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Chemotaxonomy on the leaf constituents of Thujopsis dolabrata Sieb. et Zucc.—Analysis of neutral extracts (diterpene hydrocarbon)
Biochemical Systematics and Ecology 29 (2001) 839–848
Chemotaxonomy on the leaf constituents of Thujopsis dolabrata Sieb. et Zucc.}Analysis of neutral extracts (diterpene hydrocarbon) Koetsu Takahashia,*, Shizuo Nagahamab, Takurou Nakashimaa, Hiromi Suenagab a
Faculty of Agriculture, Yamagata University, Wakaba-machi 1-23, Tsuruoka 997, Japan b Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan Received 14 June 2000; accepted 17 November 2000
Abstract The leaf terpenes in Thujopsis dolabrata from all regions in Japan were analyzed using GC and GC–MS. The results show that the major constituents of the diterpene hydrocarbon fraction in this conifer are dolabradiene, hibaene, rimuene, 13-epi-dolabradiene and abietatriene. There were wide variations in the contents of the constituents among individuals and habitats. The results also show that T. dolabrata trees from 34 habitats can be classified into three groups based on the composition of the leaf diterpene hydrocarbon. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Thujopsis dolabrata; Cupressaceae; Leaf terpenoid; Diterpene hydrocarbon; Chemotaxonomy
1. Introduction Thujopsis dolabrata Sieb. et. Zucc. (Japanese name: Asunaro) is the only species of the genus Thujopsis in Japan. It is widely distributed from Hokkaido (northern island) to Kagoshima prefecture (southern end of Kyushu island) (Hayashi, 1960). It has one variety, var. hondae Makino (Japanese name: Hiba or Hinokiasunaro), that is distributed in the northern part of Japan (Hayashi, 1960; Uehara, 1959). The main diterpene hydrocarbons in Thujopsis dolabrata var. hondae are reported to be dolabradiene and hibaene (Kitahara and Yoshikoshi, 1964a,b; Kitadani et al., *Corresponding author. Tel.: +81-235-28-2926. E-mail address: [email protected]_u.ac.jp (K. Takahashi). 0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 2 6 - 6
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K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848
1970). One paper reports that the composition of these diterpene hydrocarbons vary with latitude in the northern region of Japan along the Japan Sea (Takahashi et al., 1981). Another paper reports that rimuene, rosa-5,15-diene, and 13-epi-dolabradiene predominate in the diterpene hydrocarbons of T. dolabrata in the Kiso district (part of Nagano prefecture) (Nagahama and Tajima, 1996). It is reported that Ate, which is a famous local cultivar of T. dolabrata in the Noto district (part of Ishikawa prefecture), can be classified into two groups on the basis of the trans-communic acid content (Nagahama et al., 1996). We are conducting a chemotaxonomic study based on the diterpene hydrocarbon constitution in the leaves of Japanese Thujopsis. In this paper, we report the result of GC and GC–MS analysis of neutral extracts from leaves. 2. Materials and methods 2.1. Materials To minimize the influence of the environment, the Ohata experimental forest of the forestry agency, Aomori prefecture, was selected for sampling. The Ohata experimental forest was established in 1953 by planting nursery stocks collected from 29 natural T. dolabrata forests (habitats) in Japan. Most of the samples were collected from 168 individuals growing in this experimental forest in August 1996. The 168 individuals are representative of all but three regions in Japan. No individuals from the three regions were present in the Ohata experimental forest. Therefore, samples representative of these regions were collected from 53 individuals in seven actual habitats in three prefectures: Tottori (26 samples in 4 habitats) in November 1996, Tokushima (21 samples in 2 habitats) in March 1997 and Kagoshima (6 samples in 1 habitat) in March 1998. Table 1 lists the 34 habitats and the number of individuals from which the samples were collected for each of the habitats. Since most of the samples were collected from individuals growing in the same place, we presume that the deviation of the diterpene composition is not due to the growth environment, but to genetic factors. 2.2. Analysis of diterpene hydrocarbon Mature leaves (about 10 g) were cut using a mixer and extracted with n-hexane for one day. After evaporation of the n-hexane, the neutral extracts were chromatographed on alumina and the eluted n-hexane was analyzed using GC, TC–WAX bonded capillary 30 m, at 1908C. The identification of diterpene hydrocarbon was based on a comparison between authentic diterpene hydrocarbon and GC–MS analysis results. 2.3. Computer analysis Excel statistics 97(Win95) was used for principal component analysis and discriminant analysis.
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K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848 Table 1 Habitats of Thujopsis dolabrata where samples were collected Ohata experimental forest Population code 1 2 3 4 5 6 7 8 9
Habitat
Prefecture
Esashi Hiyama Yamashe Maeda Sado Takabayashi Ikeda Hakine Ohkuwa
Hokkaido Hokkaido Akita Akita Niigata Tochigi Gunma Tochigi Nagano
10 11
Mizukami Gunma Kawarada Ishikawa
12
Kotakagawa Hanase Shisikui Aira Nakatsu Ohata Noto Kiso Noto
13 14 15 16 17 18 19 20
Number of individuals 4 5 5 4 6 7 7 5 7 6 15
Tottori
7
Kyoto Tokushima Kagoshima Gifu Aomori Ishikawa Nagano Ishikawa
6 0 4 8 5 7 3 5
Population Habitat code
Prefecture
21 22 23 24 25 26 27 28 29
Aomori Miyagi Aomori Iwate Aomori Iwate Iwate Iwate Aomori
Okunai Akyu Souma Monma Noheji Wakayanagi Kamiarisumi Gosho Sangal
Number of individuals 6 7 8 6 9 6 7 0 3
Total 168 Tottori, Tokushima and Kagoshima prefectures Population Habitat code
Prefecture
A B C D E F G
Tottori Tottori Tottori Tottori Tokushima Tokushima Kagoshima
Tawarahara Sanbongi Betsumiya Kidaka Dewa Kunugidaira Takakuma Total
Number of individuals 10 3 5 8 10 11 6 53
3. Results and discussion Table 2 lists the composition of diterpene hydrocarbon determined by capillary gas chromatography and relative contents of the constituents for 221 typical individuals of T. dolabrata from 34 habitats listed in Table 1. Nine constituents, namely, rimuene, rosa-5,15-diene, hibaene, pimaradiene, sclarene, sandaracopimaradiene, dolabradiene, 13-epi-dolabradiene, and abietatriene were identified as listed in Table 2. The contents of these nine constituents showed wide variations among individuals and habitats. As the second step, we used a computer for principal component analysis to see the relationship between the identified nine constituents and 221 individuals. Analysis of the first and second principal components shows that the individuals can be classified into three main groups in Fig. 1. Table 3 lists factor loadings of the first and second principal components. Rimuene, sclarene, rosadiene,
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Table 2 Relative contents of diterpene hydrocarbon constituents in the leaves of Thujopsis dolabrata typical of habitats Population code Individual no. Rimuene Rosadiene Hibaene Pimaradiene Sclarene Sandaracoa1 Dolabradiene 13-epi-db2 Abietatriene Diterpene type
a b
1 5 11 18 24 30 38 44 49 53 59 65 72 80 85 90 97 106 113 127 130 138 143 150 156 162 168 175 178 186 196 207 214 216 1. Sandaracopimaradiene. 2. 13-epi-dolabradiene.
0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 24.9 0.0 0.0 0.0 0.0 29.4 0.0 0.5 27.7 0.0 0.0 0.0 1.2 0.0 45.4 45.4 46.4 54.8
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 16.2 8.2 0.0 0.0 0.0 0.0 0.0 3.6 6.5 20.6 13.0
36.7 19.6 19.1 24.5 30.4 13.8 10.8 23.7 23.4 12.4 22.2 19.2 16.0 11.8 18.1 12.4 16.5 19.2 22.6 14.4 20.1 7.8 32.2 12.1 7.3 15.7 19.0 27.9 21.0 16.3 5.4 15.3 13.2 12.9
0.0 2.5 2.1 4.1 0.0 6.8 4.1 3.8 3.1 4.9 1.9 3.5 0.0 2.7 2.4 3.8 3.1 2.9 1.8 0.9 2.6 2.7 0.8 3.0 3.3 2.2 3.8 2.4 3.8 0.7 3.6 4.6 5.7 0.3
0.0 0.0 0.6 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 2.5 6.2 2.4 1.3 4.3 1.1 0.0 1.0 4.7 2.1 2.0 4.2 0.0 0.0 0.0 0.0 0.0 13.8 4.8 3.0 6.1
0.0 1.8 2.1 1.7 0.0 1.2 1.0 1.0 1.5 1.4 0.8 1.9 0.0 1.6 1.5 1.6 2.2 1.7 2.2 0.9 2.0 1.6 0.6 3.3 3.3 2.3 2.6 2.0 1.3 2.8 3.4 2.8 3.2 3.7
56.3 68.0 67.3 56.1 63.6 71.4 75.6 63.9 66.4 74.9 67.9 69.0 77.9 71.2 60.0 67.2 45.1 60.6 42.9 76.2 44.6 42.0 28.4 0.9 0.5 71.2 64.4 57.5 60.3 65.6 0.0 0.0 0.0 3.9
5.1 5.7 6.3 4.4 4.6 5.0 6.3 5.2 4.3 5.0 4.4 4.7 5.6 4.6 4.7 4.2 3.1 6.6 24.4 5.6 22.9 3.3 28.4 56.5 35.0 6.0 5.1 6.1 5.4 6.2 0.0 0.0 0.0 2.7
1.8 2.4 2.6 6.7 1.5 1.5 2.1 1.9 1.3 1.3 2.7 1.2 1.6 5.7 7.2 8.4 3.7 4.7 5.2 2.0 5.9 8.2 7.6 5.4 10.0 2.6 1.3 2.0 1.9 3.2 24.8 20.5 8.0 2.6
D D D D D D D D D D D D D D D D RD D 13D D 13D RD 13D 13 13R D D D D D R R R R
K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848
1 2 17 21 23 25 29 3 4 24 26 28 22 6 8 7 10 5 11 18 20 9 19 16 13 12 A B C D E F 15 G
K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848
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Fig. 1. Scatter diagram of Thujopsis dolabrata based on diterpene hydrocarbon constituents in the first and second principal components.
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Table 3 Facter loadings of nine diterpene hydrocarbons in principal component analysis Diterpene hydrocarbon
First principal component
Second principal component
Third principal component
Fourth principal component
Rimuene Rosadiene Hibaene Pimaradiene Sclarene Sandaracoa1 Dolabradiene 13-epi-db2 Abietatriene
0.8608 0.7297 0.4919 0.3861 0.8016 0.6993 0.8863 0.1314 0.6844
0.1600 0.2186 0.3396 0.5523 0.0827 0.1893 0.3374 0.7759 0.2283
0.2495 0.1009 0.0625 0.5935 0.2924 0.4018 0.0195 0.5395 0.3199
0.0572 0.2992 0.7692 0.2120 0.0927 0.2265 0.2524 0.1465 0.0414
Cumulative contribution
0.452
0.597
0.716
0.813
a b
1. sandaracopimaradiene. 2. 13-epi-dolabradiene.
sandaracopimaradiene, abietatriene, and dolabradiene were representative of the first principal component and 13-epi-dolabradiene and pimaradiene were representative of the second principal component. Dolabradiene, rimuene and 13-epidolabradiene showed remarkably high values. Therefore, we examined the contents of dolabradiene, rimuene, and 13-epidolabradiene for each individual. The histograms of these three constituents did not show normal curves but showed that the individuals can be divided into two groups. For dolabradiene, there was no individual with content from 5.6 to 20.5% but the individuals could be divided into two groups with low contents (38 individuals with an average content of 0.92 and a standard deviation of 1.45) and high contents (183 individuals with an average content of 60.71 and a standard deviation of 13.11). For rimuene, there was no individual with contents from 1.8 to 14.9% but the individuals could be divided into two groups with low contents (174 individuals with an average content of 0.16 and a standard deviation of 0.37) and high contents (47 individuals with an average content of 38.04 and a standard deviation of 13.41). For 13-epidolabradiene, there was no individual with contents from 8.0 to 14.9% but the individuals could be divided into two groups with low contents (188 individuals with an average content of 4.27 and a standard deviation of 1.87) and high contents (33 individuals with an average content of 27.39 and a standard deviation of 11.50). On the basis of this result, we classified the 183 individuals with high dolabradiene contents as TypeD, the 47 individuals with high rimuene contents as TypeR, and the 33 individuals with high 13-epi-dolabradiene contents as Type13. Since two of these three constituents were found in some individuals, we classified the individuals according to whether they contain only one of the constituents or two of them. Table 4 lists the frequency of individuals thus classified. As shown in this table, there were 148 individuals (D) containing only dolabradiene as the main constituent, 26 individuals (R) containing only rimuene as the main constituent, five individuals (13) containing only 13-epi-dolabradiene as the main constituent, 21
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Table 4 Frequency of individuals with high relative contents of each main diterpene hydrocarbon constituenta Region
Prefecture
Habitat
Number of Individuals
Classification by diterpene hydrocarbon D
Hokkaido Hokkaido
Esashi (1) Hiyama (2) Tohoku Aomori Ohata (17) Ohkunai (21) Souma (23) Noheji (25) Sangai (29) Akita Yamase (3) Maeda (4) Iwate Monma (24) Wakayanagi (26) Kamiarisumi (27) Miyagi Akyu (22) Kanto Tochigi Takabayashi (6) Hakine (8) Gunma Ikeda (7) Mizukami (10) Chubu Niigata Sado (5) Ishikawa Kawarada (11) Noto (18) Noto (20) Nagano Ohkuwa (9) Kiso (19) Gifu Nakatsu (16) Kinki Kyoto Hanase (13) Chugoku Tottori Kotakagawa (12) Tawarahara (A) Sanbongi (B) Betsumiya (C) Kidaka (D) Shikoku Tokushima Dewa (E) Kunugidaira (F) Kyushu Kagoshima Aira (15) Takakuma (G) Total
4 5 5 6 8 9 3 5 4 6 6 7 7 7 5 7 6 6 15 7 5 7 3 8 6 7 10 3 5 8 10 11 4 6 221
4 5 4 3 8 9 3 5 4 6 6 7 7 7 5 7 2 6 5 7 1 1 1
13
R
13D
RD
13R
1 3
4 10
1 2 2
1 2 1
4 2 2
2
1
1 2
2 1
2 1 1 1
1 1
14
7
7 10 3 5 8 7 8 3 3
1 1 148
5
26
21
1
a
Figures in parentheses in the habitat column are population codes. D: Individual containing only dolabradiene as main constituent, 13: Individual containing only 13-epi-dolabradiene as the main constituent, R: Individual containing only rimuene as the main constituents, 13D: Individual containing dolabradiene and 13-epi-dolabradiene as the main constituents, RD: Individual containing dolabradiene and rimuene as the main constituents, 13R: Individual containing only 13-epi-dolabradiene and rimuene as the main constituents.
individuals (13D) containing dolabrudiene and 13-epi-dolabradiene as the main constituents, 14 individuals (RD) containing dolabradiene and rimuene as the main constituents, 7 individuals (13R) containing rimuene and 13-epi-dolabradiene as the main constituents.
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Fig. 2. Habitats with high contents of each diterpene hydrocarbon constituent.
The distribution of these six types (D,R,13,13D,RD,13R) shown in Fig. 1 corresponds exactly to the grouping by the principal component analysis. This shows that T. dolabrata can be classified into three types TypeD, TypeR, and Type13. We, then, examined the geographical distribution of these three types on the basis of Table 4. Fig. 2 shows the distribution. TypeD was the most common type and accounted for 82.8% of the population. This type was found mainly in the northern and western parts of Japan, namely, Hokkaido, Aomori, Akita, Iwate, Miyagi, Tochigi, Gunma, Niigata, Ishikawa, Nagano and Tottori prefectures. 86.9%(159/183) of this type was found in these prefectures. TypeR accounted for 21.3% of the population and therefore, it was not so common but was widely
K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848
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Fig. 3. Discriminant analysis for diterpene hydrocarbon constituents in the leaves of Thujopsis dolabrata. Discriminant function: Y= 0.2394X1 0.1992X2+0.0324X3 0.1940X4 0.0986X5+0.1908X6+ 0.0329X7 0.1501X8 0.3058X9+6.3536. X1, X2, X3, X4, X5, X6, X7, X8 and X9 are rimuene, rosadiene, hibaene, pimaradiene, sclarene, sandaracopimaradiene, dolabradiene, 13–epi–dolabradiene, and abietatriene, respectively.
distributed in the southern part of Japan and was found in Gunma (Mizukami), Nagano, Gifu, Kyoto, Tokushima and Kagoshima prefectures. 97.9%(46/47) of this type was found in these prefectures where TypeD was seldom found. Type13 accounted for only 14.9% of the population. It was found in the central part of Japan, namely Ishikawa, Nagano, Gifu and Kyoto prefectures. 81.8%(27/33) of this
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type was found in these prefectures. These results show that the three types have their own distribution ranges. As the final step, we used discriminant analysis. The result is shown in Fig. 3. A total of nine individuals were discriminated incorrectly between TypeD and TypeR; one individual of TypeD was discriminated as TypeR and eight individuals of TypeR were discriminated as TypeD. The discriminant ratio was 95.9% (212/221). Type13 exceeded the discriminant line between TypeD and TypeR and classified into two types (13D and13R). The result confirms that the three types, D, R, and 13 have their own distribution ranges. Thujopsis dolabrata individuals can be thus classified into three types (TypeD, TypeR, and Type13) on the basis of the contents of diterpene hydrocarbon constituents dolabradiene, rimuene, and 13-epi-dolabradiene and also their distribution corresponding to variations in the contents.
Acknowledgements We are grateful to H. Yamagami (Ohata local forestry technical officer) for his help in collecting samples at the Ohata experimental forest in Ohata in Aomori prefecture, Y. Maeda (Tottori Prefectural Forest Experiment Station researcher) for his help with the collecting trip in Tottori prefecture, and T. Maeda (Kagoshima University forestry technical officer) for his help in collecting samples at Takakuma University forest in Kagoshima prefecture. We also thank Prof. K. Ogiyama and Prof. T. Sasahara of Yamagata University for their support and Mr. K. Iuchi of Kizawa village office in Tokushima prefecture for his advice.
References Hayashi, Y., 1960. Classification and Distribution of Conifers in Japan, Norinshuppan, Tokyo, pp. 48–49, 176–179. Kitadani, M., Yoshikoshi, A., Kitahara, Y., Campello, J.P., 1970. Natural ar-Abietatriene. Chem. Pharmaceut. Bull. 18, 402–405. Kitahara, Y., Yoshikoshi, A., 1964a. The structure of dolabradiene. Tetrahedron Lett. 26, 1755–1761. Kitahara, Y., Yoshikoshi, A., 1964b. The structure of hibaene. Tetrahedron Lett. 26, 1771–1774. Nagahama, S., Tajima, M., 1996. Diterpene hydrocarbons from the leaf oil Thujopsis dolabrata var. dolabrata. Biochem. System. Ecol. 24, 49–52. Nagahama, S., Tajima, M., Nishimura, K., 1996. Chemotaxonomy of Ate (Thujopsis dolabrata) of Noto, Ishikawa prefecture. J. Jpn. Wood Res. Soc. 42, 698–702. Takahashi, K., Yasue, M., Ogiyama, K., 1981. The variation of diterpene hydrocarbon in Thujopsis dolabrata. 33rd Meeting of Japanese East–North Forestry Society, vol. 33, pp. 124–126. Uehara, K., 1959. Great Illustrated Book of Trees. Ariakeshobou, Tokyo, pp. 447–453.
Chemotaxonomy on the leaf constituents of Thujopsis dolabrata Sieb. et Zucc.}Analysis of neutral extracts (diterpene hydrocarbon) Koetsu Takahashia,*, Shizuo Nagahamab, Takurou Nakashimaa, Hiromi Suenagab a
Faculty of Agriculture, Yamagata University, Wakaba-machi 1-23, Tsuruoka 997, Japan b Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan Received 14 June 2000; accepted 17 November 2000
Abstract The leaf terpenes in Thujopsis dolabrata from all regions in Japan were analyzed using GC and GC–MS. The results show that the major constituents of the diterpene hydrocarbon fraction in this conifer are dolabradiene, hibaene, rimuene, 13-epi-dolabradiene and abietatriene. There were wide variations in the contents of the constituents among individuals and habitats. The results also show that T. dolabrata trees from 34 habitats can be classified into three groups based on the composition of the leaf diterpene hydrocarbon. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Thujopsis dolabrata; Cupressaceae; Leaf terpenoid; Diterpene hydrocarbon; Chemotaxonomy
1. Introduction Thujopsis dolabrata Sieb. et. Zucc. (Japanese name: Asunaro) is the only species of the genus Thujopsis in Japan. It is widely distributed from Hokkaido (northern island) to Kagoshima prefecture (southern end of Kyushu island) (Hayashi, 1960). It has one variety, var. hondae Makino (Japanese name: Hiba or Hinokiasunaro), that is distributed in the northern part of Japan (Hayashi, 1960; Uehara, 1959). The main diterpene hydrocarbons in Thujopsis dolabrata var. hondae are reported to be dolabradiene and hibaene (Kitahara and Yoshikoshi, 1964a,b; Kitadani et al., *Corresponding author. Tel.: +81-235-28-2926. E-mail address: [email protected]_u.ac.jp (K. Takahashi). 0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 2 6 - 6
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1970). One paper reports that the composition of these diterpene hydrocarbons vary with latitude in the northern region of Japan along the Japan Sea (Takahashi et al., 1981). Another paper reports that rimuene, rosa-5,15-diene, and 13-epi-dolabradiene predominate in the diterpene hydrocarbons of T. dolabrata in the Kiso district (part of Nagano prefecture) (Nagahama and Tajima, 1996). It is reported that Ate, which is a famous local cultivar of T. dolabrata in the Noto district (part of Ishikawa prefecture), can be classified into two groups on the basis of the trans-communic acid content (Nagahama et al., 1996). We are conducting a chemotaxonomic study based on the diterpene hydrocarbon constitution in the leaves of Japanese Thujopsis. In this paper, we report the result of GC and GC–MS analysis of neutral extracts from leaves. 2. Materials and methods 2.1. Materials To minimize the influence of the environment, the Ohata experimental forest of the forestry agency, Aomori prefecture, was selected for sampling. The Ohata experimental forest was established in 1953 by planting nursery stocks collected from 29 natural T. dolabrata forests (habitats) in Japan. Most of the samples were collected from 168 individuals growing in this experimental forest in August 1996. The 168 individuals are representative of all but three regions in Japan. No individuals from the three regions were present in the Ohata experimental forest. Therefore, samples representative of these regions were collected from 53 individuals in seven actual habitats in three prefectures: Tottori (26 samples in 4 habitats) in November 1996, Tokushima (21 samples in 2 habitats) in March 1997 and Kagoshima (6 samples in 1 habitat) in March 1998. Table 1 lists the 34 habitats and the number of individuals from which the samples were collected for each of the habitats. Since most of the samples were collected from individuals growing in the same place, we presume that the deviation of the diterpene composition is not due to the growth environment, but to genetic factors. 2.2. Analysis of diterpene hydrocarbon Mature leaves (about 10 g) were cut using a mixer and extracted with n-hexane for one day. After evaporation of the n-hexane, the neutral extracts were chromatographed on alumina and the eluted n-hexane was analyzed using GC, TC–WAX bonded capillary 30 m, at 1908C. The identification of diterpene hydrocarbon was based on a comparison between authentic diterpene hydrocarbon and GC–MS analysis results. 2.3. Computer analysis Excel statistics 97(Win95) was used for principal component analysis and discriminant analysis.
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K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848 Table 1 Habitats of Thujopsis dolabrata where samples were collected Ohata experimental forest Population code 1 2 3 4 5 6 7 8 9
Habitat
Prefecture
Esashi Hiyama Yamashe Maeda Sado Takabayashi Ikeda Hakine Ohkuwa
Hokkaido Hokkaido Akita Akita Niigata Tochigi Gunma Tochigi Nagano
10 11
Mizukami Gunma Kawarada Ishikawa
12
Kotakagawa Hanase Shisikui Aira Nakatsu Ohata Noto Kiso Noto
13 14 15 16 17 18 19 20
Number of individuals 4 5 5 4 6 7 7 5 7 6 15
Tottori
7
Kyoto Tokushima Kagoshima Gifu Aomori Ishikawa Nagano Ishikawa
6 0 4 8 5 7 3 5
Population Habitat code
Prefecture
21 22 23 24 25 26 27 28 29
Aomori Miyagi Aomori Iwate Aomori Iwate Iwate Iwate Aomori
Okunai Akyu Souma Monma Noheji Wakayanagi Kamiarisumi Gosho Sangal
Number of individuals 6 7 8 6 9 6 7 0 3
Total 168 Tottori, Tokushima and Kagoshima prefectures Population Habitat code
Prefecture
A B C D E F G
Tottori Tottori Tottori Tottori Tokushima Tokushima Kagoshima
Tawarahara Sanbongi Betsumiya Kidaka Dewa Kunugidaira Takakuma Total
Number of individuals 10 3 5 8 10 11 6 53
3. Results and discussion Table 2 lists the composition of diterpene hydrocarbon determined by capillary gas chromatography and relative contents of the constituents for 221 typical individuals of T. dolabrata from 34 habitats listed in Table 1. Nine constituents, namely, rimuene, rosa-5,15-diene, hibaene, pimaradiene, sclarene, sandaracopimaradiene, dolabradiene, 13-epi-dolabradiene, and abietatriene were identified as listed in Table 2. The contents of these nine constituents showed wide variations among individuals and habitats. As the second step, we used a computer for principal component analysis to see the relationship between the identified nine constituents and 221 individuals. Analysis of the first and second principal components shows that the individuals can be classified into three main groups in Fig. 1. Table 3 lists factor loadings of the first and second principal components. Rimuene, sclarene, rosadiene,
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Table 2 Relative contents of diterpene hydrocarbon constituents in the leaves of Thujopsis dolabrata typical of habitats Population code Individual no. Rimuene Rosadiene Hibaene Pimaradiene Sclarene Sandaracoa1 Dolabradiene 13-epi-db2 Abietatriene Diterpene type
a b
1 5 11 18 24 30 38 44 49 53 59 65 72 80 85 90 97 106 113 127 130 138 143 150 156 162 168 175 178 186 196 207 214 216 1. Sandaracopimaradiene. 2. 13-epi-dolabradiene.
0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 24.9 0.0 0.0 0.0 0.0 29.4 0.0 0.5 27.7 0.0 0.0 0.0 1.2 0.0 45.4 45.4 46.4 54.8
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 16.2 8.2 0.0 0.0 0.0 0.0 0.0 3.6 6.5 20.6 13.0
36.7 19.6 19.1 24.5 30.4 13.8 10.8 23.7 23.4 12.4 22.2 19.2 16.0 11.8 18.1 12.4 16.5 19.2 22.6 14.4 20.1 7.8 32.2 12.1 7.3 15.7 19.0 27.9 21.0 16.3 5.4 15.3 13.2 12.9
0.0 2.5 2.1 4.1 0.0 6.8 4.1 3.8 3.1 4.9 1.9 3.5 0.0 2.7 2.4 3.8 3.1 2.9 1.8 0.9 2.6 2.7 0.8 3.0 3.3 2.2 3.8 2.4 3.8 0.7 3.6 4.6 5.7 0.3
0.0 0.0 0.6 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 2.5 6.2 2.4 1.3 4.3 1.1 0.0 1.0 4.7 2.1 2.0 4.2 0.0 0.0 0.0 0.0 0.0 13.8 4.8 3.0 6.1
0.0 1.8 2.1 1.7 0.0 1.2 1.0 1.0 1.5 1.4 0.8 1.9 0.0 1.6 1.5 1.6 2.2 1.7 2.2 0.9 2.0 1.6 0.6 3.3 3.3 2.3 2.6 2.0 1.3 2.8 3.4 2.8 3.2 3.7
56.3 68.0 67.3 56.1 63.6 71.4 75.6 63.9 66.4 74.9 67.9 69.0 77.9 71.2 60.0 67.2 45.1 60.6 42.9 76.2 44.6 42.0 28.4 0.9 0.5 71.2 64.4 57.5 60.3 65.6 0.0 0.0 0.0 3.9
5.1 5.7 6.3 4.4 4.6 5.0 6.3 5.2 4.3 5.0 4.4 4.7 5.6 4.6 4.7 4.2 3.1 6.6 24.4 5.6 22.9 3.3 28.4 56.5 35.0 6.0 5.1 6.1 5.4 6.2 0.0 0.0 0.0 2.7
1.8 2.4 2.6 6.7 1.5 1.5 2.1 1.9 1.3 1.3 2.7 1.2 1.6 5.7 7.2 8.4 3.7 4.7 5.2 2.0 5.9 8.2 7.6 5.4 10.0 2.6 1.3 2.0 1.9 3.2 24.8 20.5 8.0 2.6
D D D D D D D D D D D D D D D D RD D 13D D 13D RD 13D 13 13R D D D D D R R R R
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1 2 17 21 23 25 29 3 4 24 26 28 22 6 8 7 10 5 11 18 20 9 19 16 13 12 A B C D E F 15 G
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Fig. 1. Scatter diagram of Thujopsis dolabrata based on diterpene hydrocarbon constituents in the first and second principal components.
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Table 3 Facter loadings of nine diterpene hydrocarbons in principal component analysis Diterpene hydrocarbon
First principal component
Second principal component
Third principal component
Fourth principal component
Rimuene Rosadiene Hibaene Pimaradiene Sclarene Sandaracoa1 Dolabradiene 13-epi-db2 Abietatriene
0.8608 0.7297 0.4919 0.3861 0.8016 0.6993 0.8863 0.1314 0.6844
0.1600 0.2186 0.3396 0.5523 0.0827 0.1893 0.3374 0.7759 0.2283
0.2495 0.1009 0.0625 0.5935 0.2924 0.4018 0.0195 0.5395 0.3199
0.0572 0.2992 0.7692 0.2120 0.0927 0.2265 0.2524 0.1465 0.0414
Cumulative contribution
0.452
0.597
0.716
0.813
a b
1. sandaracopimaradiene. 2. 13-epi-dolabradiene.
sandaracopimaradiene, abietatriene, and dolabradiene were representative of the first principal component and 13-epi-dolabradiene and pimaradiene were representative of the second principal component. Dolabradiene, rimuene and 13-epidolabradiene showed remarkably high values. Therefore, we examined the contents of dolabradiene, rimuene, and 13-epidolabradiene for each individual. The histograms of these three constituents did not show normal curves but showed that the individuals can be divided into two groups. For dolabradiene, there was no individual with content from 5.6 to 20.5% but the individuals could be divided into two groups with low contents (38 individuals with an average content of 0.92 and a standard deviation of 1.45) and high contents (183 individuals with an average content of 60.71 and a standard deviation of 13.11). For rimuene, there was no individual with contents from 1.8 to 14.9% but the individuals could be divided into two groups with low contents (174 individuals with an average content of 0.16 and a standard deviation of 0.37) and high contents (47 individuals with an average content of 38.04 and a standard deviation of 13.41). For 13-epidolabradiene, there was no individual with contents from 8.0 to 14.9% but the individuals could be divided into two groups with low contents (188 individuals with an average content of 4.27 and a standard deviation of 1.87) and high contents (33 individuals with an average content of 27.39 and a standard deviation of 11.50). On the basis of this result, we classified the 183 individuals with high dolabradiene contents as TypeD, the 47 individuals with high rimuene contents as TypeR, and the 33 individuals with high 13-epi-dolabradiene contents as Type13. Since two of these three constituents were found in some individuals, we classified the individuals according to whether they contain only one of the constituents or two of them. Table 4 lists the frequency of individuals thus classified. As shown in this table, there were 148 individuals (D) containing only dolabradiene as the main constituent, 26 individuals (R) containing only rimuene as the main constituent, five individuals (13) containing only 13-epi-dolabradiene as the main constituent, 21
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Table 4 Frequency of individuals with high relative contents of each main diterpene hydrocarbon constituenta Region
Prefecture
Habitat
Number of Individuals
Classification by diterpene hydrocarbon D
Hokkaido Hokkaido
Esashi (1) Hiyama (2) Tohoku Aomori Ohata (17) Ohkunai (21) Souma (23) Noheji (25) Sangai (29) Akita Yamase (3) Maeda (4) Iwate Monma (24) Wakayanagi (26) Kamiarisumi (27) Miyagi Akyu (22) Kanto Tochigi Takabayashi (6) Hakine (8) Gunma Ikeda (7) Mizukami (10) Chubu Niigata Sado (5) Ishikawa Kawarada (11) Noto (18) Noto (20) Nagano Ohkuwa (9) Kiso (19) Gifu Nakatsu (16) Kinki Kyoto Hanase (13) Chugoku Tottori Kotakagawa (12) Tawarahara (A) Sanbongi (B) Betsumiya (C) Kidaka (D) Shikoku Tokushima Dewa (E) Kunugidaira (F) Kyushu Kagoshima Aira (15) Takakuma (G) Total
4 5 5 6 8 9 3 5 4 6 6 7 7 7 5 7 6 6 15 7 5 7 3 8 6 7 10 3 5 8 10 11 4 6 221
4 5 4 3 8 9 3 5 4 6 6 7 7 7 5 7 2 6 5 7 1 1 1
13
R
13D
RD
13R
1 3
4 10
1 2 2
1 2 1
4 2 2
2
1
1 2
2 1
2 1 1 1
1 1
14
7
7 10 3 5 8 7 8 3 3
1 1 148
5
26
21
1
a
Figures in parentheses in the habitat column are population codes. D: Individual containing only dolabradiene as main constituent, 13: Individual containing only 13-epi-dolabradiene as the main constituent, R: Individual containing only rimuene as the main constituents, 13D: Individual containing dolabradiene and 13-epi-dolabradiene as the main constituents, RD: Individual containing dolabradiene and rimuene as the main constituents, 13R: Individual containing only 13-epi-dolabradiene and rimuene as the main constituents.
individuals (13D) containing dolabrudiene and 13-epi-dolabradiene as the main constituents, 14 individuals (RD) containing dolabradiene and rimuene as the main constituents, 7 individuals (13R) containing rimuene and 13-epi-dolabradiene as the main constituents.
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Fig. 2. Habitats with high contents of each diterpene hydrocarbon constituent.
The distribution of these six types (D,R,13,13D,RD,13R) shown in Fig. 1 corresponds exactly to the grouping by the principal component analysis. This shows that T. dolabrata can be classified into three types TypeD, TypeR, and Type13. We, then, examined the geographical distribution of these three types on the basis of Table 4. Fig. 2 shows the distribution. TypeD was the most common type and accounted for 82.8% of the population. This type was found mainly in the northern and western parts of Japan, namely, Hokkaido, Aomori, Akita, Iwate, Miyagi, Tochigi, Gunma, Niigata, Ishikawa, Nagano and Tottori prefectures. 86.9%(159/183) of this type was found in these prefectures. TypeR accounted for 21.3% of the population and therefore, it was not so common but was widely
K. Takahashi et al. / Biochemical Systematics and Ecology 29 (2001) 839–848
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Fig. 3. Discriminant analysis for diterpene hydrocarbon constituents in the leaves of Thujopsis dolabrata. Discriminant function: Y= 0.2394X1 0.1992X2+0.0324X3 0.1940X4 0.0986X5+0.1908X6+ 0.0329X7 0.1501X8 0.3058X9+6.3536. X1, X2, X3, X4, X5, X6, X7, X8 and X9 are rimuene, rosadiene, hibaene, pimaradiene, sclarene, sandaracopimaradiene, dolabradiene, 13–epi–dolabradiene, and abietatriene, respectively.
distributed in the southern part of Japan and was found in Gunma (Mizukami), Nagano, Gifu, Kyoto, Tokushima and Kagoshima prefectures. 97.9%(46/47) of this type was found in these prefectures where TypeD was seldom found. Type13 accounted for only 14.9% of the population. It was found in the central part of Japan, namely Ishikawa, Nagano, Gifu and Kyoto prefectures. 81.8%(27/33) of this
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type was found in these prefectures. These results show that the three types have their own distribution ranges. As the final step, we used discriminant analysis. The result is shown in Fig. 3. A total of nine individuals were discriminated incorrectly between TypeD and TypeR; one individual of TypeD was discriminated as TypeR and eight individuals of TypeR were discriminated as TypeD. The discriminant ratio was 95.9% (212/221). Type13 exceeded the discriminant line between TypeD and TypeR and classified into two types (13D and13R). The result confirms that the three types, D, R, and 13 have their own distribution ranges. Thujopsis dolabrata individuals can be thus classified into three types (TypeD, TypeR, and Type13) on the basis of the contents of diterpene hydrocarbon constituents dolabradiene, rimuene, and 13-epi-dolabradiene and also their distribution corresponding to variations in the contents.
Acknowledgements We are grateful to H. Yamagami (Ohata local forestry technical officer) for his help in collecting samples at the Ohata experimental forest in Ohata in Aomori prefecture, Y. Maeda (Tottori Prefectural Forest Experiment Station researcher) for his help with the collecting trip in Tottori prefecture, and T. Maeda (Kagoshima University forestry technical officer) for his help in collecting samples at Takakuma University forest in Kagoshima prefecture. We also thank Prof. K. Ogiyama and Prof. T. Sasahara of Yamagata University for their support and Mr. K. Iuchi of Kizawa village office in Tokushima prefecture for his advice.
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