AJCS 8(4):509-514 (2014) ISSN:1835-2707 Morphological and anatomical investigation on endemic Cota melanoloma and Cota antitaurica (Asteraceae) in Turkey Mehmet Ufuk Özbek1*, Dudu Özlem Mavi İdman2, Barış Bani3 1 Gazi University, Faculty of Sciences, Department of Biology, 06500, Teknikokullar, Ankara, Turkey Middle East Technical University, Department of Biological Sciences, 06531, Ankara, Turkey 3Kastamonu University, Faculty of Arts and Sciences, Department of Biology, 37200, Kuzeykent, Kastamonu, Turkey 2 * Corresponding author: mufukozbek@gmail.com Abstract Anthemis section Cota was accepted as separate genus recently. Genus Cota is represented by 24 taxa in Turkey. Some of them have economic importance because of their uses for various purposes such as obtaining drugs, food and dye. etc. In this study, Cota melanoloma subsp. melanoloma, C. melanoloma subsp. trapezuntica and C. antitaurica were studied morphologically and anatomically. Three of them are endemic to Turkey. C. antitaurica is only known from the type locality and critically endangered species, while C. melanoloma is distributed in only northern Anatolia. As the results, the diagnostic characteristics, synonyms, flowering and fruiting times, habitat properties, distributions and threat categories of each taxon are presented. The most important morphological characteristics for more proper discrimination the species are determined as the shape of capitula and palea, indumentum of stem and leaf. The detailed anatomical studies indicate the diagnostic characteristics as both hair and vascular bundle length and pith of the stems, hair length and length/width ratio and the type of stomata of the leaves, the layer number of parenchymatic ray cells and the cortex length of the roots. Keywords: Morphology; anatomy; Cota; endemic; Turkey. Abbreviations: Excl_exclude; inc_include; cm_centimeter; diam_diameter; mm_millimeter; µm_micronmeter; c_about; euro-Sib_ Euro-Siberian; Ir-Tur_Irano-Turanian; subsp_subspecies. Introduction Asteraceae is represented by the greatest number of taxa in Angiospermae from all over the world. It has nearly 16001700 genera and 24000-30000 species (Funk et al., 2005). Cota J. Gay is comprised of 49 species (63 taxa). A total of 24 taxa are distributed in Turkey, 10 of which are endemic to Turkey (Özbek, 2012). Cota is distributed in Mediterranean and Iran-Turan phytogeographic regions. Generally, members of the genus occur mostly in Europe (excl. North side), northern Africa, Caucasus, southern Russia and Anatolia to Afghanistan (Davis et al. 1975; Oberprieler, 2007). Recently, Anthemis sect. Cota was accepted as a generic name, Cota, by Oberprieler (2001), Greuter et al. (2003), Oberprieler et al. (2007, 2009) and Lo Presti (2010). Some species of the genus Cota have economic importance because of their uses for various purposes such as obtaining drug, food and dye etc. (Ghafoor, 2010; Öztürk et al., 2013). Genus Cota morphologically resembles Anthemis, but it differs from Anthemis by obconical, dorsiventrally flattened achenes, with prominent lateral ribs that are smooth or with 3-10 ribs on each side. The achenes of Anthemis are obovoid to obconical, circular or quadrangular in cross-section, usually with c. 10 smooth or tubercular ribs. There are few reports about the anatomy, classification and seed morphology of Cota (Metcalfe and Chalk, 1957; Zareh, 2009; Bani and Adıgüzel 2010; Ghafoor, 2010; Özbek et al., 2011). Moreover, there are some studies about chemical composition and antimicrobial activity of the closely related genus Anthemis L. (Uzel et al.,2004; Çelik et al., 2005; Uysal et al., 2005; Albayrak and Aksoy, 2012). The aim of this study is to determine the morphological and anatomical properties of Cota antitaurica (Grierson) Holub and C. melanoloma (Trautv.) Holub (including both subsp. melanoloma and subsp. trapezuntica (Grierson) Oberpr. & Greuter). However this is the first study on anatomy of any members of genus Cota in Turkey. These three taxa are endemic to Turkey. C. antitaurica is local endemic species that had not been collected since 1952. It was re-collected for the second time from its type locality by the authors in 2007. It grows in Cedrus libani and Abies cilicica subsp. cilicica forest between 1600-1900 m. C. melanoloma subsp. melanoloma which occurs on volcanic and calcareous rocks, high mountain steppe, and edge of mixed forest and open area of Quercus forest is distributed in North and Northeast side of Turkey, between altitudes of 1050-2600 m. while C. melanoloma subsp. trapezunica is scattered in Northeast Anatolia, between 800-3000 m. It prefers mountain steppes and edges of mixed forest as its habitat. 509 Table 1. Comparison of some morphological new findings. Characteristics Stems Primary segments of leaf lamina Secondary segments of leaf lamina Involucre Outer phyllaries Inner phyllaries Disc flowers Achenes Corona characters of Cota antitaurica in Flora of Turkey (Yavin and Grierson 1975) with the Flora of Turkey 25-30 cm 4-paired 3-5-paired 3-5-paired ovat, 3.5-5 x 2 mm 7-15 mm 4-5 mm 2.5-2.75 mm 0.5-0.75 mm New findings 17-33 cm 3-4-paired 4-6-paired 4-6-paired ovat, 3.5-4 x 1-1.5 mm 6-7 mm 3-4 mm 3.05-3.25 mm 0.5-0.7 mm Fig 1. Stem transverse sections. a) C. melanoloma ssp melanoloma, b) C. melanoloma ssp trapezuntica, c) C. antitaurica, ca: cambium; Co: collenchyma; Cu: cuticle; E: epidermis; h: hair; P: parenchyma; Pi: pith; VB: vascular bundle. bifurcate. Paleae 6-7 x 1-2 mm; narrowly lanceolate, acuminate at apex. Achenes obconical, brown, sometimes purple, 2.5-3 x 0.75-1 mm, prominent 10 ribbed; corona symmetric, 0.25-0.6 mm, crenulate. C. melanoloma subsp. Melanoloma ≡ Anthemis melanoloma Trautv., Bull. Soc. Imp. Naturalistes Moscou 41(1): 461 (1868). ═ Anthemis extrarosularis Freyn & Sint., Öst. Bot. Zeitschr. 44: 146 (1894). Diagnostic characters: Stem and basal leaves greyish-green; primary segments 3-4-paired, upper stem leaves obovateoblong. Habitat: Volcanic and calcareous rocks, high mountain steppe, edge of mixed forest, open area of Quercus forest, 1050- 2600 m. Flowering and fruiting time: Fl. 7-8, fr. 8-9. Phytogeographic region: Euro-Sib. element Distribution: Endemic (North and Northeast Anatolia: Kastamonu, Amasya, Sivas, Erzincan, Erzurum, Tunceli Trabzon, Bayburt, Ardahan). Threat category: LC C. melanoloma subsp. trapezuntica (Grierson) Oberpr. & Greuter, Willdenowia 33: 40 (2003). ≡ Anthemis melanoloma Trautv. subsp. trapezuntica Grierson, Notes R.B.G. Edinb. 33: 411 (1975). Diagnostic characters: Stem and basal leaves green; primary segments 5-6 -paired, upper stem leaves ovate. Habitat: High mountain steppe, edge of mixed forest, 8003000 m. Flowering and fruiting time: Fl. 7-8, fr. 8-9. Phytogeographic region: Euro-Sib. element Distribution: Endemic (Northeast Anatolia: Trabzon, Rize, Bayburt, Ardahan). Threat category: LC Results and Discussion Morphology The morphological description of each taxon is given in Flora of Turkey (Grierson and Yavin, 1975). Almost all of the morphological characteristics were expanded with the detailed investigations on our recent collections (Table 1 and 2). C. melanoloma (Trautv.) Holub Description: Perennial, rhizome woody. Stem mostly simple or numerous and branched from base, erect or ascendingerect, 15-60 cm long, 1-3 mm diam, green or greyish-green, adpressed-pilose. Sterile shoots up to 15 cm. Basal leaves (at first 5 node) reduced, 2-pinnatisect; 3.5-15 x 2-10 mm, oblong or oblong-elliptic. Median leaves 2-3 pinnatisect; 1540 x 5-30 mm, oblong or obovate-oblong; primary segments 3-6-paired, oblong-obovate, 2-15 x 0.5-7 mm; secondary segments 4-6-paired, lanceolate, 0.5-4 x 0.25-1 mm, acute at apex. Upper stem leaves 2-3 pinnatisect, 10-20 x 4-10 mm, ovate or obovate-oblong; primary segments 3-6-paired, oblong-obovate, 5-6 x 2-3 mm; secondary segments 3-4paired, lanceolate, 0.5-2 x 0.25-1 mm, acute at apex. Peduncles 6-20 cm, thickened below capitula at fruiting time. Capitula heterogamous hemispherical; 22-43 mm diam. (inc. ligules). Phyllaries imbricate, 4-seriate, pale or dark brown margined; outer phyllaries 5-6 x 2-2.5 mm, triangularlanceolate; median phyllaries 6-7 x 2.5-3 mm, oblong; inner phyllaries 6.5-7.5 x 2.5-3 mm, eliptic, obtus at apex. Ray flowers (14-)20-25(-35), white or sometimes cream; 3 toothed, lamina 10-20 x 4-7 mm, eliptic; ligula tube glabrous, 2 x 1.5 mm. Disc flowers tubular, yellow, hermaphrodite, 3-4 x 0.5-1 mm, 5-lobbed, shallowly at base; style 2-2.5 mm 510 Table 2. Comparison of some morphological characters of Cota melanoloma in Flora of Turkey (Yavin and Grierson 1975) with the new findings. Characteristics Flora of Turkey New findings Stems 25-45 cm 15-60 cm Primary segments of leaf lamina 3-6-paired 3-6-paired Secondary segments of leaf lamina 4-6-paired 4-6-paired Involucre 1.5-2 cm 1.2-2.3 cm Outer phyllaries lanceolate, 4-5 mm lanceolate-triangular, 5-6 mm Inner phyllaries oblong-elliptic, 5-7 mm elliptic, 6.5-7.5 mm Disc flowers 4-5 mm 3-4 mm Achenes 2-3 mm 2.5-3mm Corona 0.5-1 mm 0.2-0.3 mm Fig 2. Leaf transverse sections. a) C. melanoloma ssp melanoloma, b) C. melanoloma ssp trapezuntica, c) C. antitaurica, E: epidermis; h: hair; MV: midvein. C. antitaurica (Grierson) Holub ≡ Anthemis antitaurica Grierson, Notes R.B.G. Edinb. 33: 211 (1974). Description: Perennial, rhizomatous plants. Stem generally simple or branched at base, erect or erectascending, 17-33 cm long, 1-2 mm diam, lanate hairy, sparsely leafy at upwards. Steril shoots up to 7 cm. Basal leaves 2-pinnatisect, 17-40 x 5-15 mm, ovate-elliptic, petiole 7-30 (-40) mm; primary segments 3-4 paired, 3-7 x 1-2 mm, elliptic; secondary segments 4-6 paired, 1.5-2.5 x 0.5-1 mm, lanceolate, acute at apex. Median and upper cauline leaves similar, 2-pinnatisect; 8-25 x 4-15 mm, ovate-elliptic; primary segments 3-4 paired, 3-6 x 1-1.5 mm, elliptic; secondary segments 4-6 paired, 1.5-2.5 x 0.5-1 x 1 mm, lanceolate, acute at apex. Peduncles 5-17 cm long, slightly thickened below capitula at fruiting time. Capitula heterogamous, campanulate; 17-30 mm diam (inc. ligules). Phyllaries imbricate, 4-seriate, sparcely lanate, scarious margined, pale or dark brown margined; outer phyllaries 3.54 x 1-1.5 mm, ovate; median phyllaries 6-7 x 2 mm, oblonglanceolate; inner phyllaries 6-7 x 2-2.5 mm, oblonglanceolate, ciliate at apex. Ray flowers 18-22, white, 3 toothed, lamina 11-13 x 4-6 mm, elliptic; ligula tube glabrous, 1.5-2.5 x 1-1.5 mm. Disc flowers tubular, yellow, hermaphrodite, 3-4 x 0.5-1 mm, 5-lobed, shallowly at base; style 2-2.5 mm, bifurcate. Paleae 6-7 x 1-1.5 mm; oblanceolate, acuminate at apex, outer palea straw coloured, inner yellow. Achenes obconical, straw coloured to pale brown, 3.05-3.25 x 1-1.19 mm, 7 ribbed on both surfaces; corona symetric, 0.5-0.7 mm, crenulate. Diagnostic characters: Capitula campanulate, stem lanate. Habitat: Cedrus libani and Abies cilicica subsp. isaurica forest, 1600-1900 m. Flowering and fruiting time: Fl. 6-7, fr. 7-8. Phytogeographic region: Ir-Tur. Element. Distribution: Endemic. Threat Category: CR Anatomy Stem Anatomy: Generally, all the stem sections seem to have the same structure (Fig. 1). In this structure, a thin layer of cuticle covers epidermis, which makes a single layer consisting regularly arranged, rectangular cells. Four or five layers of collenchyma cells are surrounded by epidermis. Under the collenchyma cells, there is a narrow ring of parenchyma cells. Parenchyma cells cover the vascular bundles and the pith. The cambium seems to have 4 or 5 cells in all the stem’s transverse sections. The vascular bundles are all connected to each other and cover the parenchymatic cells in the pith. The main prominent difference between these three stem structures is that the piths of C. melanoloma subsp. melanoloma and C. melanoloma subsp. trapezuntica are hollowed. However, the stem pith of C. antitaurica, seems to be solidly coated with parenchymatic cells. In the stem sections of C. melanoloma subsp. melanoloma, the epidermal layer consists of single celled hairs with the average length of 50.47 ± 7.125 µm. However, in C. antitaurica, the length of single celled hairs were measured as 35.96 ± 1.935 µm. The length/diameter ratios of vascular bundles in stems show that, in C. antitaurica, the vascular bundles have nearly the same length and diameters as 148.75 ± 10.287 µm and 148.23 ± 4.191 µm respectively. In C. melanoloma subsp. melanoloma, the average length of vascular bundles is 140.91 ± 8.550 µm and the average diameter is 106.75 ± 8.149 µm. However, C. melanoloma subsp. trapezuntica has vascular bundles with an average length as 253.36 ± 9.893 µm and the average diameter as 155.76 ± 13.872 µm. Leaf Anatomy: Transverse sections of mature leaves show similar structural patterns (Fig. 2). The general appearance of leaf transverse sections show that, they seem to be isobilateral leaves. Both adaxial and abaxial epidermises have regularly arranged into irregularly shaped cells. Moreover both upper and lower sides of the leaves have stomata and 511 Table 3. The measurements of stomata. Taxa Stoma type C. melanoloma ssp Anomocytic, melanoloma Actinocytic C. melanoloma ssp Anomocytic, trapezuntica Actinocytic C. antitaurica Actinocytic Stoma length Stoma width Length/width ratio Stoma density 22.17 ± 0.769 14.93 ± 0.899 1.50 7.60 ± 2.073 27.39 ± 0.746 17.65 ± 0.989 1.55 3.00 ± 1.632 23.48 ± 0.763 16.36 ± 0.871 1.43 3.00 ± 1.001 Fig 3. Surface views of leaves. a) C. melanoloma ssp melanoloma, b) C. melanoloma ssp trapezuntica, c) C. antitaurica, s: stoma. Fig 4. Transverse sections of roots. a) C. melanoloma ssp melanoloma, b) C. melanoloma ssp trapezuntica, c) C. antitaurica, c: cortex; E: epidermis; pr: parenchymatic ray; sc: sclerenchyma; x: xylem. wide parenchymatic ray cells between the vessels and tracheids. The central parts consist of thin walled, small sized parenchyma cells, surrounded by the portion of xylem tissue. hairs. Maximum hair length of C. melanoloma subsp. melanoloma was measured as 66.32 µm. However, in C. melanoloma subsp. trapezuntica and C. antitaurica these measurements are 149.61 µm and 296.2 µm respectively. The midrib of all these leaves are triangle shaped. The abaxial surfaces of the leaves are shown in Fig. 3, and the stomatal properties with the measurements are given in Table 2. According to Table 2, the types and the length/width ratios of stomata seem to be important differences between the taxa. With the help of these characteristics, C. antitaurica can be separated from the others. However, the density of stomata seems to be the most in C. melanoloma subsp. melanoloma. Root Anatomy: Fig. 4 shows the transverse sections of roots. General anatomical characteristics of the roots are mainly the same with some minor differences. The outermost layer, epidermis, consists of thin walled and single layered parenchymatous cells. The thick walled cells of the cortex are flat and the cortex includes groups of sclerenchymatic cells. The length of the cortex tissue changes according to the taxa. The cortex length was measured as 296.43 ± 21.595 and 276.87 ± 30.521 in C. melanoloma subsp. melanoloma and C. melanoloma subsp. trapezuntica respectively. However, in C. antitaurica, the average length of the cortex is 141.178 ± 25.787. The major part of the roots is reserved by xylem elements. There are thin walled ray parenchyma cells between these xylem elements. The layer numbers of these cells are maximum 2 in both C. melanoloma subsp. melanoloma and C. melanoloma subsp. trapezuntica. However, in C. antitaurica, there are minimum 3 layers of Material and Methods Plant materials C. melanoloma subsp. melanoloma were collected from Kastamonu (A4 Kastamonu: Ilgaz Mts., Küçük Hacet hills, high mountain steppe, 2500 m, 02.08.2005, U.Özbek 1918) (Fig. 5), C. melanoloma subsp. trapezuntica were collected from Rize (A8 Rize: İkizdere, Başköy, Çermaniman hills, high mountain steppe, 2610 m, 29.07.2007, U.Özbek 2687) (Fig. 6) and C. antitaurica were collected from Adana (B6 Adana: Saimbeyli-Tufanbeyli, Bozoğlan Mts., above Obruk upland, around Karlıktepe, edge of Cedrus libani and Abies cilicica subsp. isaurica forest, 1650-1900 m, 20.06.2008, B. Bani 6120) (Fig. 7). A distribution map of C. antitaurica, C. melanoloma subsp. melanoloma and C. melanoloma subsp. trapezuntica is given in Fig. 8. Anatomical methods The investigated materials comprise our collections and deposited specimens at GAZI (Ankara). For anatomical investigations, herbarium samples were firstly boiled in distilled water to be softened. Then, these samples were placed in 70% ethyl alcohol and dehydrated with ethyl 512 Cota melanoloma subsp. melanoloma; Fig 8. melanoloma subsp. trapezuntica; Cota antitaurica. Cota alcohol solutions of increasing strength. The samples were embedded and sectioned with the help of paraffin sectioning method (Johansen, 1944). Safranine was used for staining and Entellan was used for fixing. The slices were observed by ‘Euromex FE 2025’ microscope and photographed by using a ‘Euromex CMEX DC.1300’ camera. Moreover, the leaf surfaces of each taxon were examined by 10 slides. The numbers of stomata were counted from both adaxial and abaxial epidermis and the average measurements were calculated. For measurements of epidermal architecture, 10 slides for each taxon were prepared. Average values were measured from 30 different 234 × 186 μm² samples of 4 leaves of each taxon. Fig 5. General appearance of Cota melanoloma subsp. Melanoloma. Conclusion Consequently, these three taxa can be easily distinguishable with some morphological and the anatomical characteristics. The most accurate diagnostics of them are given as follows; C. antitaurica differs from C. melanoloma by its campanulate capitula (not hemispherical), lanate stem and leaves (not adpressed-pilose) and oblanseolate paleae (not narrowly lanceolate). C. melanoloma subsp. melanoloma, differs from C. melanoloma subsp. trapezuntica by its stem and basal leaves greyish-green (not green), primary segments 3-4paired (not 5-6 paired) and upper stem leaves obovate-oblong (not ovate). Moreover, it was clearly observed from the stem sections that, C. antitaurica seems to have solid pith, while the two subspecies of C. melanoloma have hollowed pith. Furthermore, the types and the length/width ratios of stomata of the leaves and the length of the cortex tissue of the roots seem to separate C. antitaurica from the subspecies of C. melanoloma. Finally, in root anatomy, the layer numbers of parenchymatic ray cells are more the 3 in C. antitaurica, while there are a maximum of 2 layers of parenchymatic cells in C. melanoloma subsp melanoloma and C. melanoloma subsp trapezuntica. Fig 6. General appearance of Cota melanoloma subsp. Trapezuntica. Acknowledgements The authors grateful to Scientific Research Administration of Gazi University (05/2006-17) and TUBITAK (105T353) for their financial supports. Fig 7. General appearance of Cota antitaurica. References Albayrak S, Aksoy A (2012) Evaluation of antioxidant and antimicrobial activities of two endemic anthemis species in Turkey. 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