Micromorphological Traits of Balcanic Micromeria and Closely Related Clinopodium Species (Lamiaceae)

A study of the trichomes types and distribution and pollen morphology was carried out in nine Micromeria taxa (M. cristata ssp. cristata, M. cristata ssp. kosaninii, M. croatica, M. graeca ssp. graeca, M. graeca ssp. fruticulosa, M. juliana, M. kerneri, M. longipedunculata and M. microphylla) and five closely related Clinopodium species (C. dalmaticum, C. frivaldszkyanum, C. pulegium, C. serpyllifolium and C. thymifolium) from the Lamiaceae family of the Balkan Peninsula. By scanning electron microscope, non-glandular trichomes, peltate and capitate trichomes were observed on the calyx, leaves and stem of the studied species. Two subtypes of capitate trichomes were observed in Micromeria species: subtype 1 (consisting of a basal epidermal cell and an elliptically shaped head cell) and subtype 2 (consisting of a basal epidermal cell, two to three stalk cells and a round head cell). In Clinopodium species, three types of capitate trichomes were observed: subtype 1, subtype 3 (consisting of a basal epidermal cell, a short peduncle cell, and a single round head cell), and subtype 4 (consisting of a basal epidermal cell, a stalk cell, and an elongated head cell). These results support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.


Introduction
The family Lamiaceae is one of the largest families among the eudicots, consisting of more than 240 genera and more than 7200 species [1]. Many of species belonging to this family are aromatic and medicinal plants due to the presence of glandular trichomes that produce essential oil. One of the plant groups in this family is the genus Micromeria Benth. and closely related species from the genus Clinopodium L. The genus Micromeria belongs to subtribe Menthinae and tribe Mentheae (subfam. Nepetoideae). According to Briquet [2], Killick [3] and Greuter et al. [4], it is considered part of the indistinctly defined The non-glandular and glandular trichomes were observed in the studied Micromeria and Clinopodium species. Non-glandular (NG) trichomes have been observed on the leaves, calyces, and stem of both genera. These trichomes are unbranched, uniseriate, bi-cellular to multicellular and most often bent on different sides. They could be called attenuated trichomes because they gradually taper from base to tip [25]. The length of the NG trichomes is highly variable and ranges from very short trichomes, especially on the adaxial leaf surface, to very long hairs on the calyx and stem (Figures 1 and 2). The surface of these trichomes is warty (Figure 1). The main function of NG trichomes is to protect the plant from water loss. The same type of NG trichomes is present in both Micromeria and Clinopodium species. The occurrence and frequency of NG trichomes on the adaxial and abaxial sides of the leaf, the outer side of the calyx and the stem are shown in Tables 1 and 2. All Micromeria species studied have a more or less abundant coverage of NG trichomes on the adaxial and abaxial leaf surface (Figure 1). A certain exception is M. longipedunculata, in which the upper and lower leaf surfaces are less hairy. The adaxial and abaxial leaf surfaces and stem of M. graeca ssp. fruticulosa and the stem of M. cristata have the highest number of long NG trichomes forming an abundant covering.
In general, Micromeria species have a denser indumentum of attenuated trichomes on leaves than Clinopodium species. Clinopodium serpyllifolium is an exception as it has the densest NG trichomes cover of all Micromeria and Clinopodium species examined ( Figure 2). Studies by Firat et al. [22] also showed very dense indumentum on the leaves and stem of Clinopodium serpyllifolium ssp. sirnakense. The frequency of NG trichomes on the calyx of Clinopodium species is similar to this coverage of NG trichomes in Micromeria species. A slightly lower frequency of NG trichomes was observed on the calyx of C. frivaldszkyanum and C. pulegium, and on the stem of C. frivaldszkyanum. The presence of NG trichomes has been previously well documented in Micromeria [13,15,16] and Clinopodium [13,14,17,18] species, as well as in other Lamiaceae [13,14,26].
Glandular trichomes were observed on all plant parts of the Micromeria and Clinopodium species examined. These trichomes can be further divided into two main subtypes, namely, peltate and capitate trichomes. Peltate trichomes consist of a basal cell, a very short unicellular stalk and a multicellular head with a large subcuticular space ( Figure 1). These trichomes are also known from some other Lamiaceae species [13,[26][27][28][29][30][31][32]. The distribution and frequency of peltate trichomes on the studied plant parts are shown in Tables 1 and 2. Although peltate trichomes are present on all plant parts studied, they are more abundant on the abaxial leaf surface and on the calyx. They are completely absent on the adaxial side of the leaf in M. croatica and M. longipedunculata (Table 1). Peltate trichomes were previously reported in Micromeria fruticosa [13], Micromeria myrtifolia Boiss. et Hohen. [33], M. croatica [15], M. kerneri, M. juliana [16] and M. longipedunculata [34]. Husain et al. [14] showed the presence of peltate trichomes on the leaves of Clinopodium serpyllifolium, C. dalmaticum and C. thymifolium. Peltate trichomes were also detected in micrographs of C. thymifolium leaves presented by Marin et al. [35]. Mladenova et al. [19] observed these trichomes in the leaves of C. frivaldszkyanum, although they did not describe them as peltate.
The capitate trichomes can be further divided into several subtypes. Subtype 1 (C1) capitate trichomes consist of a basal epidermal cell and an elliptically shaped head cell with a larger subcuticular space. C1 trichomes are not erect but can be described as adherent to the surface (Figure 1). This type was observed on both the abaxial and adaxial sides of the leaves, on the stem and on the outer side of the calyx. They are present in all Micromeria and Clinopodium species studied (Tables 1 and 2). These trichomes were described in detail in Micromeria croatica by Kremer et al. [15]. C1 trichomes have also been observed in M. kerneri and M. juliana [16]. Although Husain et al. [14] did not mention C1 trichomes in M. longipedunculata, unclear SEM micrographs probably show just the C1 type of trichomes. On the other hand, SEM micrographs of M. fruticosa presented by Werker et al. [13] did not show C1 trichomes. According to drawings made by Koca [33], it seems that C1 trichomes are also present in Micromeria myrtifolia. C1 trichomes were also observed in Clinopodium dalmaticum, C. pulegium, C. serpyllifolium and C. thymifolium [17].
Hanlidou et al. [36] described trichomes comparable to C1 trichomes in Calamintha menthifolia Host as short and usually curved trichomes. These trichomes are regularly present in all studied Micromeria and Clinopodium species, but they cannot be considered specific only to these two genera. In addition, the SEM micrographs presented by Werker et al. [13] show C1 trichomes in Majorana syriaca (L.) Rafin., Satureja thymbra L., and Thymus capitatus (L.) Hoffmanns. et Link. However, it is evident that C1 trichomes are not as common as peltate trichomes in Lamiaceae species.
Subtype 2 capitate trichomes (C2) are composed of a basal epidermal cell, two to three stalk cells and a rounded head cell with subcuticular space (Figure 1). C2 trichomes are upright while the height of these trichomes varies from short trichomes, mainly on the abaxial side of the leaf, to quite long trichomes on the calyx and stem. However, they are often absent on the adaxial leaf side. C2 trichomes are observed only in Micromeria species studied.
The capitate trichomes of subtype 4 (C4) are upright and consist of a basal epidermal cell, a stalk cell and an elongated head cell. In these trichomes, the head cell is as narrow as the stalk cells, and only slightly enlarged above, with a subcuticular space. They resemble a finger (Figure 2). Although C4 trichomes are present in all Clinopodium species studied, they are relatively rare trichomes ( Table 2). The presence of C4 trichomes was previously observed in Clinopodium dalmaticum, C. pulegium, C. serpyllifolium and C. thymifolium by Dunkić et al. [17]. These trichomes are also visible in SEM micrographs of leaves of Micromeria fruticosa and Clinopodium thymifolium presented by Werker et al. [13] and Marin et al. [35], respectively. C4 trichomes are visible in SEM micrographs of Calamintha menthifolia, and in micrographs of Satureja montana and S. subspicata presented by Hanlidou et al. [36] and Dunkić et al. [38], respectively. Unclear micrographs presented by Al-Zubaidy et al. [40] probably show just C4 trichomes in Clinopodium vulgare L. ssp. vulgare and C. vulgare ssp. arundanum Boiss. In addition, they can be observed in SEM micrographs of Majorana syriaca, Salvia fruticosa Mill., S. officinalis L. [13], and S. divinorum Epling et Játiva [30]. Although data from the literature [13] suggest that C4 trichomes may sometimes be present in Micromeria species, our results show that they are characteristic for Clinopodium species.
The results presented here show that NG trichomes, peltate trichomes and capitate trichomes of subtype 1 are present in both Micromeria and Clinopodium taxa. On the other hand, the capitate trichomes of subtype 2 are present only in studied Micromeria taxa, while subtypes 3 and 4 are present only in Clinopodium taxa. Since the investigated taxa of the genus Clinopodium belong to the former Micromeria sect. Pseudomelissa, it can be concluded that micromorphological traits also support the recent transfer of Micromeria sect. Pseudomelissa to the genus Clinopodium.

Pollen
The pollen of all Micromeria and Clinopodium species examined is single (monad pollen) and isopolar with an elliptical equatorial outline (Figures 3 and 4).  The polar view shows a circular shape with visible ends of apertures. The pollen has six apertures (hexacolpate pollen) located in the equatorial pollen belt (zonocolpate pollen). The apertures are long and rather narrow, widest in the middle and gradually narrowing towards the poles. The margins of the apertures are clear and sharp, while the ends are narrow and pointed. The membranes are ornamented. The apocolpium is relatively small, while the mesocolpium is quite large. The pollen exine is semitectate with medium reticulate ornamentation. The reticulum meshes are unequal (heterobrochate reticulum type) with more or less smooth surfaces of muri. Only the pollen exine of M. cristata ssp. cristata and M. cristata ssp. kosaninii show uneven surfaces of the muri (Figure 3). The lumina vary in size and are narrower or about the same width as the muri, rarely wider than muri as in M. croatica (Figure 3). The shape of the lumina is irregular with obtuse angles. So far, the pollen of Micromeria marginata, M. croatica, M. longipedunculata, and M. imbricata (Forssk.) C. Chr. have been described in detail [15,21,23]. In general, there is no visible difference in pollen shape between the Micromeria and Clinopodium species studied.
The size of pollen grains according to polar and equatorial axis is shown in Table 3. According to Erdtman [20],  [15], who studied the pollen of this species from another locality. Comparable results were also obtained for the pollen of M. juliana, M. kerneri, and M. longipedunculata [16,34]. Micromeria marginata has middle-large pollen with a polar axis of 26.6 µm and an equatorial diameter of 35.2 µm [21]. According to Doaigey et al. [23], the pollen of Micromeria imbricata has a length of 34.57 and 31.33 µm (polar and equatorial axis, respectively). The smallest pollen among the investigated Clinopodium species was in C. thymifolium (23.52 µm), while the largest pollen (according to longer axis 32.47 µm) was in C. dalmaticum. The measure of polar length of Clinopodium foliosum pollen ranges from 20.45-28.6 µm and for C. menthifolium from 25.7-31.4 µm [24]. On the other hand, the equatorial width is 20.46-28.57 µm for C. foliosum and 20.9-31.43 µm for C. menthifolium [24].  The results of the ANOVA show the significant difference between most of the studied taxa in terms of pollen size (Tables 4 and 5). Although there is a significant difference between most of Micromeria and Clinopodium taxa, this difference is not clear. There is a significant difference between the closely related taxa M. cristata ssp. cristata and M. cristata ssp. kosaninii in terms of equatorial diameter. However, there is a significant difference between the closely related taxa M. graeca ssp. graeca and M. graeca ssp. fruticulosa according to the polar axis. There is no significant difference between M. croatica and M. pseudocroatica, which are one species according to Bräuchler et al. [8]. On the other hand, there is a significant difference between C. dalmaticum from Orjen Mt (Montenegro) and C. dalmaticum from Mesta River Valley (Bulgaria) for both polar and equatorial axes.

Plant Material
The survey included, depending on the point of view [8,9,12,[41][42][43][44][45], nine to ten Micromeria taxa from the Balkan Peninsula (Table 6 Samples of the stems, leaves, and flowers of ten plants per population were fixed in FAA (formalin/96% ethanol/acetic acid/water: 5/70/5/20). After three days the samples were transferred from the fixation medium to 70% (v/v) ethanol and stored in the refrigerator until analysis.
The samples for scanning electron microscopic (SEM) investigation were transferred from 70% (v/v) ethanol to 70% (v/v) acetone. Then, the samples were dehydrated from 70% (v/v) to 90% (v/v), and 100% (v/v) acetone. The dehydrated samples were subjected to critical point drying using CO 2 as the drying medium (CPD030; Bal-tec, Balzers, Liechtenstein). The samples were then sputter coated with gold (Sputter Coater, Agar Scientific Ltd., Essex, UK) and examined under the scanning electron microscope XL30 ESEM (FEI) with an acceleration voltages of 20 kV in high vacuum mode. The presence and abundance of the different trichome types was qualitatively assessed (− missing, ± rare, + present, ++ abundant, +++ extremely abundant). Pollen was collected from the anthers of several flowers per plant after critical point drying. A total of pollen from ten plants per population studied was mixed and examined under a scanning electron microscope. The length of 30 pollen grains per population was measured. Pollen size is expressed as the length of the longest axis according to Erdman [20]. Common terminology was used in describing trichomes and pollen [25,47].

Statistical Analysis
Descriptive statistics: minimum (Min), maximum (Max), mean, standard deviation (Stdev) and coefficient of variation (CV) for length and diameter of pollen grains were calculated. Polar end equatorial axis lengths of pollen grains were subjected to One-way Analysis of Variance (ANOVA). Differences between taxa were tested with Tukey's HSD post hoc tests [48]. Table 6. Details on origin and collection data of investigated Micromeria (M.) and Clinopodium (C.) taxa.

Conclusions
Non-glandular trichomes, peltate trichomes, and four subtypes of capitate trichomes were observed on the aerial parts of the Micromeria and Clinopodium taxa examined. The NG trichomes, peltate trichomes and capitate trichomes of subtype 1 (bent trichomes consisting of a basal epidermal cell and an elliptically shaped head cell) were observed in both Micromeria and Clinopodium taxa. Capitate trichomes of subtype 2 (erect trichomes consisting of a basal epidermal cell, two to three stalk cells and a rounded head cell) were observed only in Micromeria taxa, while capitate trichomes of subtype 3 (upright trichomes consisting of a basal epidermal cell, a relatively short stalk cell and a rounded celled head) and subtype 4 (upright trichomes consisting of a basal epidermal cell, a stalk cell, and a narrow and elongated head cell) were observed only in Clinopodium taxa. Such a distribution of capitate trichomes subtypes in Micromeria and Clinopodium taxa shows that, on the basis of micromorphological traits, it is possible to distinguish taxa from the former Pseudomelissa section from other Micromeria taxa. In this way, micromorphological traits support the recent transfer of Micromeria species from the section Pseudomelissa to the genus Clinopodium.
On the other hand, the pollen studies have shown that there are no significant differences between pollen of the Micromeria and Clinopodium taxa.

Conflicts of Interest:
The authors declare no conflict of interest.