COMPARATIVE MORPHOLOGY, ANATOMY AND PALYNOLOGY OF TWO SALVIA L. SPECIES (LAMIACEAE) AND THEIR TAXONOMIC IMPLICATIONS

Morphological, anatomical and palynological characteristics of Salvia glutinosa L. and S. staminea Montbret & Aucher ex Bentham and their taxonomic importance are presented. Their expanded description and phenology are also given. The features of stems, leaves, calyces, corolla and nutlets have been found to be significant to distinguish the species. Mesophyll structure, distribution of stomata on upper epidermis, size of stomata on lower epidermis, shape of vascular structure in midrib, and number of vascular bundles and presence of sclerenchymatic fibers in petiole are diagnostic characters. Moreover, pollen size and exine ornamentation are important in separating these two species.


Introduction
Salvia L. (Lamiaceae) is represented by about 1000 species displaying a remarkable diversity in growth forms, secondary compounds, floral morphology, and pollination biology.The genus is distributed extensively in three regions of the world: Central and South America (500 spp.), western Asia (200 spp.) and eastern Asia (100 spp.) (Walker and Sytsma, 2007).Turkey is a major diversity center for Salvia in Asia (Hamzaoglu et al., 2005).The first revision of Salvia L. in Turkey was made by Hedge (1982), who recognized 86 species, one hybrid and one doubtful species.Since then, six more new species and three new records have been described from Turkey.The genus Salvia has been subject to a number of studies mainly based on morphological (Hedge, 1982), anatomical (Metcalfe and Chalk, 1965;Kaya et al., 2007;Kahraman et al., 2009a, b) and palynological (Cantino et al., 1992) information.
Boissier (1875) recorded 75 species of Salvia from Turkey and placed them under seven sections using Bentham's (1833) sectional delimitation.Boissier (1875) placed Salvia glutinosa L. in the section Drymosphace and S. staminea Montbret & Aucher ex Bentham in the section Plethiosphace.Since 2005, as a part of a revision of the genus Salvia in Turkey, the present authors have carried out extensive field studies and collected a large number of specimens.Although S. glutinosa and S. staminea belong to different sections, some morphological characteristics of these two species overlap such as stem indumentum, leaf size, petiole length, inflorescence length, number of verticillasters, stamen type, and bract shape and size.Anatomical and palynological structures of these species, however, have not been studied before.The present study thus aims to clarify taxonomic importance of anatomical and palynological characters in addition to morphological ones for distinguishing these two species and their sections.

Materials and Methods
Plant specimens were collected from different localities in Turkey.The specimens have been stored in Ankara University Herbarium (ANK).
Anatomical studies were carried out on specimens kept in 70% alcohol.The paraffin method was used for the transverse sections of stem, leaf and petiole.The specimens were embedded in paraffin and then sectioned with a Leica RM2125RT rotary microtome.All sections were stained with safranin and fast green and then mounted with canada balsam.Measurements and photographs were taken using a Leica DM1000 binocular light microscope with a Leica DFC280 camera.
For palynological investigations, pollen materials were obtained from herbarium specimens.The pollen slides were prepared according to Wodehouse (1935) technique.For light microscope (LM) studies, measurements and observations were made using the Leica DM1000 binocular light microscope with the Leica DFC280 camera.The polar length, equatorial length, colpus length, exine and intine thickness for 30 pollen grains were measured under the light microscope (X 1000) and polar axis/equatorial axis (P/E) ratios were calculated.For scanning electron microscopy (SEM), the pollen grains were observed and photographed with a JEOL-6060 scanning electron microscope to determine their exine ornamentation.Pollen terminology of Punt et al. (2007) has been used.
The stems of the family Labiatae (Lamiaceae) species are quadrangular and include well-developed collenchymatous cells at the corners (Metcalfe and Chalk, 1965).Moreover, it was also reported that scleranchymatic fibers surround the vascular tissue.This report is congruent with our results observing same anatomical characteristics in the stems of S. glutinosa and S. staminea.Our results also detected thicker, undulated cuticle in S. staminea, which prefers drier habitats, opposed to S. glutinosa.The cortex layer of S. staminea is also larger than that of S. glutinosa.
Leaf anatomy: Transverse sections of the lamina and surface preparations of both epidermis of S. glutinosa (Fig. 9) showed that the upper and lower epidermis that are covered with sparsely simple hairs consisting of uniseriate oval or rectangular cells with thin and undulate cuticles.The upper epidermis cells are nearly equal to the lower.Mesophyll region (140-165 µm) consists of 2 or 3 layers of elongated palisade cells and 3 or 4 layers of nearly isodiametric spongy parenchymatic cells with large intercellular cavities (Fig. 10).The leaf is bifacial and hypostomatic.Stomata on the lower epidermis are of the diacytic type.Their length varies from 22-28 µm while their width ranges from 15-17 µm .In the midrib region, deeply crescentiform or U-shaped vascular bundles are surrounded by parenchymatic cells.There is a single large vascular bundle in the center.
Transverse sections of the lamina and surface preparations of upper and lower epidermis of S. staminea revealed (Fig. 13) that both epidermis are covered with densely eglandular hairs consisting of uniseriate, rectangular or oval cells with thin and undulate cuticles.The upper epidermal cells are larger than the lower.Mesophyll region (100-130 µm) comprises of 2-3-layered palisade cells above, 1-2-layered below and 1-2-layered spongy parenchyma cells (Fig. 14).The leaf is equifacial and amphistomatic, with diacytic type .Stomata on the lower surface show higher frequencies than those on the upper surface.On the adaxial surface, the length of stomata varies from 15-19 µm while the width of stomata ranges from 15-17 µm.On the abaxial surface, the length of stomata varies from 17-19 µm, whereas the width of stomata ranges from 15-17 µm.In the midrib region, shallow crescentiform vascular bundles are surrounded by parenchymatic cells.There are a single large vascular bundle in the center and one small bundle on each side.
Mesophyll in Salvia species is entirely parenchymatic and the midrib is surrounded by collenchymatous cells (Metcalfe and Chalk, 1965).The leaves of S. indica (Kahraman et al., 2009a) are bifacial and amphistomatic, however those of S. halophila (Kaya et al., 2007) are monofacial and amphistomatic.According to the mesophyll structure, the leaves of S. glutinosa are bifacial and hypostomatic while those of S. staminea are equifacial and amphistomatic.Moreover, the vascular bundle structure of S. glutinosa is a deeply crescentiform or U-shaped, but that of S. staminea is shallow crescentiform.Salvia glutinosa has only single large bundle in the center, whereas S. staminea has a single large vascular bundle in the center and 2 small bundles on the sides.Salvia indica (Kahraman et al., 2009a) has one large and lobed vascular bundle in the centre, however S. halophila (Kaya et al., 2007) has two large bundles.Moreover, stomata in S. glutinosa are larger than S. staminea.To sum up, the distribution of palisade and spongy parenchyma cells, the presence of stomata on the upper surface, shape and number of the vascular bundle structure, and size of stomata are taxonomically significant characters in separating the species.
Petiole anatomy: Transverse sections taken from the petiole of S. glutinosa showed the following elements (Fig. 17).The epidermal cells of both surfaces are oval and rectangular.Adaxial and abaxial epidermis cells are nearly equal in size.One to three layers of collenchyma cells are located under the epidermis.The vascular bundle surrounded by parenchymatic cells appears as a shallow arc.A large single vascular bundle is located in the middle, as well as, there are 3 or 4 small subsidiary vascular bundles in each wing.A few sclerenchyma fibers are only observed on the phloem.
Transverse sections taken from the petiole of S. staminea showed the following elements (Fig. 18).The epidermal cells of both surfaces are squarish and nearly rectangular.The adaxial epidermal cells are slightly larger than the abaxial epidermal cells.There are 2 or 3 layers of collenchyma cells under the epidermis.A broad single vascular bundle is located in the middle, as well as, there are two or three small subsidiary vascular bundles in one petiolar wing and two small bundles in the other wing.The sclerenchyma tissue is well-developed outside of the phloem and xylem.
The structure of the vascular bundles in the petiole structure of the species of Labiatae could be used as a distinctive character (Metcalfe and Chalk, 1965).In the petiole of S. glutinosa, there is one large bundle in the center and there are 3 or 4 small subsidiary vascular bundles in each petiolar wings.In S. staminea, although there is a large single vascular bundle in the center, but there are 2 or 3 small subsidiary vascular bundles in one petiolar wing and two small bundles in the other wing.Kaya et al. (2007) investigated S. halophila found 6 broad vascular bundles in its middle of the petiole and 6 small bundles on its wings.Ozdemir and Altan (2005) observed in S. huberi a single large vascular bundle in the center of the petiole and 5 small lateral bundles, 2 of which are located in one petiolar wing and 3 in the other.While in S. glutinosa only phloem is surrounded by sclerenchyma fibers, both xylem and phloem in S. staminea are surrounded by sclerenchyma fibers.Thus, number of vascular bundles and the presence/absence of sclerenchymatous tissue in the petiole may serve as taxonomically diagnostic characters.