DNA sequences of each sample have been studied.
We chose base sequences of atpB-rbcL (a segment between the atpB and the rbcL
genes on the plastid DNA) [305] and the two internal transcribed spacers (ITS1-5.8SITS2) of the ribosomal RNA gene in the nuclear genome [304]. These sequences are
noncoding, and variations therein are thought to be neutral to evolution, i.e., these
sequences indicate the history of each sample. Therefore, a combination of the
chemical aspects (isolation and structure determination) and the genetic aspects
(DNA sequences) should help in understanding diversification and evolution in a
correct manner. In this section, studies conducted on L. virgaurea and L. kanaitzensis are described briefly as examples.
The ITS (internal transcribed spacer) sequences of L. virgaurea samples collected in southwestern Sichuan Province are shown in Table 50; the sample numbers
are the same as shown in Table 1. The 11 analyzed samples could be grouped into 2
clades, A and B. These clades correspond to the L and V types, respectively, in
terms of their chemical composition. This indicates that the differences observed in
chemical composition (Sect. 3.1) originate from their genetic differences [63].
Subsequently, 41 additional samples of L. virgaurea were analyzed, which were
collected widely in northern to central Sichuan Province and partly in Gansu and
Qinghai Provinces. We found the occurrence of five chemotypes, the L, V, C, H, and
N types, although the number of samples of the C type was not very high [45, 114]
(Sect. 3.1). The ITS sequences of these samples were determined to produce three
clades, A, B, and C. Samples belonging to the new clade, Clade C, were also different from the other samples in the atpB-rbcL sequences. Correlations between the
chemotypes and the DNA clades were observed; thus, samples of chemotypes C and
H belonged to clade C, while chemotypes V and N belonged to clade B. As described
earlier, chemotypes V, H, N, and C were continuous [76], while samples belonging
to the type L (= ITS clade A) had no correlation with the other samples.
As another example, DNA sequences of L. kanaitzensis are shown in Table 51.
Twelve samples collected in Shangri-La County, Yunnan Province, and adjacent
M. Tori and C. Kuroda
Ligularia tangutica (Maximowicz) Bergmans obtained from Berlin, Germany
(sample 29), afforded 3α-angeloyloxyeremophila-9,11-dien-8-one (55), (−)-germacrene D (758), and cis-β-ocimene (875) [13].
Sample 30, L. trichocephala (Maximowicz) Matsumura et Koidzumi (from
Moscow, Russia), produced seven 1,10-epoxyfuranoeremophilanes (288, 290, 291,
293 (major constituent), 296, 300, and 303) [13]. The chemical constituents of
L. trichocephala were found to be similar to those of L. sagitta (Sect. 3.21).
Ligularia vorobierii Worosh (sample 31 from Moscow, Russia) produced nine
1β,10β,-epoxyfuranoeremophilanes (288, 290 (major constituent), 291, 293, 296,
299, 300, 302, 303) and (−)-germacrene D (758) [164]. All of the above specimens
except for samples 30 and 31 produced cis-β-ocimene (875).
4 Genetic Analyses
In our study on the chemical diversity in Ligularia species growing in the Hengduan
Mountains area of China, also DNA sequences of each sample have been studied.
We chose base sequences of atpB-rbcL (a segment between the atpB and the rbcL
genes on the plastid DNA) [305] and the two internal transcribed spacers (ITS1-5.8SITS2) of the ribosomal RNA gene in the nuclear genome [304]. These sequences are
noncoding, and variations therein are thought to be neutral to evolution, i.e., these
sequences indicate the history of each sample. Therefore, a combination of the
chemical aspects (isolation and structure determination) and the genetic aspects
(DNA sequences) should help in understanding diversification and evolution in a
correct manner. In this section, studies conducted on L. virgaurea and L. kanaitzensis are described briefly as examples.
The ITS (internal transcribed spacer) sequences of L. virgaurea samples collected in southwestern Sichuan Province are shown in Table 50; the sample numbers
are the same as shown in Table 1. The 11 analyzed samples could be grouped into 2
clades, A and B. These clades correspond to the L and V types, respectively, in
terms of their chemical composition. This indicates that the differences observed in
chemical composition (Sect. 3.1) originate from their genetic differences [63].
Subsequently, 41 additional samples of L. virgaurea were analyzed, which were
collected widely in northern to central Sichuan Province and partly in Gansu and
Qinghai Provinces. We found the occurrence of five chemotypes, the L, V, C, H, and
N types, although the number of samples of the C type was not very high [45, 114]
(Sect. 3.1). The ITS sequences of these samples were determined to produce three
clades, A, B, and C. Samples belonging to the new clade, Clade C, were also different from the other samples in the atpB-rbcL sequences. Correlations between the
chemotypes and the DNA clades were observed; thus, samples of chemotypes C and
H belonged to clade C, while chemotypes V and N belonged to clade B. As described
earlier, chemotypes V, H, N, and C were continuous [76], while samples belonging
to the type L (= ITS clade A) had no correlation with the other samples.
As another example, DNA sequences of L. kanaitzensis are shown in Table 51.
Twelve samples collected in Shangri-La County, Yunnan Province, and adjacent
M. Tori and C. Kuroda