Comparative anatomical studies on the two Stachys species ( sect . Eriostomum , subsect . Germanicae ) growing in Turkey

In this study, comparative anatomical studies on the two Stachys species (sect. Eriostomum, subsect. Germanicae) in Turkey were carried out on the plants collected from their type localities. These species are Stachys balansae Boiss. & Kotschy and Stachys carduchorum (R. Bhattacharjee) Rech.f. which are morphologically very similar to each other. Anatomical properties of Germanicae subsection was reported for the first time. In anatomical studies, the root, stem, leaf and petioles have been examined under light microscope in details; microphotographs was taken and micro-anatomical measurements of cells and tissues was done and presented in table. In anatomical results, there is almost no marked difference in anatomical structures of the roots and stems cross sections belonging to the taxa, however, anatomical differences are clear in vascular bundles present in median veins of the cross sections taken from the leaves and petioles. Anatomic properties of the two species was determined to be similar to the anatomic properties of other species of the genus Stachys.


INTRODUCTION
Stachys, with around 300 species worldwide, is one of the largest genera of Lamiaceae.The genus is concentrated in the warm temperate regions of the Mediterranean and South West Asia, with secondary centres in North and South America and Southern Africa (Bhattacharjee, 1980).
Stachys species are known in Anatolia as Ada çayı and Dağ çayı and used as sage and in popular medicines to treat genital tumours, sclerosis of the spleen, inflamematory tumours, coughs and ulcers (Potoğlu-Erkaya and Koyuncu, 2007).Teas prepared from the whole plant or leaves are used in phytotherapy, possessing sedative, antispasmodic, diuretic and emmenagogue activities (Jovanovic et al., 2008).In addition, its aerial parts are

Correct
In a previous study, we investigated the morphological and ecological features of S. balansae and S. carduchorum (Erdoğan et al., 2011).Here, we report on the anatomy of these two species.In this study, the anatomical structure of root, stem, leaf and petiole in the two species of Eriostomum section grown in Turkey is studied for the first time.
The aim of this paper was carried out in order to provide insight into anatomy two Stachys species.Moreover, a better understanding of systematics helps separate morphologically similar species from each other.

MATERIALS AND METHODS
Plant specimens of the 2 species were collected from their type localities (Table 1, Figure 1).Species collected was examined in the herbaria and were determined using the relevant literature (Ball, 1972;Bhattacharjee, 1982;Davis, 1982;Duman, 2000).The specimens are dried according to standard herbarium techniques and stored in the Herbarium of Necatibey Education Faculty, Anatomical studies were carried on specimens kept in 70% alcohol.Cross-sections of roots, stems, petioles, leaves and surface sections of leaves were taken by hand.Then, sections were stained with Floroglusin and then mounted with Gliserin-Jelatine (Yakar-Tan, 1982).Measurements and photographs of anatomic sections were taken using Olympus CX21 binocular light microscope with an Olympus Camedia camera.
For images of stomata at Scanning Electron Microscope (SEM), leaves of the dried plant samples were mounted on standard SEM pin mount stubs using a double-sided conductive carbon tape.The samples were then coated with a thin layer of gold-palladium using a Cressington 108 Auto sputter coater to reduce charging.The coated samples were imaged using a Hitachi S-4800 Scanning Electron Microscope (SEM) at an accelerating voltage of 5 to 15 kV and working distances ranging from 20 to 22 mm at University of Toledo, Ohio, USA.The SEM micrographs were then analyzed.

Root
Cross-sections taken from the root of S. balansae and S. carduchorum have revealed that the periderm layer on the outermost surface of the root is consisting of phellem by 3 to 5 layers and phelloderm and phellogen are not distinguishable.There is a multi-layered parenchymatic cortex (8 to 13 layers) under the periderm.Endodermis is one rowed and under of the endodermis pericycle is indistinguishable.Phloem is located under endodermis and 4 to 12 layered.Cambium cells are indistinguishable.The xylem is composed of vessels and tracheids.Pith rays comprise 1 to 3-rowed rectangular cells.Pith region of S. balansae consist of xylem elements whereas S. carduchorum consist of polygonal or orbicular parenchymatous cells (Figure 2).

Stem
Cross-sections taken from the stem have exhibited a monolayer epidermis covered by a thin cuticle.The epidermis is composed of oval, cubic or rectangular cells.There are glandular and eglandular hairs (simple and unbranched, 1 to 4 cells) on epidermis.On young stems are seen diacytic type stomatas.Underneath the epidermis, multilayered collenchyma cells are located at the corners (7 to 13 layers) and there are 1 to 2 rows of chlorenchyma cells between them.Beneath this is situated the cortex which consists of 2 to 6 layers of oval, ovate or orbicular parenchymatous cells.Endodermis under the cortex is one rowed.Pericycle is indistinguishable.Sclerenchymatic cell clusters (1 to 4 layers) are situated in beneath endodermis.Phloem is 3 to 6-layered and consists of oval and rectangular cells.Cambium is indistinguishable.The xylem is composed of trachea and tracheids.Trachea is orbicular or ovoid while tracheids are polyhedral.Rays are 1 to 2 rowed.The pith is large and comprised of hexagonal or orbicular parenchymatic cells with intercelular spaces in the centre of stem (Figure 3).

Leaf
The epidermis is composed of a single layer cells and rectangular or oval cells.Adaxial epidermis is larger than lower and covered with a thick undulate cuticle.There are glandular and eglandular hairs (simple and unbranched, 1 to 2 cells) and on the surfaces of both epidermis.Stomata type is diacytic and occurs on the surfaces of both epidermis being more abundant on the lower surface.Stomata is located on the same level with epidermal cells.Leaf is isobilateral.Mesophyll occurs palisade 2 seriate beneath adaxial epidermis and 1 to 2 seriate above abaxial epidermis, at the median region of mesophyll is located spongy parenchyma that is 2 to 5midrib that is located to beneath the epidermis is seen collenchyma.Parenchyma that is located beneath collenchymas is shaped polygonal and oval and 3 to 6 layered at adaxial surface and 3 to 9 layered at adaxial surface.Vascular bundle is surrounding by parenchyma cells.The xylem faces towards the adaxial surface while phloem faces the abaxial surface.The phloem tissue is 3 to 8 layered and surrounded by parenchymatic cells (Figure 5a and c).In the phloem cells of S. balansae are not seen sclerenchymatic cells while phloem cells of S. carduchorum are seen (1 to 2 rowed) (Figure 4).

Petiole
A cross section of the petioles shows that the adaxial surface is concave and slightly curved and the abaxial surface is convex and acute.There are eglandular (simple, 1 to 5 cells) and glandular hairs on both adaxial and abaxial surface.The epidermis is composed of oval and rectangular cells 1 layered.Collenchyma is situated under epidermis.It is 3 to 8 layered on abaxial surface, 1 to 2 layered underadaxial surface and 4 to 12 layered the ends of the petiolar wings.Parenchyma cells are hexagonal oroval shape.They are 2 to 5 layered on nearly flat and abaxial surface is broadly convex while adaxial surface of S. carduchorum deeply concave (V-In the midrib, adaxial surface of S. balansae is shaped) and abaxial surface rounded-convex. 1 to 3 rowed in adaxial surface and 2 to 4 rowed in abaxial surface of collateral vascular bundle is located in the midrib region.seriate and composed of polygonal or rounded cells.The adaxial surface and 4 to 10 layered on abaxial surface.In the midrib, adaxial surface of concave-curved or deeply concave (V-shaped); abaxial surface is convex or rounded-convex.In the middle region of petiole, there are one (S.carduchorum) or two (S.balansae) collateral large vascular bundles crescent shape and one small subsidiary collateral bundle at the ends of petiolar wings.The phloem tissue is 3 to 8 layered and surrounded by    . carduchorum (c, d). ab, abaxial epidermis, ad, adaxial epidermis, co, collenchyma, p, parenchyma, mb, median bundle, acb, accessory bundle, sc, sclerenchyma, ph, phloem, x, xylem.parenchymati cells.In the phloem cells of S. balansae are not seen sclerenchymatic layer while phloem cells of S. carduchorum are seen (1 to 2 layered).The xylem is composed trachea and tracheids.Xylem faces towards the adaxial surface while phloem faces the abaxial surface (Figure 6).

DISCUSSION
In this study, two taxa belonging to the same subsection (Germanicae) and morphologically difficulty in distinguishing from each other is investigated.Moreover, anatomical properties of Germanicae subsection have *ad, Adaxial surface (upper epidermis); *ab, abaxial surface (lower epidermis).been reported for the first time.Our anatomical results are similar to Metcalfe and Chalk (1950) and Salmaki et al. (2011).The results obtained from anatomical investigation carried out on two species were generally similar.No important differences observed in root and stem anatomical features of S. balansae and S. carduchorum.However, Pithregion (root) of S. balansae consist of xylem elements while S. carduchorum consist of polygonal or orbicular parenchymatous cells.
Of the anatomy of leaf is appeared two significant differences.The first; in the midrib, adaxial surface of S. balansae is nearly flat and abaxial surface is broadly convex while adaxial surface of S. carduchorum deeply concave (V-shaped) and abaxial surface roundedconvex.The second; in the phloem cells of S. balansae are not seen sclerenchymatic cells while phloem cells of S. carduchorum are seen.
Of the anatomy of petiole is appeared significant difference.In the midrib, adaxial surface of S. balansae is slightly concave-curved and abaxial surface is convex while adaxial surface of S. carduchorum deeply concave (V-shaped) and abaxial surface rounded-convex.Besides, large vascular bundles in the middle region of petiole are one bundle of S. carduchorum and two bundles of S. balansae.Moreover; in the phloem, cells of S. balansae are not seen sclerenchymatic layer while phloem cells of S. carduchorum are seen.
Comparative anatomical characters of stem, leaf and petiol are given (Table 2 to 4).In addition to comparative micro-anatomical measurements, various tissues of S. balansae and S. carduchorum are presented in Table 5.
Selected micrographs of common indumentum types of stem, leaf and petiole of S. balansae and S. carduchorum, are presented in Table 6 and Figure 7. Two basic types of trichomes can be distinguished: Glandular and non-glandular trichomes.Nonglandular trichomes are unbranched and unicellular (Figure 7g and j) or multicellular (Figure 7f).Glandular trichomes are two types: Capitate and peltate.Capitate trichomes consist of short stalked (single cell base, short single cell neck, and

Table 1 .
Collection data of S.balansae and S. carduchorum from Turkey.
Table 1 is mixed with table 2 and Table 1 isn't in proof.I added Table 1 following.

Table 5 .
Comparative micro-anatomical measurements various tissues of S. balansae and S.carduchorum.

Table 6 .
Characterisation of the trichome types of S. balansae and S. carduchorum.