Peltate Glandular Trichomes in Relation to Their Parameters, Essential Oil Amount, Chemotype, Plant Sex and Habitat Characteristics in Thymus pulegioides

Abstract

The parameters and plant habitat characteristics of glandular trichomes could allow for faster and cheaper identification and selection of more essential oil-rich wild aromatic plants for further cultivation. This study aimed to establish relationships between the parameters of peltate glandular trichomes and essential oil content in commercially potential Thymus pulegioides in relation to plant sex, chemotype, and habitat characteristics. In total, 124 T. pulegioides plants belonging to different chemotypes and sexes and collected from 23 natural habitats were analysed. Essential oils were extracted by hydrodistillation, and a light microscope was used to investigate parameters of peltate glandular trichomes in upper and lower leaf epidermises. For investigation of the dynamics of the parameters of peltate glandular trichomes, T. pulegioides were growing in open ground under the same environmental conditions. Results demonstrated that the essential oil percentage in phenolic chemotype plants was higher than in plants of a non-phenolic chemotype. Females and hermaphrodites did not significantly differ according to essential oil percentage. Cover abundance of T. pulegioides negatively affects the density and diameter of peltate glandular trichomes and the essential oil percentage in T. pulegioides. The parameters of peltate trichomes in the upper leaf epidermis could be anatomical markers, helping to select T. pulegioides with higher essential oil contents from natural habitats as promising candidates as new crops.

1. Introduction

Although the collection of wild aromatic plants (which are most often also medicinal plants) is still an important method of obtaining raw material of these plants, it can cause the loss of their genetic diversity and habitat destruction [1]. Therefore, to meet the growing demand, to preserve genetic resources, and to guarantee the sustainable use of wild aromatic plants, the domestication and the cultivation of their wild forms are important. The content of essential oil, which is greatly influenced by the genetic characteristics of the plant, is one of the initial criteria for selecting aromatic plants from their natural habitats for their future cultivation [2].

Species of the Thymus (Lamiaceae) genus are pharmaceutically valuable essential oil-bearing medicinal and aromatic plants, many of which are commercially important as sources of secondary metabolites and phytochemicals [3,4]. The intraspecific chemical polymorphism—when plants of the same species accumulate essential oils with different chemical compositions—as well as the sexual dimorphism (gynodioecy)—when females (i.e., male-sterile individuals) coexist with hermaphroditic individuals in natural populations—is typical for species of this genus [5,6]. Plants of Thymus species possess two types of glandular trichomes—peltate and capitate. Literature data suggest that the peltate glandular trichomes (consisting of one basal epidermal cell; a wide, short stalk; and several secretory head cells) are more associated with the synthesis of essential oils in various members of the Lamiaceae genus because they produce most of monoterpenes and sesquiterpenes, the main compounds of essential oils [7,8]. When describing the raw material of Thymi herba in the European Pharmacopoeia, only peltate glandular trichomes are also described as secretory trichomes made up of secretory cells, the cuticle of which is generally raised by secretion to form a globule to avoid bladder-like covering [9]. Additionally, microscopic observations of peltate glandular trichome distribution and abundance in distinct plant organs of Thymus albicans showed their highest density in leaves, which is significantly distinct from their density in calyces, corollas, and bracts [10]. The morphological and chemical differences between females and hermaphrodites and different chemotypes in species of the Thymus genus have been studied [6,11]. However, the differences in the anatomical characteristics related to essential oil accumulation have not been investigated either between females and hermaphrodites or between different chemotypes.

Essential oil-bearing large thyme (Thymus pulegioides L.), widely occurring in Central Europe, Scandinavia, the Baltic region, and Southeast England, is a medicinal and aromatic plant characterised by different pharmaceutical properties, with a high intraspecific chemical polymorphism [12,13,14,15] and strongly expressed sexual dimorphism with high female frequency in natural habitats [16]. Due to the chemical polymorphism inherent to the species, it is possible to select wild-growing T. pulegioides clones belonging to different chemotypes with characteristic biologically active chemical compounds [16]. One of the initial selection criteria is the essential oil content, which, like other species of the Thymus genus, is highest during the flowering stage and varies significantly between different plants [9,17].

Determining the preliminary essential oil content in a plant not by classic hydrodistillation but by calculating its predicted content from the parameters of the peltate glandular trichomes would be a faster and cheaper method (it could be performed in nature with a portable microscope). Establishing the relationships between essential oil content and the parameters of peltate glandular trichomes and habitat characteristics could be helpful in order to facilitate the selection of T. pulegioides that accumulate more essential oil. Therefore, this study aimed to establish the relationships between the diameter and density of peltate glandular trichomes and essential oil content in relation to plant sex, chemotype, and some habitat characteristics.

2. Materials and Methods

2.1. Essential Oil Content, Chemotype, and Sex of Thymus pulegioides Plants

In total, 124 blooming T. pulegioides plants were randomly collected from 23 natural habitats (5–7 plants each, excluding one habitat with 4 investigated plants) (

Table 1. Characteristics of Thymus pulegioides habitats (F-B—vegetation class Festuco-Brometea erecti 1943; T-G—vegetation class Trifolio-Geranietea sanguinei 1961; M-A—vegetation class Molinio-Arrhenatheretea elatioris 1937; *—anthropogenic plant community; **—cover abundance of all Thymus pulegioides growing in habitat according to Braun-Blanquet scale [20]; ***—number of chemically and anatomically investigated Thymus pulegioides individuals in habitat.

No.	Locality	Coordinates WGS-84	Class of Vegetation	Relief	Inclination, °	Exposition	Lightning, %	Total Herb Cover, %	Thymus pulegioides **	Thymus pulegioides ***
1	Vilnius dist., Uosininkai	54.714975, 25.587978	M-A	wavy	–	–	95	90	1	4
2	Vilnius dist., Dunojai	54.620109, 25.708344	T-G	wavy	–	–	80	80	2	6
3	Vilnius dist., Vindžiūnai	54.563248, 25.703089	F-B	wavy	–	–	80	80	1	5
4	Vilnius dist., Grikieniai	54.777020, 25.114203	T-G	slope	30	S	100	98	2	7
5	Širvintos dist., Kernavė	54.879947, 24.846071	T-G	plane	–	–	100	85	+	7
6	Vilnius dist., Trečiokiškės	54.833401, 24.996916	M-A	wavy	–	–	98	90	2	5
7	Vilnius, Visoriai	54.753797, 25.261059	M-A	wavy	–	–	98	90	3	5
8	Elektrėnai mun., Paaliosė	54.795416, 24.885738	F-B	slope	10	S	90	80	1	6
9	Trakai dist., Meiriškės	54.725080, 24.878361	F-B	slope	20	S	100	95	1	7
10	Trakai dist., Maušiškės	54.605374, 24.869349	M-A	wavy	–	–	100	95	+	5
11	Trakai dist., Onuškis	54.522103, 24.616383	M-A	slope	20	SE	100	85	+	5
12	Trakai dist., Rūdiškės	54.512941, 24.783850	T-G	plane	–	–	100	90	1	5
13	Vilnius dist., Nemenčinė	54.908972, 25.322706	M-A	slope	30	S	100	95	+	5
14	Vilnius dist., Paberžė	55.016576, 25.401064	M-A	wavy	–	–	100	90	1	5
15	Molėtai dist., Dubingiai	55.091495, 25.432930	M-A	slope	15	SW	100	90	1	5
16	Vilnius, Mažieji Gulbinai	54.777692, 25.290565	T-G	plane	–	–	100	90	1	5
17	Vilnius dist., Minkeliai	54.947612, 25.489782	M-A	slope	10	SE	100	85	+	5
18	Vilnius dist., Bratoniškės	54.853805, 25.377456	*	plane	–	–	60	75	1	5
19	Šilalė dist., Rubinavas	55.285243, 22.811440	F-B	slope	10	SE	100	85	2	6
20	Šilalė dist., Medvėgalis	55.374361, 22231996	T-G	slope	30	SW	100	95	+	6
21	Šilalė dist., Kaltinėnai	55.340161, 22.260123	T-G	slope	10	S	100	70	+	5
22	Telšiai dist., Rainiai	55.574721, 22.174702	M-A	plane	–	–	100	80	1	5
23	Tauragė dist., Skaudvilė	55.406632, 22.598877	T-G	slope	20	S	100	85	1	5

). Figure 1 demonstrates T. pulegioides growing wild in an investigated natural habitat. Eighty investigated individual plants were female, and 44 were hermaphrodites. A plant’s sex was established by the morphology of the flowers: a female individual has a pistil in the flowers only, while a hermaphrodite has both pistils and stamens. Also, 79 individuals of T. pulegioides belonged to a group of phenolic chemotypes, and 45 individuals belonged to a group of non-phenolic chemotypes. Each chemotype has a specific odour [18,19], which enables the attribution of T. pulegioides individuals to the phenolic or non-phenolic chemotype group. Thus, the assignment of each T. pulegioides plant to one of several chemotype groups was performed organoleptically based on the odour of its aboveground parts in the following way: (1) for the group of phenolic chemotypes—according to the presence of the odour of monoterpene phenols thymol and carvacrol (both these isomers smell alike); (2) for the group of non-phenolic chemotypes—according to the presence of the odour of a sweet rose with a specific citrus tang or an “ester” odour with a lavender scent.

Essential oils were isolated by hydrodistillation in a Clevenger-type apparatus (Simax, Praha, Czech Republic) in 2 h from all 124 T. pulegioides plants [9]. The essential oils were isolated from dried above-ground parts of each plant separately in 2–3 replicates; the contents of essential oils were recalculated as percentages of dry matter (% v/w).

2.2. Analysis of Peltate Glandular Trichomes

The density and diameter of peltate glandular trichomes in the lower and upper leaf epidermis were investigated separately in all 124 T. pulegioides plants. The impress method and an ECLIPSE Ci-S Nikon light microscope (Nikon Europe B.V.) were used to investigate anatomical structures. Both sides of 5–7 fresh leaves, collected from the middle of different flowering stems of each plant, were spread with a thin layer of colourless nail polish, and the formed films were viewed with a light microscope. To assess the density of peltate glandular trichomes in plants, 30–50 view fields (6–7 view fields per leaf) on each leaf side in each plant were examined under a microscope. To assess the diameter of peltate glandular trichomes in the plants, 50 peltate glandular trichomes from each plant’s lower and upper epidermis (7–10 peltate glandular trichomes in each analysed leaf) were investigated. The view fields used for analysis of the density and diameter of peltate glandular trichomes were chosen to avoid the margins, apex, and big veins of leaves.

2.3. Analysis of Habitats

A total of 23 different habitats of T. pulegioides were described in Lithuania (Table 1). The distances between habitats were not less than 20 km. The study was conducted in 16 m² habitat fields according to the methodology of Braun-Blanquet [20]. Plant communities were distinguished according to the vegetation classification systems of J. Balevičienė et al. [21]. Relative lightening of the habitats was defined visually.

The soil samples were collected from each habitat separately and dried at room temperature. Each sample of topsoil was prepared in the following way: 5 subsamples (subject to the area of habitat; each subsample ~100 g) were taken from a depth of 10–15 cm (from the plant rhizosphere) according to the envelope principle with a distance of 1 m from the central point of habitat and mixed (homogenized). The content of mobile phosphorus was estimated photoelectrocolorimetrically; the content of mobile potassium by flame photometry; the contents of mobile manganese, mobile iron, mobile calcium, mobile sulphur, and mobile magnesium by atomic absorption spectroscopy; and the soil pH electrometrically using a 1 M KCl solution. Soil analysis was performed at the Institute of Agriculture of the Lithuanian Research Centre for Agriculture and Forestry.

2.4. Dynamics of Density and Diameter of Peltate Glandular Trichomes

Eight plants of T. pulegioides belonging to different chemotypes and grown in the open ground of the field collection of the State Scientific Research Institute Nature Research Centre (Vilnius, Lithuania) were used for the investigation of the dynamics of the density and diameter of peltate glandular trichomes in leaves. These plants were grown under the same environmental conditions and not watered or fertilised. The diameter and density of peltate glandular trichomes were measured from the start of vegetation to the start of flowering every two weeks in the same way as described above.

2.5. Statistical Analysis

A t-test was used for the analysis of differences in pairs of various parameters of plants and plant groups (phenolic and non-phenolic chemotypes and females and hermaphrodites), Pearson correlation was used for analysis of correlation relationships between various parameters of plants, Spearman correlation was used for analysis of correlation relationships between parameters of plants groups, and the Kruskal–Wallis test was used for analysis of differences between habitats and habitat groups (belonging to three phytocoenological vegetation classes) according to the parameters of T. pulegioides and their habitats. The letter r denotes the correlation coefficients, and the letter N denotes the sample sizes (the number of individual T. pulegioides plants or the number of habitats, depending on the context of the content). Statistical data processing was carried out with STATISTICA^® 7 and MS Excel 2016 software.

3. Results

3.1. Characteristics of Thymus pulegioides Habitats

Half of the investigated T. pulegioides habitats were located on south, southeast, or southwest slopes with 10–30° inclination (Table 1). Most habitats had good light, varying between 90% and 100%, and only three had light below 90%. Total herb cover in habitats varied from 70% to 98% and significantly positively correlated with habitat illumination (r = 0.42, p < 0.05, N = 23). According to the Braun-Blanquet scale, the value of cover-abundance of T. pulegioides was usually 1, and only one habitat reached a value of 3. Almost half of the investigated T. pulegioides habitats belonged to the grassland communities of phytocoenological vegetation class Molinio-Arrhenatheretea, a little less to the Trifolio-Geranietea vegetation class, and the least to the Festuco-Brometea vegetation class; one habitat in Vilnius district was anthropogenic (Table 1). Variation of soil pH and amounts of some micro- and macroelements in soils of the investigated habitats are presented in

Table 2. Soil composition and pH of Thymus pulegioides habitats. A sample of topsoil of each habitat was prepared from 5 subsamples, which were taken from the plant rhizosphere according to the envelope principle at a distance of 1 m from the central point of the habitat and mixed. Therefore, the values presented in the table represent averages.

No.	Locality	pHKCl	Phosphorus, mg/kg	Potassium, mg/kg	Manganese, mg/kg	Iron, mg/kg	Calcium, mg/kg	Magnesium, mg/kg	Sulphur, mg/kg
1	Vilnius dist., Uosininkai	7.2	76	72	116	544	8696	768	3.0
2	Vilnius dist., Dunojai	6.0	92	61	54.4	369	856	93	3.5
3	Vilnius dist., Vindžiūnai	6.0	115	78	72.7	477	1116	179	4.1
4	Vilnius dist., Grikieniai	6.7	57	122	176	1092	4776	1076	4.3
5	Širvintos dist., Kernavė	4.8	174	102	85.8	743	644	86	2.2
6	Vilnius dist., Trečiokiškės	7.1	116	78	122	547	6212	1096	7.9
7	Vilnius, Visoriai	7.2	119	86	139	777	12,768	1704	5.1
8	Elektrėnai mun., Paaliosė	6.8	132	52	101	785	1153	241	1.6
9	Trakai dist., Meiriškės	7.4	115	82	84.9	436	20,880	1252	2.2
10	Trakai dist., Maušiškės	7.3	310	63	96.1	501	9396	1412	5.0
11	Trakai dist., Onuškis	7.5	387	92	73.6	794	16,784	1624	3.1
12	Trakai dist., Rūdiškės	6.3	278	96	142	2560	4382	333	2.5
13	Vilnius dist., Nemenčinė	6.1	82	51	50.4	440	974	148	3.4
14	Vilnius dist., Paberžė	6.7	134	118	102	767	3588	1072	1.8
15	Molėtai dist., Dubingiai	7.0	115	92	122	703	3026	746	3.2
16	Vilnius, Mažieji Gulbinai	5.0	212	105	88.2	998	782	108	2.0
17	Vilnius dist., Minkeliai	7.6	141	58	103	766	8864	1792	3.3
18	Vilnius dist., Bratoniškės	6.5	202	79	105	700	1445	222	5.5
19	Šilalė dist., Rubinavas	7.0	180	53	142	808	4336	255	6.7
20	Šilalė dist., Medvėgalis	7.5	179	61	95.9	611	12,672	412	6.8
21	Šilalė dist., Kaltinėnai	5.1	91	78	59.5	611	661	91	5.2
22	Telšiai dist., Rainiai	7.4	110	101	87.2	937	19,900	1084	4.8
23	Tauragė dist., Skaudvilė	6.1	28	85	51.8	270	1657	148	5.4

.

3.2. Essential Oil Amounts of Thymus pulegioides in Different Habitats, Chemotypes, and Sexes

The essential oil percentage in the studied T. pulegioides plants was 0.82 ± 0.23% (N = 124) and varied between individuals within the limits of 0.12–1.68%. The average essential oil percentages of T. pulegioides investigated in different habitats are presented in Figure 2: the highest average essential oil percentages (1% and more) were determined in T. pulegioides of habitat nos. 11, 12, 18, and 19 and the lowest in those of habitat no. 21. However, only habitat no. 12 significantly (p < 0.05) differed from habitat no. 2, as well as habitat no. 21 significantly differing from habitat nos. 12 and 18, according to the essential oil percentage in T. pulegioides.

Statistically significant (p < 0.05) correlations were found between the average essential oil percentage of T. pulegioides in the habitat and the mobile potassium and mobile iron content in habitat soil (r = 0.58% and r = 0.57%, respectively; N = 23), as well as between the average essential oil percentage of T. pulegioides in the habitat and the cover abundance of T. pulegioides in the habitat (r = −0.48, N = 23). Although the average essential oil percentage of T. pulegioides in habitats belonging to grassland communities of the Festuco-Brometea vegetation class was 0.1% higher than in habitats belonging to grassland communities of the Trifolio-Geranietea vegetation class (0.85 ± 0.07% and 0.76 ± 0.33%, respectively), this difference was not statistically significant.

The essential oil percentage in T. pulegioides plants of the phenolic chemotypes was higher and significantly (p < 0.05) differed from the essential oil percentage in T. pulegioides plants of non-phenolic chemotypes (

Table 3. Essential oil percentage, as well as the density and diameter, of peltate glandular trichomes in the upper and lower leaf epidermis in different chemotypes and sexes of Thymus pulegioides. The t-test demonstrated that only plants of phenolic chemotypes significantly (p < 0.05) differed from plants of no-phenolic chemotypes according to essential oil percentage (marked with different letters in the table). Significant differences in parameters between other groups were not detected.

Parameter		Chemotypes		Sexes
		Phenolic (N = 79)	Non-Phenolic (N = 45)	Females (N = 80)	Hermaphrodites (N = 44)
Essential oil, %	Means ± SD	0.90 ± 0.32 a	0.67 ± 0.31 b	0.85 ± 0.34	0.76 ± 0.3
	Min	0.18	0.12	0.12	0.21
	Max	1.68	1.54	1.68	1.54
Density of peltate glandular trichomes in upper epidermis (in mm2)	Means ± SD	7.0 ± 1.8	7.2 ± 1.6	7.2 ± 1.6	7.4 ± 1.9
	Min	3.3	4.3	3.3	4.1
	Max	10.2	11.7	10.9	11.9
Density of peltate glandular trichomes in lower epidermis (in mm2)	Means ± SD	8.0 ± 2.1	8.1 ± 1.7	8.0 ± 2.1	7.5 ± 2.0
	Min	4.3	4.4	3.7	3.4
	Max	13.2	10.7	13.2	11.4
Diameter of peltate glandular trichomes in upper epidermis, µm	Means ± SD	64.7 ± 3.6	63.3 ± 4.5	64.1 ± 4.1	64.2 ± 3.9
	Min	52.2	51.1	51.1	53.3
	Max	73.7	74.2	73.7	74.2
Diameter of peltate glandular trichomes in lower epidermis, µm	Means ± SD	61.0 ± 5.5	59.6 ± 4.1	59.9 ± 3.4	61.6 ± 7.0
	Min	47.5	48.9	47.5	48.9
	Max	101.0	69.9	67.3	101.0

). Meanwhile, T. pulegioides females and hermaphrodites did not significantly differ according to the essential oil percentage, although in females, the average essential oil percentage was higher than in hermaphrodites (Table 3).

3.3. Density of Peltate Glandular Trichomes in Thymus pulegioides Growing in Different Habitats and Belonging to Different Chemotypes and Sexes

The peltate glandular trichome density was 7.0 ± 1.7 and 8.0 ± 2.0 trichomes in mm² (N = 124) and varied from 3.3 to 11.7 and from 4.3 to 13.2 trichomes in mm² in the upper and lower leaf epidermis of T. pulegioides, respectively. Although the upper and lower leaf epidermises significantly (p < 0.05) differed according to the peltate glandular trichome density, a significant correlation was not established between the peltate glandular trichome density in the upper and lower epidermis. Essential oil percentage positively correlated with peltate glandular trichome density in the upper and lower leaf epidermis. However, it was significant only in the upper epidermis (r = 0.34, p < 0.05, N = 124) (Figure 3A).

Figure 4 represents the average density of peltate glandular trichomes of investigated T. pulegioides growing in different habitats. Although average values of peltate glandular trichome density in the upper and lower leaf epidermis of T. pulegioides growing in some habitats differed by as much as two times, only habitat nos. 18 and 6 differed significantly (p < 0.05) in peltate glandular trichome density in the upper epidermis.

Significant correlations of peltate glandular trichome density with amounts of soil elements have not been established. The average density of peltate glandular trichomes in both T. pulegioides leaf epidermises in the habitat negatively correlated with habitat illumination and cover abundance of T. pulegioides in the habitat. Between the illumination of the habitat and the average density of peltate glandular trichomes in the lower epidermis, this relationship was significant (r = −0.42, p < 0.05, N = 23). T. pulegioides plants growing in habitats belonging to the Molinio-Arrhenatheretea elatioris, Festuco-Brometea, and Trifolio-Geranietea vegetation classes did not differ significantly in the density of peltate glandular trichomes in either the upper or lower epidermis.

In non-phenolic chemotypes, the peltate glandular trichome density in the upper and lower epidermis was higher than in phenolic chemotypes (Table 3). However, these differences were not significant. In phenolic and non-phenolic T. pulegioides individuals, significant (p < 0.05) positive correlations were found between essential oil percentage and density of peltate glandular trichomes in the upper and lower epidermis; these relationships were somewhat stronger in plants of phenolic chemotypes.

Females and hermaphrodites of T. pulegioides did not significantly differ in the peltate glandular trichome density in either the upper or lower epidermises. In females and hermaphrodites, the density of peltate glandular trichomes was higher in the lower epidermis than in the upper epidermis (Table 3). In both female individuals and hermaphrodites, statistically significant (p < 0.05) correlations were established between essential oil percentage and peltate glandular trichome density only in the upper leaf epidermis; this relationship was stronger in hermaphrodites than in female individuals (r = 0.23, N = 44 and r = 0.52, N = 80, respectively).

3.4. Diameter of Peltate Glandular Trichomes in Thymus pulegioides Growing in Different Habitats and Belonging to Different Chemotypes and Sexes

The diameter of peltate glandular trichomes was 64.2 ± 4.0 µm and 60.5 ± 5.0 µm (N = 124) and varied from 51.1 µm to 74.2 µm and from 47.5 µm to 101.0 µm in the upper and lower leaf epidermis of T. pulegioides, respectively (Figure 5). We established statistically significant correlations between the diameter of peltate glandular trichomes in the upper epidermis and the diameter of peltate glandular trichomes in the lower epidermis (r = 0.19, p < 0.05, N = 124). Also, significant (p < 0.05) positive relationships between the essential oil percentage in T. pulegioides and the diameter of peltate glandular trichomes both in the upper and lower epidermis were detected (r = 0.33 and r = 0.24, respectively, N = 124) (Figure 3B). Statistically significant relationships between the density and diameter of peltate glandular trichomes were not detected.

Diameters of peltate glandular trichomes in T. pulegioides growing in different habitats are represented in Figure 6. According to the diameter of peltate glandular trichomes in the lower epidermis, habitat nos. 2 and 3 differed significantly from habitat nos. 14 and 16, and habitat no. 2 also differed significantly from habitat nos. 17 and 23. According to the diameter of peltate glandular trichomes in the upper epidermis, habitat no. 3 differed significantly from habitat nos. 9, 12, and 16, and habitat no. 2 significantly differed from habitat no. 9.

A significant negative correlation was found between the average diameter of peltate glandular trichomes in the upper leaf epidermis of T. pulegioides and the mobile sulphur content of the soil in the habitat (r = −0.43, p < 0.05, N = 23). Like the density, the average diameter of peltate glandular trichomes in both leaf epidermises of T. pulegioides negatively correlated with the cover abundance of T. pulegioides in the habitat. T. pulegioides plants growing in habitats belonging to the Molinio-Arrhenatheretea elatioris, Festuco-Brometea, and Trifolio-Geranietea vegetation classes did not differ significantly according to the diameter of peltate glandular trichomes in the upper and lower epidermis.

As in the case of the peltate glandular trichome density, the peltate glandular trichome diameter in the upper epidermis did not differ significantly from the diameter of peltate glandular trichomes in the lower epidermis in T. pulegioides plants of phenolic chemotypes, but the parameters of these indicators, unlike in the case of peltate glandular trichome density, were higher in T. pulegioides plants of phenolic chemotypes than in no-phenolic chemotypes (Table 3).

In both sexes of T. pulegioides plants, the diameter of peltate glandular trichomes was higher in the upper epidermis than in the lower epidermis (Table 3), but females did not differ significantly from hermaphrodites in either the diameter of peltate glandular trichomes in the upper epidermis or the diameter of peltate glandular trichomes in the lower epidermis. Both females and hermaphrodites were found to have significant (p < 0.05) positive correlations between the essential oil percentage and the peltate glandular trichome diameter of both leaf epidermises.

3.5. Dynamics of Density and Diameter of Peltate Glandular Trichomes During Leaf Vegetation

After evaluating the dynamics of peltate glandular trichome density in T. pulegioides during the period from the beginning of leaf development to plant flowering, it was found that at the beginning of plant vegetation (in May), the density of peltate glandular trichomes in the upper leaf epidermis was higher than their density in the lower leaf epidermis (but did not differ significantly) or was it the same. Later, the density of peltate glandular trichomes in the upper leaf epidermis began to decrease and, before and during flowering, was lower than in the lower leaf epidermis; during flowering, the density of peltate glandular trichomes in the lower epidermis differed significantly (p < 0.05) from the density in the upper epidermis (Figure 7).

At the early beginning of vegetation (in the first half of May), the larger diameter of peltate glandular trichomes was characteristic of peltate glandular trichomes in the lower leaf epidermis. Later, starting in the second half of May, the situation changed, and until the flowering of the plants, the larger diameter was always recorded in peltate glandular trichomes of the upper epidermis; in the second half of May, the difference in the diameter of peltate glandular trichomes between the two epidermises was the highest and statistically significant (p < 0.05) (Figure 8).

The raw material preparation of all plants of the Thymus genus is underway only during flowering, since this is when the plants accumulate the most essential oil and produce the largest aboveground biomass [9]. Therefore, dynamic changes of density and diameter in peltate glandular trichomes of Thymus pulegioides were made only prior to the flowering period.

4. Discussion

Among the studied T. pulegioides individuals, the essential oil percentage varied widely, from 0.12% to 1.68% (Figure 2). T. pulegioides is widespread almost throughout Europe, and intraspecific variation in essential oil content has been observed in many countries; for example, the essential oil content in T. pulegioides plants growing in Romania varied within the range of 0.7–1.1% [22], in Italy—0.9–1.1% [23], in Croatia—0.60–1.31% [24], in Portugal—it reached up to 1.8% [25], and in Kosovo—up to 1.58% [26]. Intraspecific differences in essential oil content may be influenced not only by the genetic characteristics of the plants but also by various abiotic environmental factors [27]. The studied T. pulegioides habitats were located in meadows that belonged to the Molinio-Arrhenatheretea elatioris, Trifolio-Geranietea sanguinei, and Festuco-Brometea erecti phytocoenological vegetation classes (Table 1). Also, habitats were characterised by good lighting and different soil pH and chemical composition values (Table 1 and Table 2). However, results showed that although the essential oil percentage of T. pulegioides in habitats belonging to the Festuco-Brometea vegetation class was 0.1% higher than in habitats belonging to the Trifolio-Geranietea vegetation class, this difference was not statistically significant.

Meanwhile, the essential oil percentage of T. pulegioides in the habitats significantly (p < 0.05) correlated with the contents of mobile potassium and mobile iron in the habitat soil (r = 0.58 and r = 0.57, respectively, N = 23). It is not for nothing that T. pulegioides plants in habitat no. 12, where soil contained the highest (2–9 times more than in other habitats) mobile iron and high mobile potassium contents, were distinguished by the highest average essential oil percentage (Table 2 and Figure 2). According to literature data, the chemical composition of the soil can influence the synthesis of essential oil, but this effect may differ for different plant species. For example, potassium can affect the essential oil content differently, even in plants belonging to the same Lamiaceae family: when Thymus mastichina and Origanum vulgare plants were fertilized with potassium fertilizers, the essential oil content increased [28,29,30]. However, no reliable correlations were found between the amount of potassium in the soil and the amount of essential oil in Origanum compactum [31]; additionally, fertilization with potassium did not stimulate the synthesis of essential oil in Ocimum basilicum [32]. The previous studies also did not establish a reliable correlation between the essential oil content in T. pulegioides plants and the potassium content in habitat soil [33]. Chemical elements are required to synthesise and regulate enzymes involved in synthesising many essential oils’ chemical compounds [34,35,36]. The same chemical element can be part of several enzymes responsible for the course of genetically programmed biosynthetic pathways of one or another compound. For example, manganese is important as a cofactor for synthesising terpene precursors and monoterpenes [37,38]. The same chemical element can affect the essential oil contents of different plant species differently through the genetically determined composition of volatile compounds synthesised in them. Also, interpreting the results of studies conducted in natural habitats is more complicated than interpreting the results of experiments conducted under controlled conditions, as some unknown habitat factors may have an additional influence. The results demonstrated that, although unreliably, the average percentage of T. pulegioides essential oil in habitats was negatively correlated only with the mobile sulphur amount in the soil. Negative correlations were also found between the mobile sulphur content in the soil and the studied peltate glandular trichome parameters (density and diameter). This suggests that mobile sulphur in the soil, negatively affecting the parameters of peltate glandular trichomes, may indirectly influence the essential oil percentage in T. pulegioides plants.

A statistically significant (p < 0.05) negative correlation between the average essential oil percentage in T. pulegioides in the habitat and the cover abundance of T. pulegioides in habitat (r = −0.48, N = 23) indicates that plants of this species accumulate lower amounts of essential oil in habitats with higher cover abundance of their own. This could be related to the intraspecific competition of plants in the habitat. Since this phenomenon negatively affects plant development, it may also suppress the development of peltate glandular trichomes. It is not for nothing that a negative relationship (although statistically insignificant) was also found between the cover abundance of T. pulegioides in the habitat and parameters of peltate glandular trichomes (density and diameter). Meanwhile, when T. pulegioides raw material is collected in natural habitats, it is recommended not to do so in habitats with a high cover abundance of T. pulegioides: due to possibly greater intraspecific plant competition, T. pulegioides in such habitats may accumulate lower amounts of essential oil. Also, for the same reason, it is recommended that optimal distances between plants be determined in crops of this species.

Although the significant correlation between the density of peltate glandular trichomes in the upper and lower epidermis was not established, the number of peltate glandular trichomes per unit area in the two leaf epidermises differed: the average density of peltate glandular trichomes in the lower leaf epidermis was significantly (p < 0.05) higher than in the upper epidermis (8.0 ± 2.0 and 7.0 ± 1.7 trichomes in mm², respectively; N = 124). Meanwhile, the diameter of peltate glandular trichomes was significantly higher in the upper epidermis. Similar trends were found in other Lamiaceae species: a higher number of glandular trichomes in the lower leaf epidermis was found in Thymus serpyllum, Ocimum campechianum, O. basilicum, Mentha × piperita, and Salvia nemorosa, and a larger diameter of glandular trichomes was found in the upper epidermis in T. serpyllum, T. vulgaris, M. × piperita, Salvia officinalis, and Origanum majorana [23,39,40,41,42,43]. It is believed that essential oil accumulating in the glandular trichomes helps the plant defend itself against pests. Tiny insect pests (for example, aphids and whiteflies) that attack plants, protecting themselves from the sun, rain, and other environmental factors, also usually settle on the undersides of leaves [44]. Therefore, a higher density of glandular trichomes in the lower leaf epidermis may be a better way for the plant to protect itself directly from pests. However, the plants use volatile essential oils to repel pests (and herbivores) at a distance, too. In this case, the glandular trichomes located in the upper leaf epidermis are more useful. Perhaps this is why significant positive correlations were found between the essential oil percentage and the density and the diameter of peltate glandular trichomes in the upper leaf epidermis (Figure 3). However, it should be kept in mind that, although the peltate glandular trichomes in Thymus albicans plants were most abundant in leaves, a few of them were also in bracts, calyces, and corollas [10]. Also, a little essential oil is also accumulated in capitate trichomes (which consist of only one or a few head cells) [8]. Therefore, established correlations in T. pulegioides should be slightly adjusted.

However, as shown in an investigation of the dynamics of peltate glandular trichome density and diameter during T. pulegioides leaf development, the density of peltate glandular trichomes is not always higher in the lower epidermis, and the diameter of peltate glandular trichomes is not always greater in the upper leaf epidermis: until the second half of May, the density and diameter of peltate glandular trichomes were higher in the upper and in lower leaf epidermis, respectively (Figure 7 and Figure 8). The number of glandular trichomes is closely related to plant ontogeny, and variations in their number on the upper and lower sides of leaves can be associated with changes in leaf orientation on the stem [39,45]. The density of glandular trichomes in M. × piperita at the beginning of leaf development, when the leaves grow vertically relative to the stem (orthotropic orientation) and the lower epidermis is not protected from direct sunlight, was higher in the upper epidermis of the leaf. Later, as the orthotropic orientation of the leaves changed to a plagiotropic orientation and more sunlight fell on the upper epidermis, the density of glandular trichomes in the upper epidermis steadily decreased, and in the lower epidermis, it reached a maximum when the leaves fully matured and exceeded the density of glandular trichomes in the upper epidermis [39]. Therefore, changes in leaf orientation during development, accompanied by the influence of light, could possibly inhibit the development of peltate glandular trichomes in the upper epidermis and promote them in the lower epidermis in T. pulegioides. This statement is also strengthened by the negative relationships found in our study between the density of peltate glandular trichomes and T. pulegioides habitat illumination, which, in the lower epidermis, was significant (R = −0.42, p < 0.05). A lower number of glandular trichomes at higher light intensity was found in Perilla ocymoides (Lamiaceae) plants, too [46].

As glandular trichomes mature, their secretory cells begin synthesising volatile compounds and secrete them into the subcuticular space, where they are stored. Therefore, the amount of essential oils synthesised by glandular trichomes can influence the increase in the diameter of glandular trichome heads [47,48]. This is confirmed by the significant (p < 0.05) positive correlation between the amount of essential oil and the diameter of peltate glandular trichomes in the upper and lower leaf epidermis of T. pulegioides (R = 0.33 and R = 0.24, respectively). Light positively affects the amount of essential oil synthesised in plants of the Thymus genus [49]. Therefore, at the beginning of vegetation, while T. pulegioides leaves were in an orthotropic position, the peltate glandular trichomes of the lower leaf epidermis received more direct sunlight and could synthesize essential oil more intensively; this could explain why the diameter of the peltate glandular trichomes was higher in the lower epidermis of the leaves in early May. Later, as the position of the leaves changes relative to the stem and the upper epidermis receives more light, the diameter of peltate glandular trichomes in the upper leaf epidermis increases (Figure 8).

5. Conclusions

This study showed that the density and/or diameter of peltate glandular trichomes in the upper leaf epidermis could be anatomical markers, helping to select in natural habitats with T. pulegioides individuals with higher amounts of essential oil more quickly and cheaply. Based on the established, reliable positive correlations, it is likely that individuals with a higher density and diameter of peltate glandular trichomes in the upper leaf epidermis accumulate more essential oil. However, this proposed method for rapid screening of essential oil-rich wild T. pulegioides plants based on peltate glandular trichome characteristics, due to the weak obtained correlation, can be considered more a trend than a significant conclusion, and it cannot be applied to other essential oil-bearing plants. Also, it would be valuable to extend observations to additional Thymus species, as well as different structural types of glandular trichomes and other parts of plants (for example, corollas and calyces) in which peltate glandular trichomes are also found, although in smaller quantities. A negative impact of high T. pulegioides cover abundance in natural habitats on the amounts of essential oils accumulating in T. pulegioides plants was detected; therefore, it is recommended that optimal distances between plants be determined in future crops of this species.