An investigation of the anatomy, palynology and trichome types of Phlomis olivieri (Lamiaceae)

Introduction

The genus Phlomis L. is one of the largest genera of subfamily Lamioideae (Lamiaceae) with more than 100 recognized species distributed in Asia, south Europe and north Africa that have been divided into two main sections: Phlomis and Phlomoides (Moench) Briq.(Rechinger, 1982). Section Phlomis was subdivided by Bentham (1834) into three subsections: Dendrophlomis Benth., Gymnophlomis Benth. and Oxyphlomis Benth. The diagnostic character for separating sections is corolla shape. Corolla in sect. Phlomis have a curved upper lip and a trifid lower lip with large median and smaller lateral lobes as opposed to the presence of a straight upper lip and a trifid lower lip with subequal lobes in sect. Phlomoides (Azizian and Moore, 1982). The genus represented by nearly 19 species in Iran including Ph. olivieri Benth. which grows wildly in the north, northwest, west and centre of Iran (Rechinger, 1982; Jamzad, 2012).

The taxonomic value of the indumentum as well as its implication in systematics and phytogenetics are well known in Lamiaceae and in the related families Acanthaceae, Bignonicaceae, Scrophulariaceae and Verbenaceae (Abu-Asab and Cantino, 1987; Ahmad, 1974, 1978; Cantino, 1990; El-Gazzar and Watson, 1968, 1970; Elias and Newcombe, 1979; Gairola et al., 2009; Mathew and Shan, 1983; Metcalfe and Chalk, 1972; Rahn, 1992). Trichome micromorphology has been suggested to be useful in the phylogeny reconstruction (Abu-Asab and Cantino, 1987) and has been widely used in the systematics of Lamiaceae (Cantino, 1990; Kaya et al., 2000; Erken, 2005; Satil et al., 2007; Dinç and Özturk, 2008; Güvenç and Duman, 2010; Celep et al., 2011) as well as specific and subspecific levels (Bruni et al., 1987; Giuliani et al., 2008; Bini Maleci et al., 1992; Sebebe and Harley, 1992; Servettaz et al., 1992).

The importance of pollen morphology within the Lamiaceae has been proven as supplementary data to classification (Abu-Asab and Cantino, 1992, 1993, 1994; Dinç et al., 2009). A comprehensive overview of the pollen survey within subfamily Lamioideae sensu Erdtman (1945) has been published by Abu-Asab and Cantino (1994). Moreover, some further examinations of pollen have been carried out, e.g. by Sebebe and Harley (1992: for Stachys L.), Dönmez et al. (1999: for Teucrium L.), Satil et al. (2005: for Thymus L.), Dinç and Özturk (2008: for Stachys sect. Ambleia Benth.), Dinç et al. (2009: for Lallemantia L.) and Asadollahi et al. (2014: for Salvia L.). The pollen morphology generally supported the segregation of some genera of Lamiaceae such as Marrubium L., Phlomis and Stachys. However, its taxonomic value for infrageneric classification varies in the family Lamiaceae.

Up to now, some studies on Phlomis have been conducted (Bech, 1963; Azizian and Cutler, 1982; Azizian and Moore, 1982). But neither of them exactly investigated the anatomy, palynology and trichome tpyes of Ph. olivieri. Therefore, the present study aims to investigate the trichome types, palynology and anatomy of Ph. olivieri as an important rangeland plant of Iran.

Materials and Methods

Specimens of Phlomis olivieri were prepared from fresh material collected from various wild populations from west of Iran and herbarium specimens. Voucher specimens (Table 1) were deposited in the herbarium of the University of Isfahan. Anatomical studies were performed using an average of 40 fresh specimens kept in 70% ethanol. All sections were made from the stems, leaves, petioles, calyces and bracteoles using commercial razor blades. Sections were stained with Methyl blue and carmine and mounted on the slides using Canada balsam. Samples were studied using an Olympus microscope model BX-41 light microscope with 40X to 400X magnifications. For identification of trichome types, trichomes were obtained from surface of stems, leaves, petioles, calyces and bracteoles were studied using an Olympus microscope model BX-41 light microscope with 40X to 400X magnifications. For palynological study, pollen grains were obtained from herbarium samples. The pollen slides were prepared according to Wodehouse (1935) technique. For LM investigation, measurements and observations were made using the Olympus microscope model BX-41 LM. For the scanning electron microscopy (SEM), the pollen grains were observed and photographed with a KYKY-3200 SEM to determine their exine ornamentation. Also pollen terminology has been used based on Punt et al., 2007.

Results

Phenology and habitat

Phlomis olivieri is a perennial herb species distributed in Iran and Iraq, and flowering in June to August. This plant grows on mountainous regions, adjacent to rocky slopes, steppe vegetation and the overgrazed rangeland soils of Irano-Turanian region and Hyrcanian district of Iran (Jamzad, 2012) and could be as one of important destroyed rangeland indicator together with Stachys inflata Benth. (Mozaffarian, 2005) (Figure 1).

Figure 1. General appearance of Phlomis olivieri (yellow flower) together with Stachys inflata

Anatomical characteristics

Stem

The stem was clearly quadrangular. The epidermis was covered by a thin cuticle. The epidermis consisted of a single layer of oval, squarish and rectangular cells. Underneath the epidermis, multi-layered collenchyma cells were located at the corners of the stem. The cortex was composed of 2-5 layers of irregular oval and rectangular parenchymatic cells with intercellular spaces. Vascular bundles were next to each other. Those located at the corners were slightly bigger in size than the others. Cambium was not visible. Primary and secondary xylem could be differentiated. Tracheae in the secondary xylem were denser and larger than in the primary xylem. The pith was large and comprised of hexagonal or orbicular parenchymatic cells with intercelular spaces in the centre of stem (Table 2, Figure 2).

Leaf

Transverse section of the lamina indicated that the upper and the lower epidermis were covered with a nearly thick cuticle layer. The thickness of both epidermises cuticles was nearly equal. Both epidermises consisted of 1 layered oval and rectangular cells. Upper epidermal cells were larger than lower epidermal cells or nearly equal to them. The mesophyll was composed of elongated rectangular palisade parenchyma and spongy parenchyma cells. The palisade parenchyma was 1 layered. The spongy parenchyma cells were 3-4 layers. The spongy parenchyma cells had intercellular spaces. Transverse section of the midrip showed that the adaxial surface was concave and the abaxial surface was convex. The epiderm was 1 layer. There was one large vascular bundle in the center that was surrounded by parenchymatic cells (Table 2, Figure 3). Stomata are diacytic.

Figure 2. Cross-section of the stem of Phlomis olivieri. e. Epidermis; cp. Cortex parenchyma; ph. Phloem; sx. Secondary xylem; px. Primary xylem; p. Pith. Scale Bar= 100 μm

Figure 3. Cross-sections of the leaf blades of Phomis olivieri. ab. Abaxial epidermis of midrip; ad. Adaxial epidermis of midrip; le. Lower epidermis; ue. Upper epidermis; vb. Vascular bundle. Scale Bar = 200 μm

Petiole

The adaxial surface was concave and the abaxial surface was convex. Both epidermises were one layer and covered with a thick cuticle layer. The epidermal cells of both surfaces were more or less rectangular to oval. Under epidermis, 2-7 collenchyma layers had placed, as well as collenchyma density at the corner was more than other parts. Parenchyma cells were hexagonal or orbicular. There were two collateral vascular bundles in the centre and two small subsidary bundles in the corners of petiole (Table 2, Figure 4).

Calyx

The both epidermises were one layer and covered with a thick cuticle layer. The upper epidermal cells were nearly longer than the lower epidermal cells. Parenchyma cells consisted of 4-5-layer cells. A single layer of elongated continuous fiber cells was observed under parenchyma cells. A one small parenchyma bundle was visible in middle of fiber cells. The expanded parenchyma cells had located under fiber cells (see Table 2).

Bracteole

The upper and lower epidermises were one layer and covered with a thick cuticle layer. 3-4 parenchyma layers had placed after vascular bundles. The fiber cells were observed among of xylem cells (see Table 2).

Figure 4. Cross-section of the petiole of Phlomis olivieri. ab. Abaxial epidermis; ad. Adaxial epidermis; co. Collenchyma; h. Hair; p. Parenchyma; svb. Subsidiary vascular bundles; vb. Vascular bundles. Scale Bar: 100 μm

Trichome morphology

Five different trichome types on the stems, leaf blades, petioles, calyces and bracteoles of Phlomis olivieri were observed: peltate, capitate glandular, stellate, unicellular simple and dendroid trichomes (Figure 5). Capitate glandular and peltate trichomes could be distinguished by head size and stalk length (Ascensão and Pais, 1998). In the capitate trichome, the length of the stalk should be more than half the height of the head, whereas peltate trichomes were short with a uni- or bicellular stalk and a large secretory head with 4 to 18 cells arranged in one or two concentric circles (Werker et al., 1985). Type 1 is the typical peltate glandular trichome and consisted of a basal epidermal cell, a very short unicellular stalk and a round multicellular secretory head consisting of four or eight cells. Type 2 was a capitate glandular trichome composed of a basal epidermal cell, unicellular to bicellular stalk of variable length and a large, unicellular or bicellular secretory head. Type 3 was a non-glandular (stellate) trichomes consisted of several basal epidermal cells, and branched at the tip of the stalk cell to form star-shaped that divided to two groups: group 1 (sessile or short stalked stellate trichomes) and group 2 (long stalked trichomes). The First group trichomes did not have stalk or have short stalk but the second group trichomes had long stalk to subdivided into three subgroups: stalks are uni-, bi- and multiseriate. Type 4 was dendroid trichomes that this kind of trichomes was branched along the stalk cell and the branching was along the biserrate long stalk cells. Type 5, this type was composed of unicellular simple trichomes that were observed nearly in all of parts. The sessile or short stalk and multiseriate stalked stellate trichomes were on the adaxial surface of leaf but uni- and biseriate stellate trichomes mostly were observed on the abaxial leaf surface. The density of the stellate (especially, sessile and multiseriate) trichomes were in abundance on the abaxial surface of calyx. The glandular trichomes were present in abundance on the abaxial surface of leaf. The most density of glandular trichomes was observed on the adaxial surface of petiole and the most density of stellate trichomes was observed on the adaxial surface of bracteole (Table 3). The dendroid trichomes were only observed on abaxial surface of calyx. The dendritic trichomes were reported for the first time in this species.

Figure 5. Types of trichome in Phlomis olivieri. A. Unicellular simple; B. Stellate; C. Capitate glandular; D. Peltate glandular; E. Dendroid. Scale Bar = 10 μm

Pollen characteristics

The pollen grains were tricolpate. Their shape was protate-spheroidal. The dimensions of polar axis and equatorial axis were 38.15-41.26 and 26.05-29.45 µm, respectively. The ratio P/E was 1.39-1.48. The exine thickness was 2.95-3.15µm. Exine ornamentation was reticulate and perforate (Figure 6, Table 4).

Figure 6. General appearances of the pollen grains in Phlomis olivieri

Discussion

The Phomis olivieri is an important rangeland plant in Iran also the anatomical characters, palynology and trichome types of this species were exactly examined. This is the first report from the anatomical characters of calyx and bracteole in this species. Metcalfe and Chalk (1972) pointed out that the structure of the vascular bundles in the petiole of the species of Lamiaceae could be used as a diagnostic character. In the petiole of this species, there is two-lobed vascular bundle in the centre and 1 small subsidiary bundle in petiolar wings. Therefore, the anatomical properties of the petiole may be useful characters for distinguishing and separation of this species from other species in the genus. Four vascular bundles were observed in bracteole. The number of vascular bundles in bracteole could be considered as a remarkable character. Five main types of trichomes were observed. Our findings about trichomes types are not consistent with Azizian and Cutler (1982). Our study about trichomes showed the presence of dendroid trichomes on abaxial surface of calyx. The dendroid trichomes are reported for the first time in Ph. olivieri from Iran whereas its importance remains unsound.

Acknowledgements

The first author would like to thank Ms. Hesarkhani and Mr. Safikhani for their kind assistance and guidance.