Comparative anatomical studies of some Teucrium sect. Teucrium species: Teucrium alyssifolium Stapf, Teucrium brevifolium Schreb. and Teucrium pestalozzae Boiss. (Lamiaceae)

Abstract Teucrium alyssifolium Stapf (endemic), Teucrium pestalozzae Boiss. (endemic) and Teucrium brevifolium Schreb. are three closely related taxa in Teucrium sect. Teucrium. The obtained data from the anatomical studies revealed that these three taxa represent the general anatomical characteristics of the Lamiaceae family. Leaves, anatomical features such as thick cuticle, abundant trichomes, rich palisade parenchyma layer in the mesophyll provide evidence that these three species are xeromorphic structures. Leaf and stem anatomy showed that the taxa have generally similar anatomical features. However, cuticle layers, epidermis cells size, indumentum density, mesophyll types, palisade parenchyma occupied in the mesophyll, presence of spherocrystals in leaves and parenchyma, collenchyma and sclerenchyma layers in stems show differences amongst the taxa. Anatomical characters of leaf and stem of these taxa are examined for the first time in this study.


Introduction
The genus Teucrium L. has approximately 300 species all over the world. Teucrium's cosmopolitan distribution is mainly concentrated in Europe, North Africa and in the temperate parts of Asia (Ecevit-Genç et al. 2015. Teucrium is a large and polymorphic genus which is represented by 49 taxa (36 species) in Turkey. There are 18 endemic taxa (Govaerts 1999, Duman 2000, Dönmez 2006, Parolly and Eren 2007, Dönmez et al. 2010, Dinç et al. 2011a, 2011b, Dirmenci 2012, Özcan et al. 2015a, Vural et al. 2015, Dinç and Doğu 2016. The classification of Teucrium is based on sections. The main characters in the separation of sections are the calyx-shape and flower arrangement (Davis 1982;Navarro et al. 2004;Dinç et al. 2008;Özcan et al. 2015a;Vural et al. 2015). Especially, leaf anatomy is important for the classification of the genus (Dinç et al. 2009). Also, absence or presence of trichomes and their types on the nutlets and vegetative parts are very important for classifying the species (Dinç et al. 2011a, Ecevit-Genç et al. 2015.
Teucrium species have traditionally been used in Turkey for abdominal pain, stomach-ache, common cold, high fever, antipyretic, rheumatic pain and as an antidiabetic (Sezik et al. 2001, Aksoy-Sagirli et al. 2015. T. alyssifolium is a narrowly distributed endemic species. It is classified as a 'Conservation Dependent (LR/cd)' category of IUCN and it is a source of polyphenols and flavonoids and has confirmed antioxidant activities (Semiz et al. 2016). T. pestalozzae is an endemic species and its essential oil is characterised with β-caryophyllene (27.6%) and germacrene D (13.8%) as major constituents (Baser et al. 1997). Spathulenol and δ-cadinene are the main compounds of T. brevifolium essential oil and it has shown anti-tumour activities, a selective cytotoxicity on large lung carcinoma (IC 50 value of 80.7 μg/ml) (Menichini et al. 2009).
Pollen morphology supplies useful data at the taxonomic level in Teucrium (Navarro et al. 2004, Marzouk et al. 2017. Oybak and İnceoğlu (1988) studied pollen morphology of some Turkish Teucrium members. They found out that the species belonging to different sections had different pollen type and pollen shape, while pollen grain size and apocolpia size were the main characters used for distinguishing the species. Especially, T. alyssifolium could be easily separated from the other species of the sect. Teucrium according to pollen data.
There are several studies on Teucrium anatomy (Lakusic et al. 2006, Dinç et al. 2008, 2011a, 2011b, Dehshiri and Azadbakht 2012, Dinç and Doğu 2012, Doğu et al. 2013, Özcan 2013, Özcan and Eminagaoglu 2014, Ruiters et al. 2016). However, the anatomy of T. pestalozzae, T. brevifolium and T. alyssifolium has not been investigated. In our previous studies, we investigated the nutlet and leaf micromorphol-ogy of some species belonging to the sect. Teucrium in Turkey (Ecevit-Genç et al. 2015. In the present study, we report on the anatomical features of the leaves and stems of T. alyssifolium, T. brevifolium and T. pestalozzae. The aim of this paper is to understand the anatomy of these three Teucrium species. Also, a better understanding of systematics helps the distinction of morphologically closely related taxa from each other.

Materials and methods
T. pestalozzae samples were collected from Antalya, T. brevifolium and T. alyssifolium samples were collected from Muğla provinces in Turkey (Figures 1, 3, 5). Voucher specimens are stored in the Herbarium of the Faculty of Pharmacy, Istanbul University (ISTE). Data about habitats of each investigated species are given in Table 1. Permanent microscopic preparations were made of plant materials fixed in 70% alcohol during the field studies. Cross-sections of the plant leaves and stems were taken manually and stained with Sartur solution (Çelebioğlu and Baytop 1949). Several slides were made and photographed for each species with an Olympus BH-2 and Canon A 640 digital camera.

Results
The anatomy of the collected specimens were assessed by examination of leaf and stem cross sections (Figures 2,4,6). This is the first study about the anatomical features of the leaves and stems of T. alyssifolium, T. brevifolium and T. pestalozzae.

T. alyssifolium Stapf Leaf anatomy
The epidermis at the both surfaces of the leaves is single layered. The epidermis consists of single-layer, ovoid or rectangular cells which are covered by thick cuticula. The upper epidermis cells are larger than the lower ones. Both leaf surfaces are covered by glandular and non-glandular trichomes. Also, the upper epidermis is covered with  lower-density trichomes than the lower epidermis. The spherocrystals occur in the upper epidermis cells of the leaf in T. allysifolium. Leaves are isolateral. The mesophyll is differentiated into 1 layered palisade and 2-3-layered spongy parenchyma. The palisade parenchyma cells are under the upper and lower epidermis. Their shapes are cylindrical in transverse section. The palisade parenchyma occupies about 60-65% of the mesophyll. The spongy parenchyma cells, ovoid or circular, are located between the palisade tissues. Both parenchyma tissues contain starch grains. The midrib has 3-4 layered collenchyma and 1-2 layered parenchyma below the lower epidermis. The vascular bundle is located in the central part of the midvein. Vascular bundles are collateral. The xylem layer is just below the collenchyma. 1-2 layered parenchyma and 5-6 layered collenchyma are located under the phloem (Figure 2A).

Stem anatomy
The stem is quadrangular shaped. The epidermis consists of single-layer, ovoid or rectangular cells which are covered by thick cuticula. There are glandular and non-glandular trichomes on the epidermis. Collenchyma with a single layer of cells between the corners but 4-5 layers of collenchyma at the corner of the stem. The cortex, consisting of 3-4 layered ovoidal parenchymatous cells, is located under the collenchyma. The endodermis is conspicuous as a single layer. The vascular tissue is surrounded by 1-2 layers of sclerenchyma fibres. The cambium is indistinguishable. Phloem and xylem members are conspicuous. The pith is present at the middle of the stem and it is completely filled by orbicular parenchymatic cells ( Figure 2B, C).

Leaf anatomy
The epidermis in both surfaces of the leaves is single layered. It is consists of single-layer, ovoid or rectangular cells which are covered by cuticula. Both surfaces are covered by a thick cuticula layer, with dense indumentum built of glandular and non-glandular trichomes. The upper epidermal cells are as large as the lower ones. Spherocrystals are observed in both epidermis cells. Leaves are dorsiventral. Palisade parenchyma has two layers and palisade parenchyma cells shapes are cylindrical in transverse section. The palisade parenchyma occupies about 60% of the mesophyll. Spongy parenchyma consists of four or five layers and their cells are ovoid or circular.  Starch accumulated in both spongy and palisade parenchyma. Midrib has 5-6 layered collenchyma and 1-2 layered parenchyma below the lower epidermis. The collateral vascular bundle is located in the central part of the midvein. The xylem layer is found under the collenchyma. 1-2 layered parenchyma and 2-3 layered collenchyma are located under the phloem ( Figure 4A).

Stem anatomy
The stem is rectangular shaped. The epidermis consists of single-layer, ovoid or rectangular cells which are covered by thick cuticula. It is covered by glandular and nonglandular trichomes. Underneath the epidermis, 6-7 layers of collenchyma are located at the corners, 3-4 layered collenchyma is located between the corners. Beneath the collenchyma, 5-6 layered rectangle shaped parenchymatous cells are located. Starch grains are also present in the parenchymatous cells. Endodermis and cambium are inconspicuous. 2-3 sclerenchymatic cell clusters are situated at the corners above the phloem. The pith is present in the middle of the stem and is completely filled by orbicular parenchymatic cells ( Figure 4B, C).

Leaf anatomy
The epidermis in both surfaces of the leaves is single layered. It is consists of single-layer, ovoid or rectangular cells which are covered by cuticula. The upper epidermis cells are larger than the lower ones. The upper cuticle layer is slightly thicker than the lower ones.
Both surfaces are covered by glandular and non-glandular trichomes. Also, trichomes are abundant on the lower epidermis of leaves and sparse on the upper epidermis of leaves. Leaves are dorsiventral. The spherocrystals occur in the upper epidermis of the mesophyll. Mesophyll is differentiated into 2-layered palisade and 5-6-layered spongy parenchyma. Palisade parenchyma cells are cylindrical shaped in transverse section. The palisade parenchyma occupies about 50-55% of the mesophyll.
The spongy parenchyma cells are ovoid or circular. Both parenchyma tissues densely contain starch grains. Midrib has 5-6 layered collenchyma and 1 layer of parenchyma below the lower epidermis. The collateral vascular bundle is located in the central part of the midvein. The xylem layer is found under the collenchyma. 1-2 layered parenchyma and 4-5 layered collenchyma are located under the phloem ( Figure 6A).

Stem anatomy
The stem is rectangular shaped. The epidermis consists of single-layer, rectangular cells which are covered by cuticula. There are glandular and non-glandular trichomes on the epidermis. Underneath the epidermis, there is collenchyma with 1-2 layers between the corners but 7-8 layers of collenchyma at the corner of the stem. The cortex, consisting of 5-6 layers of ovoid shaped parenchymatous cells, is located under the collenchyma. 1-2 layers of sclerenchyma fibres are located above the phloem. The cambium is indistinguishable. Phloem and xylem members are conspicuous. The pith is present in the middle of the stem and is completely composed of orbicular parenchymatic cells ( Figure 6B, C).

Discussion
Sect. Teucrium is one of the eight Teucrium sections distributing in Turkey. The members of this section are perennial and shrubs or subshrubs. Leaves are entire to deeply dissected (in T. orientale subspecies, T. parviflorum, T. pruinosum) and revolute at the lower surface.
Flowers borne in racemes or spreading panicles or axillary in upper leaves. Peduncles/pedicels are 1-3-flowered. Calyx not gibbous, obconical-campanulate, teeth ± equal and triangular (Ekim 1982).  Three species showing the characteristic features of the section Teucrium, investigated in the present study, have some significant distinguishing characters. Especially the size of parts of the flowers are very distinctive. T. alyssifolium easily differs with its leaf and bract shape, flower, filament and calyx size from T. brevifolium and T. pestalozzae in their natural habitats (Table 2). T. alyssifolium is dwarf, suffruticose and T. brevifolium and T. pestalozzae are shrublet plants. Stamens of T. alyssifolium are longer than its lips, stamens subequal to lip in T. pestalozzae and slightly shorter than lips in T. brevifolium. T. alyssifolium has the shortest and T. brevifolium has the longest stems.
In this study, morphologically related three taxa belonging to the sect. Teucrium have been investigated. Moreover anatomical features of these three species have been reported for the first time. Our results showed the general anatomical characteristics of three Teucrium species as well those reported by Metcalfe and Chalk (1950) and Dinç et al. (2008Dinç et al. ( , 2009Dinç et al. ( , 2011aDinç et al. ( , 2011b, Lakusic et al. (2010), Doğu et al. (2013), Özcan (2013).
The results of the present study revealed that there were differences amongst the leaf anatomy of these three taxa ( Table 2). The cuticle layer is on both sides and is of equal thickness to the epidermis for T. alyssifolium and T. brevifolium leaves. However, the upper cuticle layer of T. pestalozzae leaves is slightly thicker than the lower ones. The upper epidermis cells are larger than the lower ones as T. alyssifolium and T. pestalozzae, but both epidermis cells are the same size as in T. brevifolium. T. alyssifolium and T. brevifolium have a more dense indumentum than T. pestalozzae. Also, the indumentum of T. brevifolium has the same density on both sides, but the surface of the lower leaves of the other two species is denser than the upper ones. Mesophyll is dorsiventral in T. brevifolium and T. pestalozzae but isolateral in T. alyssifolium. The mesophilic organisation is an important distinguishing character for T. alyssifolium. Mesophyll types may be a good distinctive character in different species but sometimes it can be the same in some closer species (Erdoğan et al. 2012).
The palisade parenchyma shows a slight difference in mesophyll amongst the studied taxa. However, these differences can be based on different ecological conditions. Collenchyma layers are different in midrib amongst these studied taxa. The spherocrystals occur in the upper epidermis of the leaf in T. alyssifolium and T. pestalozzae and both epidermis of the leaf in T. brevifolium. According to Metcalfe and Chalk (1950), druse and simple crystals are generally seen in dicotyledon plants. Absence or presence of the crystals and their density are used to distinguish the genera and their species (Salimpour et al. 2009, Güvenç andKendir 2012). However, spherocrystals and raphides are less common crystal types for dicotyledons. Spherocrystals and raphides have a diagnostic value for dicotyledons (Dinç et al. 2008, 2013, Ruiters et al. 2016). According to Dinç et al. (2008Dinç et al. ( , 2009 and Ruiters et al. (2016), spherocrystals are an interspecific classification of sect Teucrium. Our results supported their observations. Some characteristics of the leaf anatomy which indicates of xeromorphy have been reported before in previous studies (Metcalfe and Chalk 1983, Lakusic et al. 2010, Dinç et al. 2008). According to the results of our study, the three of taxa have leaves with xeromorphic features such as cuticula layer thickness, dense trichomes and a high proportion of the palisade parenchyma in the mesophyll.
In conclusion, this study shows that leaf and stem anatomy have a diagnostic value in the distinction of these three closely related Teucrium species in sect. Teucrium. Anatomical characters contribute to the separation of three species with the morphological characters.

Collenchyma cells layers of midrib
3-4 layered on the lower surface, 5-6 layered on the upper surface 5-6 layered on the lower surface, 2-3 layered on the upper surface 5-6 layered on the lower surface, 4-5 layered on the upper surface Stem Length 3.5-9.0 cm 30-110 cm 15-18 cm
Polium species in Turkey are not conspicuously rectangular. Parenchyma, collenchyma and sclerenchyma layers have some differences amongst the stem of studied taxa. Endodermis is conspicuous only in T. alyssifolium. Three studied taxa display general characteristics of Lamiaceae anatomy.