MICROMORPHOLOGICAL AND ANATOMICAL CHARACTERS OF THE TURKISH ENDEMIC Marrubium trachyticum Boiss. (LAMIACEAE)

In the present study, micromorphological characters of stem, leaf, calyx and mericarp of the Turkish endemic Marrubium trachyticum Boiss were investigated using scanning electron microscopy (SEM) and the anatomy of root, stem and leaf using light microscopy (LM). Stellate trichomes with unequal rays and branched hairs were observed on the stem, leaf and calyx. The distributions and densities of glandular trichomes on these vegetative organs were less than the eglandular trichomes. The mature mericarps of the species were ovate in shape and sculpturing pattern was penta-hexagonal colliculate. According to the anatomical results, M. trachyticum has secondary root structure. The stem has a distinct collenchyma layer. The stem is surrounded by oval or rectangular epidermal cells with a thick cuticle and is quadrangular in shape. The leaves are bifacial (dorsiventral) with anomocytic stomata in both the upper and the lower surfaces.


Introduction
The genus Marrubium L. belongs to the Lamiaceae family and includes annual and perennial herbs. The species of the genus have an important distribution in Irano-Turanian and Mediterranean phytogeographic regions. The genus comprises about 40 taxa throughout the world (Hedge 1992) and is represented in Turkey by 21 taxa of which 12 are endemic (Cullen 1982, Davis et al. 1988, Ekim et al. 2000, Aytaç et al. 2012. It is thought that Turkey is the main centre of diversity for the genus Marrubium (Akgül & Ketenoğlu 2014). M. trachyticum Boiss., growing in steppe, slopes and fields inside the altitude range from 900 to 2500 m a.s.l. (Cullen 1982), is one of the endemic species of the genus in Turkey and has been included in the Red Data Book of Turkish Plants in near threatened (LR-nt) status (Ekim et al. 2000).
Marrubium has been used as a traditional medicine for asthma, pulmonary infections, inflammation, and hypotension and also as pain reliever (Meyre-Silva & Cechinel-Filho 2010). Furthermore, there are some reports on Marrubium species about their effects on reducing oxidative stress and inflammatory reactions due to their high amount of polyphenol and flavonoid contents (Yousefi et al. 2013(Yousefi et al. , 2014 (Cantino 1990, Navarro & El-Qualidi 2000. Glandular hairs are widely distributed over the aerial reproductive and vegetative organs of members of the family and their structures have been investigated in a number of studies (Bosabilidis 1990, Ascensao et al. 1999, Kaya et al. 2003. However, non-glandular trichomes are more common than glandular trichomes within Lamiaceae (Cantino 1990). The aim of this study is to investigate the detailed characteristics of micromorphological structures of stem, leaf, calyx and mericarp of M. trachyticum using SEM and its anatomy of root, stem and leaf using light microscopy.

Materials and Methods
Plant samples were collected from May to July 2014 from natural populations in the vicinity of Bağcılı village in Çorum, Turkey. The specimens were dried according to standard herbarium techniques and stored in the Ondokuz Mayıs University, Faculty of Art and Science Herbarium (OMUB). The taxonomical descriptions of the plants were made according to Flora of Turkey (Cullen 1982).
Dried stem, leaf, calyx and mericarp samples were mounted directly on stubs using double-sided adhesive tape for SEM investigations. The stubs were coated with gold for 5 minutes and then observed and photographed in JEOL-JSM 7001 Scanning Electron Microscope. The terminology of Cantino (1990) and Koul et al. (2000) was followed for micromorphological investigations.
Anatomical investigations were performed using an average of 30 fresh specimens which were kept in 70% alcohol. Transverse sections of roots, stems and leaves and surface sections of leaves were used to make permenant/temporary slides and the slides were viewed and photographed using a Nikon Coolpix 5200 digital camera. All anatomical measurements were done in computer media based on the photographs with the help of the Image J program. Stomatal index was calculated according to the method described by Meidner & Mansfield (1968) for both surfaces of the epidermis.

Micromorphological characteristics
Observation of stem using SEM showed that eglandular and glandular trichomes are present on stems of M. trachyticum (Fig. 1A-B). Lanate eglandular trichomes, in particular, are more abundant at the base of the stem. The head of glandular trichomes with short stalk are composed of a spheric cell (Fig. 1B).
Leaf surface is more or less densely covered with eglandular stellate trichomes and eglandular trichomes on the abaxial surface are longer than the adaxial surface ( Fig. 1C-D). Glandular trichomes are rare on both side of leaf surfaces. The leaves of the investigated species are amphistomatous and with anomocytic stomata (Fig. 1C). The mean number of stomata per mm 2 of leaf surface is 15.01±3.01 on adaxial epidermis and 25.64±4.89 on the abaxial epidermis (Table 1). Stomata are more abundant on the abaxial side of the leaf. The upper and lower epidermis consist of cells with strongly sinous walls ( Fig.  1C-D).
SEM observation showed that the throat of calyx has more greyish stellate trichomes (Fig. 1E). However, these trichomes are more sparsely on the calyx teeth (Fig. 1F). The distribution and density of the glandular trichomes on calyx are less than eglandular ones (Fig. 1E-F).

Root anatomy
The root structure in M. trachyticum is more or less uniform. In cross-sections, the periderm layer on the outermost surface is multilayered ( Fig. 2A). Cortex consists of 11-12 layers of oval or rectangular parenchymatous cells. Cambium and phloem cells are distinguishable. The thickness of phloem layer is 59.99±8.27µm ( Table 2). The xylem consists of vessel members and tracheids. The xylem rays are composed of 7-8 rowed rectangular cells. The pith is completely filled with xylem elements.

Stem anatomy
Cross-section of the stem is clearly quadrangular in shape. The epidermis consists of oval or rectangular cells and is covered by a thick cuticle. There are one-celled or multicellular non-glandular or glandular hairs on the epidermis (Fig. 2B). The collenchyma tissue consisting of 10-12 layers of ovoidal cells is located underneath the epidermis. The cortex tissue is composed of ovoidal and quadrangular cells with thin walls and the thickness of this layer is 130.70±23.31µm (Fig. 2B, Table 2). Beneath the cortex parenchyma, small groups of phloem sclerenchyma cells are located above the phloem and vascular cambium is indistinguishable. Xylem elements are thick-walled. The mean diameter of vessel elements is 20.20±4.35µm ( Table 2). The pith consists of large and cylindrical parenchymatic cells and pith cells become smaller towards the central part of the stem (Fig. 2B).  In transverse sections, the upper and the lower epidermises comprise of uniseriate, quadrangular and oval cells. However, lower epidermal cells are larger than upper epidermis cells (Table 2). Both epidermises are covered with non-glandular and glandular hairs (Fig. 2C). 2-3 layers of collenchyma cells are present in the midrib located between the upper and the lower epidermis. The leaves are bifacial (dorsiventral) (Fig. 2D). Palisade tissue is composed of 1-2 layered cylindrical cells with e plenty of chloroplasts. Spongy parenchyma with large intercellular spaces are round or irregular in shape and the thickness of this layer is 39.71±2.56µm (Fig. 2D, Table  2). Vascular bundles are collateral type. In their surface views, the leaves are amphistomatic and epidermal cells have wavy walls (Fig. 2E). Adaxial stomata length and width is higher than the abaxial stomata dimensions ( Table 2). The number of stomata per mm 2 in lower surface is higher than in upper surface (Table 1).

Discussion
In this study, micromorphological and anatomical properties of M. trachyticum studied by light and scanning electron microscopies were determined to be useful characters.  (2015) and Ahvazi et al. (2016) indicating that the stellate and branched hairs were found to be in Lamiaceae family members. However, in the present study, the distributions and densities of glandular trichomes on stem, leaf and calyx of M. trachyticum are less than the non-glandular trichomes. It was previously reported that non-glandular trichomes are more common than glandular trichomes in Lamiaceae (Cantino 1990 Marrubium trachyticum was found to have the same general root anatomy characteristics as in other members of the Lamiaceae family. In root cross sections, the protective tissue was comprised of periderm and the center of the root was composed of tracheary elements. It was previously determined that the pith rays of some species within Lamiaceae, e.g. Lamium lycium Boiss. Anatomical studies showed that the leaves of the M. trachyticum was bifacial (dorsiventral). Palisade parenchyma cells were presented in upper surface of the leaf and spongy parenchyma cells had large intercellular spaces in the lower surface. These leaf anatomy characteristics coincide with those previously reported by Akgül et al. (2008) and Büyükkartal et al. (2016) for some other Marrubium species. In leaf cross-sections, vascular bundles were collateral type in M. trachyticum and the phloem surrounded xylem. Our observation showed that M. trachyticum has amphistomatic leaves. Akgül (2004) and Tüylü et al. (2017) also reported that the leaves of the some other Marrubium species are amphistomatic. In surface sections of leaf, anomocytic type stomata were observed with higher numbers in abaxial surface of the leaf.
In conclusion, some characteristic micromorphological and anatomical features of M. trachyticum were reported in detail. The results presented here have the potential to contribute to further taxonomic studies of the genus.