PoPulation variability in Thymus glabrescens Willd. from Serbia: morPhology, anatomy and eSSential oil comPoSition

In five indigenous populations of Thymus glabrescens Willd. collected in the region of Banat (Serbia), the variability on leaf morphological traits, leaf and stem anatomy, and composition of the essential oil was studied. The major component in the studied populations was either thymol or γ-terpinene. Distinct differentiation of populations with respect to chemical composition of essential oils might be related to spatial distribution of the studied populations. No correlations between morphology, anatomy, and essential oil yield and composition were determined. Both capitate and peltate glandular trichomes were found on calyces, whereas the latter were noticed on the abaxial and adaxial leaf surface.


INTRODUCTION
Thymus L. is one of the most important genera as regards the number of species (more than 200) within the family Lamiaceae.This genus belongs to the tribe Mentheae, subfamily Nepetoideae (M o r a l e s , 1986).That there are serious difficulties in the taxonomical interpretation of the taxa belonging to the genus Thymus owing to the high variability of populations with respect to many morphological and micromorphological traits, as well as the composition of secondary compounds (D a j i ć -S t e v a n o v i ć and Š o š t a r i ć , 2006).This variabily is caused both by environmental factors and genetic variation due to frequent hybridization leading to variable chromosome number and expressed gynodioecy, a sexual polymorphism in which natural populations contain two type of plants -females and hermaphrodites (T h o m p s o n , 2002).
volatiles, the bacterial cell membrane has been proposed as the primary target of their antimicrobial action (M i t i ć -ć u l a f i ć et al., 2005).Spasmolytic and antioxidant activities (M i g u l et al., 2004; S a cc h e t t i et al., 2005) have also been reported for the phenolic oil extract of the plants.
Thymus glabrescens Willd.belongs to the section Serpyllum, subsection Isolepides (J a l a s , 1972).It is a perennial herbaceous plant, distributed in Central and eastern europe as well as in Asia, inhabiting open dry meadows, grasslands, and rocks with sunny exposure (J a l a s , 1972).In Serbia, the species has a scattered distribution being found on the slopes of Mt.Fruška Gora, in the Deliblato Sands, throughout Šumadija, and in Southwest Serbia (D a j i ć -S t e v a n o v i ć and Š o š t a r i ć , unpublished data).
Despite broad scientific interest in the biology, taxonomy, chemotypes, and related biological activity of secondary metabolites of Thymus species, there is a general lack of information regarding Th. glabrescens, with the exception of two reports from the Western Balkans (K a r u z a -S t o j a k o v i ć et al., 1989;Kustrak, 1990) and one from Romania (K i s g y ö r g y et al., 1983).We therefore studied the morphological, anatomical, and essential oils of autochthonous populations of Th. glabrescens.The results are here present as a first report on comparative morphology, anatomy, and essential oil composition of this species from Serbia.

Collection of plant material and study site
Th. glabrescens was collected during the flowering period of June 2004 in the Banat region of Northeast Serbia (Table 1).Voucher specimens were determined in accordance with the Flora of Serbia (D i k l i ć , 1974) and Flora europaea (J a l a s , 1972) and deposited at the Department of Botany, Faculty of Agriculture, University of Belgrade, Belgrade.

Morphology and anatomy
The following morphological features of leaves were analyzed: length (mm), width (mm), and the length/ width ratio (N = 30) in all of the collected populations.
In analysis of leaf (N = 45) and stem (N = 30) anatomical traits, plant material was fixed in FAA, subjected to the standard paraffin procedure, and microtome sectioned using a LeICA SM 2000 R microtome, after which sections 7-10 µm thick were stained with safranine and aniline blue.Image analysis was done with LeICA IM1000 software.
In order to describe the number of glandular trichomes on leaves, 30 leaves were taken from individual plants and bright-field microscopy was conducted using a LeICA XTL-3400 D stereo-microscope.For scanning electron microscopy (SeM), small pieces of dry leaves and calyces of 10 plants were sputter-coated with gold for 180 sec at 30 mA using a BAL-TeC SCD 005 instrument and viewed with a JeOL JSM-6460L Velectron microscope at an acceleration voltage of 20 kV.

Isolation and analysis of essential oils
Air dried aerial parts were subjected to hydrodistillation for 3 h using a modified Clevenger-type apparatus with a water-cooled oil receiver to reduce hydrodistillation artifacts.The oil obtained was dried over anhydrous sodium sulfate and stored at 4-6°C until analyzed.
GC-FID analysis was carried out in a Hewlett Packard 5890 II gas chromatograph equipped with FID, a split-splitless injection system (split ratio of 1:30), and a 25 m x 0.32 mm HP-5 fused silica capillary column (film thickness: 0.52 μm).The carrier gas was H 2 , the flow rate 1 ml/min.Oven temperature was programmed from 40 o C to 260 o C at a 4 o C/ min linear rate; injector and detector temperatures were maintained at 250 o C and 300 o C, respectively.The injection volume was 1 μl of 1 % (w/v) essential oil in ethanol.GC-MS analyses were performed with a Hewlett Packard G 1800C GCD Series II (GC-eID) instrument fitted to a 30 mm x 0.25 mm HP-5 MS capillary column (film thickness: 0.25 μm) using He (1 ml/min) as the carrier gas, temperature of the transfer line being maintained at 260 o C. essential oil components were identified by matching their mass spectra with published data (A d a m s , 1989) and by consulting libraries of mass spectra (Wiley and NIST/NBS).

Data processing
Analysis of variance (ANOVA and Duncan multiple range test) and cluster analysis (based upon euclidean distances) of all surveyed populations of Th. glabrecens were performed using STATISTICA software, version 7.0.

leaf morphology
The population variability of leaf morphology showed that most of the studied populations differed significantly from each other (p<0.001) with the respect to leaf length, leaf width, and the leaf length/width ratio (Table 2).Maximal leaf length and width were determined for P3 and P4, respectively, minimal leaf length and width for P1 and P5, respectively.The highest and lowest leaf length/ width ratios were found for P3 and P1, respectively.
The number of secretory glands was practically equal on the abaxial and adaxial leaf surfaces (Table 2) when calculated as the mean for all populations (8.75: 8.76).Nevertheless, among the surveyed populations, the number of glands on both leaf surfaces differed to a great extent.The highest number of secretory glands was determined for P1, followed by P2, and the lowest for P4.
Based upon all tested morphology variables, three distinct clades were determined, one comprising P1, P2, and P5, the other with P3, whereas population P4 was found to be the most distant from all other populations, probably due to the significantly lowest average number of glandular trichomes on both the upper and lower leaf epidermis (Fig. 1).

leaf and stem anatomy
Leaf anatomical features measured in two different regions (at the central midrib and near the apex) showed that all characters were significant for discrimination of populations (ANOVA, Table 3).The highest and lowest leaf depths were found for P4 and P1, respectively.The height of upper and lower epidermal cells was greatest in P4 and P3, respectively.The height of palisade tissue was greatest in P4 and smallest in P1.
Stem measurements showed that only rib width was not significant for population discrimination.The highest and lowest stem widths were recorded for P4 and P5, respectively, whereas maximal and minimal central cylinder widths were found for P4 and P5, respectively.Based on all measured variables of leaf and stem anatomy, the grouping of populations was in two clades (Fig. 2): the first was made up of P1 and P5 and the second of all the other populations.

Description of secretory structures (SeM microscopy)
As in other species of the family Lamiaceae, Th. glabrescens is characterized by the presence of glandular trichomes, which are generally classified as either capitate (clavate) or peltate (subsessile), based on their morphological characteristics (We r k e r , 1993; Ascensao et al., 1998).Capitate glandular trichomes consist of one or two cells that sit atop a    chomes were determined (Fig. 3).Capitate glands were noticed on calyces only, but there they were very dense (Fig. 3a).Peltate trichomes were found on both surfaces of the leaf, as well as on the calyces (Fig. 3, b, c, d).

essential oil composition
The composition of essential oils in Th. glabrescens populations from Serbia was characterized by the presence of a total of 52 components (Table 4).The highest yield of essential oil was found in P2 (0.80%), the lowest in P5 (0.25%).The average essential oil yield for all populations was 0.60 ± 0.22%.
Average values of each component (Table 4) indicated that the major components were thymol (36.55%), γ-terpinene (22.32%) and p-cymene (14.03%).A certain variability of essential oil composition was observed in the analyzed populations of Th. glabrescens.While thymol was the major component in populations P2, P4, and P5, γ-terpinene was dominant in P1 and P3.
Based on all identified components of the essential oil, Th. glabrescens populations were grouped into two clades, the first made up of P4 and P5, the second of P1 and P3 and the close to them P2 (Fig. 4).Such grouping might be related to spatial distribution of the populations, populations P1, P2, and P3 being found in the central part of the Banat region and populations P4 and P5 in the southern part of the study area (Table 1).
Regarding the presence of particular chemical compounds in populations of Th. glabrescens (Table 5), it was found that the dominant compounds were monoterpene hydrocarbons (especially in P1, P2, and P3), followed by phenols (thymol was present in all populations except P3), aromatic hydrocarbons, and phenolic ethers.
Previously reported results on essential oil composition in Th. glabrescens from Croatia indicated that the major components were 1,8-cineole (29.4%); myrcene, camphene, α-pinene, β-pinene, and thymyl acetate (14.3%); and carvacrol, p-cimene and thymol, depending on the population (K u s t r a k et al., 1990).For Th. glabrescens from Bosnia and Herzegovina, the main component was α-terpinyl acetate (32%), followed by terpenene-4-ol, thymol, myrcene, and α-pinene components (K a r u z a -S t o j a k o v i ć et al., 1989).Our results indicate the existence of one major chemotype within populations of Th. glabrescens from Serbia -the thymol chemotype -but two "sub-chemotypes" could be distinguished, the strict thymol sub-type and the γ-terpinene sub-type.However, the presence of thymol, carvacrol, γ-terpinene, and p-cimene cannot be considered independently, since all these terpenes are closely connected in biogenetic processes, where γ-terpinene and p-cimene are known as precursors in the biochemical pathway of phenols (S t a h l -B i s k u p , 2002).

Fig. 1 .
Fig. 1.Cluster analysis of populations of Th. glabrescens based on traits of leaf morphology.

Fig. 2 .
Fig. 2. Cluster analysis of populations of Th. glabrescens based on traits of leaf and stem anatomy.

Fig. 4 .
Fig. 4. Cluster analysis of populations of Th. glabrescens based on essential oil composition.
essential oils containing large amounts of thymol have been shown to possess high antioxidant activity (F a r a g et al., 1989; A e s c h b a c h et al. 1994; L o ž i e n é et al.

Table 1 .
Geographic position and habitat description for studied populations of Th. glabrescens

Table 2 .
Morphological traits in populations of Thymus glabrescens (leaf length and width expressed in mm, number of secretory glands expressed per mm2 of leaf surface).Abbreviations: LL: leaf length; LW: leaf width; L/W ratio: leaf length/width ratio; No. glands ULE: number of glands on upper leaf epidermis; No. glands LLE: number of glands on lower leaf epidermis.

Table 3 .
Anatomical traits of leaf and stem in populations of Th. glabrescens (μm).Abbreviations: Upper Epid H: upper leaf epidermis height; Lower Epid H: lower leaf epidermis height; Palisade H: palisade height; CC Width: central cylinder width; variables without asterisk refer to measurments performed at the central leaf midrib; variables with asterisk refer to measurements near the leaf apex.
In Th. glabrescens, both types of glandular tri-