Bot. Bull. Acad. Sin. (1995) 36: 247-251

Hsiao and Lin — Chemotaxonomy of Clerodendrum

A chemotaxonomic study of essential oils from the leaves of genus Clerodendrum (Verbenaceae) native to Taiwan

J. Y. Hsiao1 and M. L. Lin

Department of Botany, National Chung Hsing University, Taichung, Taiwan, Republic of China

(Received January 11, 1995; Accepted July 11, 1995)

Abstract. Fifty-four mature-leaf samples of nine Clerodendrum taxa native to Taiwan were collected from various locations on the island. Each sample was steam-distilled, and the resulting essential oil was analyzed by gas chromatography. The relationship among taxa was analyzed by cluster analysis of the gas chromatogram data (463 peaks). We assumed that peaks with the same retention time on different chromatograms were the same compound. The cluster analysis indicated a congruence between morphological and chemical relationships at the intraspecific level. All intraspecific taxa were linked as distinct clusters. At the interspecific level, there was congruence and discrepancy between morphological and chemical relationships. The results of the present study support the taxonomic treatment in which the white-flowered variant of C. paniculatum is recognized as a variety of the species. The results also support the existence of C. trichotomum var. fargesii and C. trichotomum var. ferrugineum as intraspecific taxa of C. trichotomum.

Keywords: Chemotaxonomy; Clerodendrum; Essential oils; Taxonomy; Verbenaceae.


The genus Clerodendrum (Verbenaceae) is widely distributed in the tropics and subtropics, with a few species extending into the temperate regions. The species number has been estimated to be five hundred and sixty (Moldenke, 1971) and five hundred and eighty (Munir, 1989). The genus was first described by Linnaeus in 1753, based on the type species Clerodendrum infortunatum from India. In 1763, Adanson changed the Latinized form, Clerodendrum, into the Greek form, Clerodendron. In 1942, Moldenke re-adopted Linnaeus' original Latinized form, and the practice has been followed by most taxonomists. The genus comprises small trees, shrubs (sometimes climbing), and (rarely) herbs. Morphologically it is characterized by opposite and exstipulate leaves, tetragonal or terete stems, terminal or axillary cymose inflorescence or panicles, hypogynous bisexual flowers, persistent calyx, cylindrical corolla-tube with spreading 5-lobes at the top, epipetalous and exserted stamens, long-exerted style and short-bifided stigma, imperfectly 4-celled ovary, exalbuminous seeds, and endocarp separating into 2 to 4 stony pyrenes.

The species of Clerodendrum native in Taiwan are distributed from the coasts to low-to-middle altitudes in the mountains. The treatments by taxonomists are diverse (e.g. Hayata, 1912; Kanehira, 1917; Li, 1963; Moldenke, 1971; Hsiao, 1978; Liu and Liao, 1981; Hue and Chen, 1982; Liu et al., 1988). The indisputable taxa includes

C. canescens Wall., C. cyrtophyllum Turcx., C. inerme (L.) Gaertner, and C. philippinum Schauer forma multiplex (Sweet) Moldenke. Among the more controversial taxa, several authors (Moldenke, 1971; Hsiao, 1978; Liu et al., 1988) treated C. trichotomum Thunb. as comprising three varieties, while others (Li, 1963; Liu and Liao, 1981) recognized the existence of only two varieties, with the exclusion of C. trichotomum var. ferrugineum. Hue and Chen (1982) recognized the species without any intraspecific division. Another controversial taxon is the white-flowered variant of C. paniculatum L., which was published as a new variety by Hemsley in 1895 and named C. paniculatum var. albiflorum Hemsley. Hsieh (1973), however, lowered its rank to a form and named it C. paniculatum L. forma albiflorum (Hemsl.) Hsieh. The existence of C. intermedium Cham. in Taiwan is questionable even though it was treated as a native species by Hsiao (1978) and Liu (1981). In the present study, a plant close to the description of C. intermedium, and having leaves without lobes, was collected from Kenting Botanic Garden (Lin 1050). This solitary specimen is thought to be a cultivated plant, and this species is not included in the present study.

The information gathered from essential-oil studies has proved to be of value in the taxonomic and evolutionary investigations of plants (Von Rudloff, 1975; Southwell and Stiff, 1990; Brophy and Clarkson, 1992; Brothy et al., 1994). The purpose of the present study is to re-evaluate the relationships among native taxa of Clerodendrum in Taiwan, based on their essential-oil compositions. It is expected that these data will shed light on the natural relationships of the taxa.

1Corresponding author.

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

Materials and Methods


Mature-leaf samples and voucher specimens of 54 plants belonging to 9 native taxa of Clerodendrum were collected from various locations in Taiwan (Table 1). Voucher specimens are on deposit at TCB (Herbarium, National Chung Hsing University).

Capillary Gas Chromatography

For each sample, 250 grams of fresh mature leaves were steam-distilled to obtain the essential oil. The essential oils were analyzed by capillary gas chromatography with a Hitachi G3000 gas chromatograph equipped with a flame ionization detector (FID) and a Hitachi D2000 recorder. A 60-meter DB-5 fused silica capillary column 0.25 mm in diameter (J & W, USA.) was used for the separation of essential oil components. We injected 0.2 µl of each essential oil sample. The GC separating conditions were as follows: (N2 carrier-gas flow rate) make-up gas, 30 ml min-1, capillary column, 0.76 ml min-1; H2 flow rate, 30 ml min-1; air-flow rate, 300 ml min-1; column pressure, 1 kg cm-2; split ratio, 1/10; injection-port temperature, 260°C; detector temperature, 260°C; oven temperature, 60°C to 230°C at 2°C per min, then 230°C for 50 min.

Numerical Taxonomic Analysis

Nine taxa were treated as OTUs (operational taxonomic units). The values of gas chromatogram peaks were recorded as integrated-area percentages and were used as taxonomic characters. The peak values were averaged over the samples used, and this value was used in subsequent cluster analysis. The average taxonomic distances between each OTU-pair was calculated, and the resulting distance matrix was used in a UPGMA (unweighted pair-group method using arithmetic averages) cluster analysis (Sneath and Sokal, 1973). The formula for average taxonomic distance is

where n represents character number, and Xi and Xj represent the average percentage data of OTU i and OTU j, respectively. The numerical taxonomic analysis was performed using NTSYS-pc (version 1.80) software (Rohlf, 1993).

Results and Discussion

We identified 463 peaks, with the assumption that peaks with the same retention time on different chromatograms were the same compound. The data on these 463 peaks for each of the 54 samples can be found in Lin (1992). The first 5 major peaks and their percentages for each taxon are listed in Table 2. Based on the composition of the 5 major peaks, C. canescens, with peaks 379 and 455 as the first and second major peaks, is a distinct taxon. All other taxa have peaks 79 and 126 as their first and second major peaks, except for C. paniculatum, in which the rank order is reversed. The unusually high percentage of peak 455 in C. canescens is unique among the studied taxa. Clerodendrum inerme is distinct from the other taxa, in having peak 434 as its 5th major peak. Clerodendrum cyrtophyllum has a very high percentage (35.21%) of peak 79, and peak 47 is the 5th major peak.

Table 1. Sources of the samples studied.

Taxa Collector's Locality (County)

field No.

C. canescens Lin 916 Longtang (Ilan)

Lin 921 Hoping (Taichung)

Lin 933 Chungho (Taichung)

C. cyrtophyllum Lin 226 Longtang (Ilan)

Lin 228 Longtang (Ilan)

Lin 231 Chunghsingling (Taichung)

Lin 232 Chunghsingling (Taichung)

Lin 236 Dahan Forest Road (Pintung)

C. inerme Lin 704 Dadu (Taichung)

Lin 714 Sutiping (Taitung)

Lin 723 Baisawuan (Pintung)

Lin 724 Baisawuan (Pintung)

Lin 726 Wuanlitung (Pintung)

Lin 727 Wuanlitung (Pintung)

C. paniculatum Lin 136 Tienbien (Taichung)

Lin 140 Chuchi (Chiayi)

Lin 145 Chuchi (Chiayi)

Lin 148 Chuchi (Chiayi)

Lin 1001 Kantou (Yunling)

Lin 1011 Dahan Forest Road (Pintung)

Lin 1020 Tienbien (Taichung)

Lin 1051 Kanting (Pintung)

C. paniculatum Lin 157 Hsinhua (Tainang)

var. albiflorum Lin 187 Chushan (Nantou)

Lin 1501 Kanting (Pintung)

Lin 1521 Tienbien (Taichung)

C. philippinum Lin 305 Danshue (Taipei)

f. multiplex Lin 306 Danshue (Taipei)

Lin 309 Danshue (Taipei)

Lin 316 Tienli (Taichung)

Lin 332 Chushan (Nantou)

Lin 333 Tienli (Taichung)

Lin 336 Kantou (Yunling)

Lin 337 Kantou (Yunling)

Lin 342 Yangmingshan (Taipei)

C. trichotomum Lin 820 Ginshan (Taipei)

Lin 822 Ginshan (Taipei)

Lin 830 Ginshan (Taipei)

Lin 851 Ginshan (Taipei)

Lin 852 Ginshan (Taipei)

Lin 853 Ginshan (Taipei)

C. trichotomum Lin 862 Kanting (Pintung)

var. fargesii Lin 863 Kanting (Pintung)

Lin 864 Liduo (Pintung)

Lin 866 Liduo (Pintung)

Lin 867 Gankuo (Pintung)

Lin 868 Dahan Forest Road (Pintung)

Lin 894 Yuoshu (Nantou)

Lin 895 Yuoshu (Nantou)

C. trichotomum Lin 827 Wushuo (Nantou)

var. ferrugineum Lin 828 Wushuo (Nantou)

Lin 844 Ali (Taitung)

Lin 845 Ali (Taitung)

Lin 893 Zunchuguan (Nantou)

Hsiao and Lin — Chemotaxonomy of Clerodendrum

Table 2. Five major peaks and their percentages in the taxa studied.

Taxon 1 2 3 4 5

C. canescens 379a (9.70%)b 455 (7.62%) 79 (6.40%) 126 (5.37%) 43 (4.85%)

C. inerme 79 (14.91%) 126 (5.34%) 379 (3.62%) 32 (2.47%) 434 (1.94%)

C. cyrtophyllum 79 (35.21%) 126 (6.57%) 379 (4.49%) 43 (2.81%) 47 (2.13%)

C. philippinum multiplex 79 (40.60%) 126 (13.52%) 379 (7.37%) 43 (2.77%) 84 (2.60%)

C. paniculatum 126 (14.50%) 79 (14.07%) 379 (4.00%) 46 (3.98%) 113 (2.89%)

C. paniculatum albiflorum 79 (22.82%) 126 (14.56%) 379 (5.82%) 113 (5.24%) 44 (2.73%)

C. trichotomum 79 (14.40%) 126 (12.38%) 409 (11.36%) 379 (5.88%) 43 (3.17%)

C. trichotomum fargesii 79 (15.24%) 126 (8.49%) 379 (6.83%) 409 (6.01%) 215 (4.47%)

C. trichotomum ferrugineum 79 (14.72%) 126 (9.37%) 379 (8.35%) 409 (7.98%) 32 (6.10%)

aPeak number.

bPercentage of total peak area.

Table 3. Average taxonomic distances of the OTU-pairs.

C. canescens 0.0000

C. cyrtophyllum 1.4127 0.0000

C. inerme 0.6664 0.9909 0.0000

C. paniculatum 0.7530 1.0822 0.5254 0.0000

C. paniculatum var. albiflorum 1.0010 0.7502 0.6694 0.4567 0.0000

C. philippinum f. multiplex 1.7044 0.5034 1.2958 1.2781 0.8920 0.0000

C. trichotomum 0.8272 1.1405 0.6449 0.5731 0.7175 1.3302 0.0000

C. trichotomum var. fargesii 0.6799 1.0205 0.4162 0.5216 0.6407 1.2618 0.4149 0.0000

C. trichotomum var. ferrugineum 0.7198 1.0830 0.5230 0.5579 0.6913 1.3089 0.3461 0.3548 0.0000

This is unique for this species. Clerodendrum philippinum forma multiplex, similar to C. cyrtophyllum, also has an exceptionally high percentage of peak 79 (40.6%). Its 5th major peak (peak 84), however, is not in the list of major peaks of any other taxa. Clerodendrum paniculatum has roughly the same percentages of peaks 126 and 79, while its white-flowered variant has higher percentage of peak 79. The 4th and 5th major peaks of these two taxa are also different. The first 4 major peaks of C. trichotomum and its two variants are similar, except that the order of the 3rd and 4th major peaks is different in C. trichotomum. The 5th major peak of each of these three taxa are also different from each other.

The average taxonomic distances of the OTU-pairs are listed in Table 3. The phenogram from the cluster analysis is shown in Figure 1. In this phenogram, C. cyrtophyllum and C. philippinum forma multiplex form a distinct group, while the rest of the taxa form another group. The similarity of the total composition of the essential oil of C. cyrtophyllum and C. philippinum forma multiplex is also reflected in their five major components—the first 4 major peaks of these two taxa are similar (Table 2). In the other group, C. canescens is the most distinct taxon. This is not unexpected, because it possesses distinct major components. The essential oil composition

Figure 1. Phenogram based on 463 essential oil characters.

of C. inerme is close to that of C. trichotomum and its two varieties. As expected, C. trichotomum and its 2 varieties link together to form a cluster. Along with their morphological relationship, the essential oil composition of C. trichotomum is closer to that of C. trichotomum var. ferrugineum than to that of C. trichotomum var. fargesii. Clerodendrum trichotomum var. fargesii is the most isolated taxon among these three taxa, but the linkage level of this cluster indicates that these three taxa have the most-

Botanical Bulletin of Academia Sinica, Vol. 36, 1995

similar essential oil compositions among the 9 taxa studied. As expected, C. paniculatum and its white-flowered variant also form a cluster, but the linkage level of this cluster is higher than those of C. trichotomum and its two varieties. In a previous study of C. paniculatum and its white-flowered variant (Hsiao and Lin, 1990) we concluded that the morphological and chemical evidence supported the taxonomic treatment in which the white-flowered variant is treated as a variety of C. paniculatum instead of as a form. The results of the present study reinforce this conclusion, and we suggest that the scientific name C. paniculatum var. albiflorum Hemsl. be adopted.

The results of the present study show that there is a strong congruence between morphological and chemical relationships at the intraspecific level. In the essential oil phenogram, three intraspecific taxa of C. trichotomum and two intraspecific taxa of C. paniculatum are clustered as distinct groups. At the interspecific level, there is congruence and discrepancy between morphological and chemical relationships. Stenzel et al. (1988) studied the phenetic relationships of 129 Clerodendrum taxa, based on 52 morphological characters, using the methods of cluster analysis and minimum spanning tree. They concluded that these taxa could be divided into two subgenera and seven groups. With the exception of C. paniculatum var. albiflorum and C. trichotomum var. ferrugineum, the subjects of the present study were included in their study. In their results, C. cyrtophyllum and C. inerme belonged to group 1; Clerodendrum philippinum, C. trichotomum, and C. trichotomum var. fargesii belonged to group 2; Clerodendrum paniculatum belonged to group 3; Clerodendrum canescens belonged to group 4. All these taxa are members of the so-called `subgenera B'. In the present study, we reveal that C. cyrtophyllum is closer to C. philippinum forma multiplex than to C. inerme, and C. inerme is closer to the members of C. trichotomum than to C. cyrtophyllum. The members of C. paniculatum link to the group that comprises C. inerme and members of C. trichotomum. The essential oil composition of C. canescens is distinct, though it has relationships with members of C. paniculatum, C. trichotomum, and C. inerme. Several observed discrepancies between morphological and chemical relationships in the studied taxa may be the result of the difference in evolutionary directions of these two categories of characters.

Acknowledgment. This study was supported by grant NSC-80-0211-B-005-06 from the National Science Council, Republic of China.

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Hsiao and Lin — Chemotaxonomy of Clerodendrum