ANATOMY AND TRICHOME MICROMORPHOLOGY OF STACHYS SCARDICA ( GRISEB . ) HAYEK ( LAMIACEAE )

The anatomy and micromorphology of the vegetative organs and calyx of Stachys scardica (Griseb.) Hayek were investigated using light (LM) and scanning electron microscopy (SEM). The analysis of the anatomical structure of stem, leaf and leaf petiole showed the presence of an additional adaxial phloem in the vascular bundle of the petiole that was recorded exclusively in species belonging to the subgenus Betonica. On the surface of studied plant parts, three types of trichomes were found: simple nonglandular uniseriate multicellular, nonglandular branched elongated and glandular peltate trichomes. The present study shows that certain micromorphological and anatomical features of Stachys taxa are valuable taxonomic characters.

Stachys scardica is an erect, hirsute, perennial herb with ovate and oblong-lanceolate leaves.The leaf margin is crenate-serrate or dentate.Leaf indumentum is composed of multi-branched nonglandular trichomes and peltate glandular trichomes.It grows on mountain and subalpine meadows and scrub, on serpentine or limestone.Distribution on the Balkan Peninsula includes Albania, Bulgaria, Greece and the southern parts of ex Yugoslavia (Ball, 1972).
According to the available literature data, the anatomy and trichome micromorphology of S. scardica have not yet been examined.In this work, we investigated the anatomy of the vegetative organs, and morphology and distribution of trichomes on the stem, leaves and calyx of S. scardica using a light microscope (LM) and scanning electron microscope (SEM).The type of indumentum is described and classified.The general classification scheme and the terminology follow Radford et al. (1974) and Roe (1971).

Plant material
Aerial parts of the analyzed plants were collected at the flowering stage in July 2010 on Mt.Kopaonik.A voucher specimen has been deposited in the Herbarium of the Institute of Botany and Botanical Garden "Jevremovac", University of Belgrade, Faculty of Biology, Serbia (BEOU), voucher No. 16828.

Light microscopy (LM)
Small segments of stem, leaf and calyx were fixed in FAA and further processed using the standard paraffin method (Jensen, 1962).For the light microscopy analysis, 5µm-thick sections were cut on a Reichert microtome and stained with safranin and alcian blue.
The sections were then observed on a LM LEICA DMSL and photographed using a LEICA DC300digital camera.

Scanning electron microscopy (SEM)
Small segments of dry stem, leaves and calyx were coated with a thin layer of gold (ion sputtering coating) in a BALTEC-SCD 005 Sputtering Device.Observations were carried out on a JEOL JSM 6460 LV scanning electron microscope at 20 kV.

RESULTS
S. scardica is a perennial herb with a four-sided stem and ovate-lanceolate leaves.The indumentum of vegetative organs of S. scardica comprised three types of trichomes: simple nonglandular uniseriate multicellular trichomes, nonglandular branched elongated trichomes (stellate -branched), and glandular peltate trichomes.Nonglandular branched trichomes were found on all vegetative organs of S. scardica, playing a protective role against herbivores.Glandular peltate trichomes were observed on the stem, leaves and calyx.SEM micrographs of S. scardica aerial parts are presented in Fig. 2. The capitate glandular trichomes were not found on the surface of S. scardica organs.

Stem anatomy and indumentum
The herbaceous stem of S. scardica in cross section has a square form with pronounced angles.The stem is covered with a one-layered epidermis and thin cuticle.Collenchyma is single-layered among the angles, but 6-7 layers of collenchyma are observed below the epidermis at the angles.The rest of the cortex is composed of several layers of compact parenchyma.The pericycle is cylindrical, made of sclerenchyma elements, interrupted in a few places by several parenchyma cells.The cambium is in the form of a ring and produces the phloem and xylem elements, also in a ring form.The central part of the stem is built up of large parenchymatous cells with small intercellulars (Fig. 1.A).On the stem surface, simple multicellular uniseriate trichomes are present, as well as specific branched trichomes -multicellular dendroid hairs,  with three multicellular uniseriate branches above the base (Fig. 2. A, B).Glandular peltate trichomes have a short one-celled stalk and head of four cells, of various diameters (Fig. 2. B).Cuticle rupture was not observed using SEM, and secretion seems to remain trapped in the intact subcuticular spaces unless external factors cause their rupture.

Leaf anatomy and indumentum
S. scardica has ovate-lanceolate cauline leaves, with a crenate margin, cordate at the base, green above, grayish-green below.The mesophyll is differentiated into palisade and spongy tissues.The palisade parenchyma consists of two layers of densely packed, elongated cells.Spongy globular-shaped parenchyma cells are 3-4 layered with small intercellular spaces (Fig. 1.D). Vascular bundles are colateral, without cambium, surrounded by a parenchymatic bundle sheath.The main vein is well developed with pronounced lower side and has one-layered collenchyma located below the upper and lower epidermis (Fig. 1.C).
The leaves of S. scardica are petiolate, with the petiole up to 10 mm long.The adaxial surface of the petiolus is plane with a visible curve on the ends (V shaped) and the abaxial surface is convex (Fig. 1.E.).The transverse section of petiolus of S. scardica shows a one-layered epidermis, subepidermal collenchyma cells in the angles, numerous densely packed parenchymatous cells, one large central vascular bundle and two lateral vascular bundles.Epidermal roundshaped cells are covered with a thin cuticle.Simple nonglandular uniseriate multicellular trichomes and nonglandular branched elongated trichomes are present on the epidermis.Ground tissue consists of dense parenchyma cells, with the largest cells around the central vascular bundle.The vascular bundle is collateral, without cambium, containing xylem and two phloem, of which the adaxial phloem is more developed (Fig. 1.F). Tracheal elements are very well developed.The sclerenchyma enveloped the adaxial phloem and xylem (Fig. 1.F).Small lateral vascular bundles contained xylem and phloem and were surrounded by sclerenchyma more developed around the phloem.
The leaf showed dorsiventral symmetry with larger adaxial and smaller abaxial rectangular epidermal cells covered with a thin cuticle (Fig. 1.D).Both leaf sides bear numerous nonglandular trichomes and peltate glandular trichomes (Fig. 2. C, D, E).Multicellular uniseriate trichomes, as well as branched trichomes, are situated on both leaf sides.On the abaxial leaf surface, branched stellate trichomes are situated along the veins, and between the veins (Fig. 2. C).The base of stellate trichome is large and different numbers of multicellular uniseriate branches are divided from the base.The peltate trichomes with 4-celled heads, as well as stomata, are observed on the abaxial leaf surface (Fig. 2. D).The adaxial leaf surface is covered with branched nonglandular and peltate trichomes.From the base of the branched trichome mostly unicellular branches emerge (Fig. 2. E).The peltate trichomes have 4-celled heads (Fig.

E).
Sepal leaves, about 8-10 mm long, are also covered with different types of trichomes on both sides (Fig. 2. F, G, H).The calyx is characterized by the presence of unusual branched trichomes (Fig. 2. G); it is very hairy and covered with simple unicellular short trichomes (Fig. 2. F, H) and densely distributed branched trichomes (Fig. 2. G).The observed number of branches was two, six, or eight.The branches are multicellular, uniseriate, warted, with pointed apex.The outer surface of the calyx is densely covered by numerous stellate hairs and by small peltate glandular trichomes.From the inner side of the calyx very long simple uniseriate trichomes were observed to emerge (Fig. 2. H).

DISCUSSION
The herbaceous stem of S. scardica showed the typical four-angled form and structure of Lamiaceae members, covered with a one-layered epidermis with trichomes, well-developed collenchyma at the angles of the stem, cambium and conducting tissues in ring form and central parenchymatous tissue.The leaves are ovate-lanceolate, comprising the epidermis covered with trichomes, mesophyll and vascular bundles.The petiole is triangle-shaped, with epidermis, not differentiated mesophyll and vascular bundles surrounded by sclerenchyma.The petiole anatomy showed variability in different Lamiaceae species (Gupta and Bhambie, 1980;Akçin et al., 2011).The distinct feature of S. scardica and S. officinalis, which belong to the subgenus Betonica, is the presence of an additional adaxial phloem in the central vascular bundle.This type has not been found in any investigated species from the Stachys subgenus (Bhattacharjee, 1980).Various types of trichomes were observed on all analyzed organs of S. scardica, which is a characteristic feature of Lamiaceae members.In Lamiaceae, nonglandular and glandular trichomes are distinguished, with peltate and capitate trichomes as the basic types of glandular trichomes (Cantino, 1990;Fahn, 2000;2002).The glandular trichomes' structure can vary widely among species, as does the composition of the essential oils they produce for protection of the plant aerial parts against herbivores and pathogens (Werker, 1993).The function of nonglandular trichomes depends on their morphology, the organ on which they are situated and their direction and orientation.When nonglandular trichomes are densely spread all over the plants, they may serve as a mechanical barrier (Werker, 2000).Apart from having different functions in plants, the trichomes' microcharacters could be of taxonomical significance.Some authors, in studying the micromorphology of Stachys trichomes, considered that the characters of taxonomic interest were the presence of glandular and nonglandular trichomes, thickness of the cell walls, number of cells, presence of few-armed or branched, or vermiform trichomes, orientation of trichomes in relation to the epidermal surface, presence of papillae on trichome surface, etc. (Salmaki et al., 2009).
The other investigated species of Stachys varied in presence and types of nonglandular and glandular trichomes.Falciani et al. (1995), Giuliani and Maleci Bini (2008), Giuliani et al. (2008), Giuliani and Mal-eci Bini (2012) and Venditi et al. (2013) have studied the micromorphology and distribution of trichomes of Stachys species grown in Italy and described different glandular and nonglandular trichomes.The typical peltate trichomes with large heads of variable numbers of secreting cells and small capitate trichomes (subsessile) are present on the leaves and calyces of S. officinalis subsp.officinalis, S. alopecuros subsp.alopecuros and S. annua.S. germanica subsp.germanica and S. germanica subsp.salviifolia possess peculiar peltate trichomes with well-developed stalk on the leaves and calyces (Giuliani and Maleci Bini, 2008).In S. recta subsp.serpentini and S. recta subsp.recta only one type of glandular trichome was found, a short-neck capitate trichome (Giuliani et al., 2008).S. alopecuros subsp.divulsa had peltate and two types of capitate trichomes (Venditti et al., 2013).In the study of differential characters between Stachys subgenus Betonica and Stachys subgenus Stachys, Giuliani and Maleci Bini (2012) found that Stachys subgenus Betonica possessed only peltate, while Stachys subgenus Stachys has different types of large capitate hairs, which was confirmed in our study.In addition, the peltate trichomes of the subgenus Betonica show an unusual secretion composed of flavonoids and essential oils (Giuliani and Maleci Bini, 2012;Marin et al., 2004), which can also support the view of various authors that within the Stachys genus two subgenera should be recognized (Betonica and Stachys) or even treated as a separate genera.Turkish species S. balansae and S. carduchorum (sect.Eriostomum) possess unbranched nonglandular trichomes, unicellular or multicellular and capitate and peltate glandular trichomes (Erdogan et al., 2011).The micromorphological study of the species S. cydni and S. yildirimlii showed that the type of trichomes may contribute to the separation these species.The branch number of the trichomes and cell number of the branches differ significantly between the two species (Dink and Ozturk, 2008).
In an investigation of the trichome morphology of the Lebanese Stachys species, Beyrouthy et al. (2009) found 21 types of trichomes on different organs of six species, but only four types were found on all studied species.Among these types, the unicellular warted nonglandular trichomes were mentioned, as was found in the S. scardica calyx.They suggest that nonglandular trichomes are more important than glandular trichomes in the differentiation of Stachys species.
In the comprehensive study of Iranian Stachys taxa, Salmaki et al. (2009) observed numerous types of trichomes that showed considerable variability among different species, but were constant among different populations of one species, and therefore, afford valuable characters in the delimitation of sections and species.Rezakhanlo and Talebi (2010) described 24 trichome types in Iranian S. lavandulifolia.The obtained results confirmed the usefulness of indumentum characters in taxonomic identification at different infrageneric levels, the importance of which was previously recognized (Abu Asab and Cantino, 1987).Trichome morphology is an essential character for delimitation of the section Ambleia Bentham and Zietenia Gled.(Benth) in the genus Stachys, while the section Ambleia is characterized by dendroid indumentum and is isolated from other sections of Stachys by this feature (Bhattacharjee, 1980).The morphology and distribution of glandular and nonglandular trichomes may be considered as distinctive characters at the subspecies level.This is consistent with the taxonomic classification of S. recta subsp.serpentini as a subordinate taxon of S. recta (Giuliani et al., 2008).
The pubescence of S. scardica may contribute to its adaptation to the environmental conditions where this species usually grows, and protection from various external factors such as extensive light, UV irradiation, extreme temperatures and herbivores.Analyzing the results obtained from the investigation of trichomes of other Stachys species, S. scardica was the only one to possess branched stellate nonglandular trichomes compared to other species from the subgenus Betonica.In addition, the capitate glandular trichomes are absent from the vegetative organs of this species.Results of an investigation into essential oil and flavonoids (Grujic Jovanovic et al., 2004;Marin et al., 2004) are in accordance with previous investigations and classification of this genus (Bhattacharjee, 1980).
Throughout the history of the classification of Stachys, different opinions have been expressed about the status of Betonica.The Betonica group is characterized by prominent sterile rosettes and usually unbranched flowering shoots.The leaf margins are deeply crenate to serrate.The only constant diagnostic morphological feature of the subgenus Betonica is the presence of sessile flowers and bracteoles with a hardened base.Among anatomical features, the presence of adaxial phloem in the petiole, and nutlets without pitted thickenings in the sclerenchyma are constant characteristics of the subgenus Betonica (Bhattacharjee, 1984).The structure of the vascular bundles of the petiole in species belonging to the subgenus Betonica, sections Betonica and Macrostachya was specific.The adaxial phloem that was recorded exclusively in the species S. officinalis and S. scardica appeared as a valuable differential character, since it did not occur in the vascular bundles of the petiole of species from the subgenus Stachys.Leaf anatomy provides valuable characters that are useful in subgeneric classification as well as species discrimination in Stachys.The most important diagnostic characters are the shape of leaf transverse section, length of ventral and dorsiventral axis, number of median bundles in the petiole, number of cell layers of palisade and spongy parenchyma, type and thickness of collenchyma as well as trichome type (Salmaki et al., 2011).Delimitation of the Stachys from its related taxon, Betonica, has been a comprehensive investigated.Bentham (1848), Boissier (1879), Reichinger (1982) and Krestovskaya and Vassilyeva (1997) excluded Betonica from Stachys.Some other authors included it in the genus (Briquet, 1897;Ball, 1972;Bhattacharjee, 1980).Bhattacharjee (1980) presented a new infrageneric classification for the genus in the Old World.
The results of indumentum investigation support the classification of Betonica and Stachys as distinct subgenera or even genera, rather than the classification of some authors who consider them to be sections, at the same level as other sections proposed within the genus Stachys.From our investigation, it was revealed that the anatomical structure of the leaf petiole supports the delimitation of these two related taxa and, consequently, could be regarded as a valid taxonomic character.

Fig. 1 .
Fig. 1.Anatomy of S. scardica.A -cross section of stem of S. scardica; B -cross section of petal leaf; C, D -cross section of leaf; E, Fcross section of petioles.