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Numerical Taxonomic Studies on Some Species of the Genus Thymus L. (Labiatae) in Turkey



Tulay Aytas Akcin
 
ABSTRACT

The similarities of some species of the genus Thymus L. (Labiatae) from Turkey, were studied using the morphological and chemical characters. Chemical analyses were carried out by gas chromatography/mass spectrometers. For each taxa, 20 quantitative characters and 130 volatile oil substituents were determined and the data subjected to numerical taxonomic analyses. The results exhibited that the dendrograms obtained from morphological and chemical data were similar. To the results, the closest taxa were T. sipyleus Boiss. and T. leucotrichus Hal. Likewise, T. praecox Opiz., T. longicaulis C. Presl. and T. thracicus Velen. were closely related species. However, T. pseudopulegioides Klokov and Des. Shost was distinct from the other taxa.

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Tulay Aytas Akcin , 2006. Numerical Taxonomic Studies on Some Species of the Genus Thymus L. (Labiatae) in Turkey. Asian Journal of Plant Sciences, 5: 782-788.

DOI: 10.3923/ajps.2006.782.788

URL: https://scialert.net/abstract/?doi=ajps.2006.782.788

INTRODUCTION

The genus Thymus L. contains some hundreds of species which distributed over nearly the whole of the world (Klokov et al., 1954).The Mediterranean region can be described as the center of the genus (Stahl-Biskup and Saez, 2002). Jalas (1980) reported that the genus Thymus L. includes 37 species in Turkey and 14 of which are endemics. First description of the genus was by Linneaus (1753) in his book Species Plantarum. In the revision by Jalas (1971b) the genus were divided into two subgenera: Coridothymus and Thymus. In this revision, some of the species found in the sect. Pseudotymbra or Serphyllum were scattered in sect. Hypodromi (Jalas, 1971b).

Thymus L. is an aromatic plant with increasing importance in food processing (Letchamo et al., 1994). Thymus species have been used for more than 2000 years as medicinal herbs and many of them are still in use (Tzakou and Constantinidis, 2005). The genus Thymus L. called “thyme” is one of the plants used as a folk medicine in Turkey (Başer et al., 1991a). Volatile oil constituents of thyme are used as anticeptics, antispazmodics and fungusidal (Meriçli, 1986a; Çingi et al., 1991).The anticeptic, antioxidative, insectecidal, preservative and anaesthetic properties of thyme are due to their biologically active substances, such as thymol, carvacrol, linalool, geraniol and other volatiles in the essential oil (Van-Den Broucke and Lemli, 1983). Recent studies have showed that Thymus species have strong antibacterial, antifungal, antiviral, antiparasitic, spasmolytic and antioxidant activities (Stahl-Biskup and Saez, 2002). The aromatic and medicinal properties of the genus Thymus have made it one of the most popular plants throughout all of the world (Nickavar et al., 2005).Although thyme is useful in the food and aroma industries and nowadays a serious drug in phytotherapy, both the taxonomy and the chemistry of the genus appear quite complex (Zarzuelo and Crespo, 2002).

There are many works on thyme flavonoid aglycones and glycosides (Hernandez et al., 1987; Tomas-Barberan and Wollenweber, 1990; Rustaiyan et al., 2000; Stahl-Biskup and Saez, 2002).Previous studies suggested that the excreted flavonoids were very useful taxonomic markers within the genus, since different flavonoid patterns were found among various species of the genus ( Hernandez et al., 1987).It was also determined that the thyme species collected from similar geographical localities exhibited slight differences in the relative amounts of each compound (Tomas-Barberan and Wollenweber, 1990). Meriçli (1986 a) reported that some species of Thymus L. contained oil dots at different quantity varying from one sampling locality to other. Yamaura et al. (1989) noted that the synthesis of essential oils in aromatic plants might be influenced by environmental factors, such as temperature, photoperiods and water stress. It was reported that the thyme species showed chemical polymorphism (Schmidt et al., 2004). Tzakou and Constantinidis (2005) suggested that further studies are needed, which should cover the essential oils from different individual plants or specimens from various geophraphical regions.

Table 1: Arithmetic averages of 20 quantitative characters in investigated Thymus L. taxa

The main objective of this study was to classify some Turkish Thymus L. species by using numerical taxonomic methods and to compare the results obtained from the morphological and chemical data.

MATERIALS AND METHODS

Specimens belonging to different taxa of Thymus L. were collected from Black Sea Region, Turkey during the period between May and August of 1995-1997.The specimens were identified by referring to the information in the Flora of Turkey and the East Aegean Islands, Vol.7 (Davis, 1982). Descriptions of all species were made using both fresh material and herbarium specimens. For each specimen, forty-nine distinctive morphological characters were determined. The maximum and minimum boundaries of twenty quantitative characteristics were measured and their arithmetic averages were calculated (Table 1).

The essential oil content was analysed by a Hewlett-Packard Gas Chromatography (GC) and Gas Chromatography/mass Spectrometers (GC/MS) as described by Tanker (1973) and Başer et al. (1991b). The operation conditions were as follows: initial column temperature was programmed to 220°C at 4°C min-1 and 240°C at 1°C min-1. Carrier gas was helium with a flow rate of 1 mL min-1. Individual components were identified by comparing with mass spectra of the authentic samples and with the Wiley/NBS Registry of mass spectral data library.

For numerical analyses, the SPSS program (version 8.0) was applied (Wolf and Whitkus, 1987; Reiseberg et al., 1987). The average taxonomic distances between pairs of OTUs were computed using Euclidean distance method (Sneath and Sokal, 1973; Doğan et al., 1992). Arithmetical averages of the twenty morphological characters were used. The volatile oil components of each taxa was assumed as present or absent to produce data matrix showing the similarities among the taxa. UPGMA (Unweighted Pair Group Method using Arithmetic averages) trees obtained from the two analyses (i.e., morphological and chemical) were compared with each other.

RESULTS AND DISCUSSION

The phenogram resulting from UPGMA using the averages of twenty morphological quantitative characters is presented in Fig. 1. From the morphological data it appears that two species, T. sipyleus and T. leucotrichus are the most similar taxa at 96.0% similarity level. SPSS program clearly suggested that two main clusters were apparent, one comprising T. sipyleus and T. leucotrichus; the other grouping T. praecox, T. thracicus, T. longicaulis and T. pseudopulegioides. In the second grouping, T. pseudopulegioides formed basal node to this grouping.

Actually, these results are basically similar to phylogenetical data in Flora of Turkey (Davis, 1982). However, T. leucotrichus differs from T. sipyleus in that it has leaf with revolute margins and longer hairs.

Fig. 1: Phenogram based on morphological characters

Table 2: The compounds detected in the volatile oils of five Thymus L. taxa

The numerical analyses of morphological characters showed that T. praecox, T. thracicus and T. longicaulis were interpreted as related species. Morphological evidences also supported these similarities. Particularly, T. thracicus and T. longicaulis are so similar in morphology that some members of T. thracicus are sometimes assigned as T. longicaulis (Jalas, 1971a). In the present study, it was found that some of the morphological characteristics, such as leaf shape and whether or not leaf margins are revolute were found to be useful to distinguish these Thymus L. species. T. thracicus differs from T. longicaulis in that it has flat leaf margin and oblanceolate leaf shape. In addition, Jalas (1971 a) suggested that characteristics, such as length of calyx and upper calyx teeth were also usable diagnostic characters for these species. Actually, in T. longicaulis the length of calyx teeth is 2.5-4.2 (-4.7) mm and the length of upper calyx teeth is 0.5-1.3 (-1.5) mm. In comparison in T. thracicus these characters are 4.2-5.5 mm and 1.2-1.8 mm, respectively. Therefore, it was found that these quantitative characters could be useful for distinguishing the two species in addition to other morphological characteristics mentioned above. The results of UPGMA analyses showed that T. pseudopulegioides was not similar to other taxa. These results agree with the fact that this taxon can be clearly distinguished from the other taxa based on morphological characters. T. pseudopulegioides is different from the other taxa despite the fact that the stem is hairy along the four edges and dimensions of cauline leaves (Table 1). It seems that UPGMA dendrogram using quantitative characters supports the morphological evidences.

Gas Chromatography (GC) and Gas Chromatography/ Mass Spectrometers (GC/MS) analyses of the volatile oils of five taxa of Thymus L. led to determination of one-hundred thirty different compounds (Table 2). Twelve of these compounds (i.e rosefuran, pentacosene, phytol, tricyclene, 3-nananone, perillene, myrtenal, cubebene, carvone, pinovarvone, nonyl acetate, cis carveol ) were present in less than 0.02%. Thus, they were excluded from the data analyses. Thymol, which is characteristic essential oil of Thymus L. genus, was not found in T. sipyleus volatile oil. The chemical composition of the volatile oil from T. sipyleus showed that carvacrol was the lowest compounds of the oil with percentages of 0.15%. These results were also supported by Tanker (1973), as he pointed out that some of Thymus L. species with a lemon- like odour contains no thymol. Tanker (1973) and Meriçli (1986 a, b) suggested that the genus Thymus L.; except for T. sipyleus that has lemon-like odour, was characterized by its thymol odour due to thymol and carvacrol. The main components of the essential oil of T. sipyleus were β-caryophyllene (11.66% ), linalool (9.14%), 1,8-cineole (7.46%), caryophyllene oxide (5.20%) and α-terpineole (3.08%). Citral-a (neral) and citral-b (geranial) were also determined in only volatile oil of T. sipyleus suggesting that it can be distinguished from the other species based upon the chemical characters.

Morphological similarities of T. longicaulis and T. thracicus have also been evident from the chemical features. T. thracicus oil was rich in p-cymene (25.44%), thymol (15.69%), carvacrol (18.21%) and γ-terpinene (11.08%). The main components of the essential oil of T. longicaulis were α-terpnyl acetate (37.98%), thymol (16.76%), p-cymene (15.01%) and β-bisabolene (3.54 %).

Fig. 2: Phenogram based on chemical characters

Thymol, the main constituent of the essential oil, was found in high concentrations in T. longicaulis and T. thracicus volatile oil, whereas it was determined at lower amount in T. praecox volatile oil. On the other hand, T. longicaulis and T. thracicus are also similar to each other because of the higher thymol concentrations.

T. pseudopulegioides is distinguished from all of the other taxa by having carvacrol methyl ether at higher concentrations (4.57%). Similarly aromadendrane, neryl acetate, (E)-α-bisabolene and cadelene, were also only found in T. pseudopulegioides volatile oil. These results agree with the fact that T. pseudopulegioides is different from the other taxa with regard to chemical characters.

UPGMA dendrogram using chemical components for each taxon (Fig. 2) resulted in different from that obtained using quantitative characters (Fig. 1).It was unfortunate that T. leucotrichus could not be included in chemical based UPGMA analyses because it had essential oil very low concentration. Phenetic analyses based on chemical characters indicated that the most similar taxon were T. thracicus and T. longicaulis with respect to volatile oil components. In the phenogram, two main clustering were apparent, one formed by T. thracicus and T. longicaulis and the other formed by T. sipyleus and T. praecox. However, T. pseudopulegioides was placed at the base of four and was sister taxon to the two maining clusters.

T. sipyleus was closely related to T. praecox in UPGMA dendrogram based on chemical characters (Fig. 2) whereas it was the most similar to T. leucotrichus in morphological based UPGMA analyses (Fig. 1).This discrepancy is due to fact that T. leucotrichus could not be included in the present numerical classification based upon chemical characters because it had lower content of volatile oil.

The results suggested that the essential oil oils of Thymus L. taxa are not always correlated with the classification. On the other hand, morphologically related species may be share different compounds. For instance, α-terpnyl acetate was present in T. longicaulis at higher concentrations (37.98%) while it was absent from T. thracicus. This contention was also supported by Hernandez et al. (1987), as he pointed out that the volatile oils may be altered by environmental conditions. Previous studies have reported that the essential oil contents of thyme species were different depending on the locality (Adzet and Martinez, 1981; Tomas-Barberan et al., 1988; Tzakou and Constantinidis, 2005). The present study is also in accordance with this contention. The results supported an influence of ecological factors on the excretion of essential oils.

UPGMA dendrogram based on quantitative characters is more reliable and reflects the possible relationships of taxa. A comprehensive study covering all of the Thymus L. species seems to be necessary to construct a satisfactory classification.

ACKNOWLEDGMENTS

I am indebted to Dr. Muhammet Dalcı for supervising this research. I am also grateful to Dr. K. Hüsnü Can Başer, Dr. Mine Kürkçüoğlu and other collegues for determining the GC/MS.

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