Bangladesh J. Plant Taxon. 28(2): 295‒309, 2021 (December) DOI: https://doi.org/10.3329/bjpt.v28i2.57128 © 2021 Bangladesh Association of Plant Taxonomists COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS (MILL.) HAYEK (ASTERACEAE) IN TURKEY BURCU YILMAZ ÇITAK*, EMRAH ŞIRIN, HÜSEYIN DURAL AND KUDDISI ERTUĞRUL Department of Biology, Faculty of Science, University of Selçuk, Konya, Turkey Keywords: Morphoanatomy; Cyanus; Numerical analysis; Plant systematics. Abstract The identification of species based on anatomical characters is valuable to investigate their taxonomic status, phylogeny and understand their autoecology. The current study analysed the stem and leaf properties of 20 species of the subgenus Cyanus (Mill.) Hayek in Turkey. The Cyanus species exhibited xeromorphic anatomical features, such as tight palisade parenchyma and induced spongy parenchyma, which indicated adaptations to the arid soil and a forest ecosystem in which they occurred, as well as to high light intensity and precipitation. Anatomical features such as the midrib shape and number of vascularization patterns differed among the species. Similar anatomical characters were analysed for the investigated species. These properties can provide an important database for future studies including the phylogeny within the subgenus Cyanus. Introduction The group Cyanus (Mill.) Hayek of the family Asteraceae was first described as a genus (Miller, 1754), and then later reduced to a section of the genus Centaurea L., a position that was accepted by some taxonomists (Candolle, 1838; Bentham, 1873; Stefanov, and Gheorghiev, 1931; Wagenitz, 1975). However, by some other taxonomists, it was recognized as a subgenus of Centaurea based on molecular evidence (Susanna and Garcia-Jacas, 2009; Hilpold et al., 2014a, 2014b). However, some botanists also recognized Cyanus as a separate genus based on morphological evidence (Soják, 1972; Greuter, 2003, 2008; Hellwig, 2004; Wagenitz, 2006; Olšavská et al., 2009, 2011; Stoyanov, 2016; Negaresh, 2018). In Turkey, the subgenus Cyanus was rearranged as a subgenus (Ertuğrul et al., 2018) as a result of molecular studies (Susanna and Garcia-Jacas, 2009; Hilpold et al., 2014a, b). The florets of the subgenus Cyanus are blue or purplish (rarely creamy or pale pinkish), which are extremely unusual colours in the subtribe Centaureinae (Boršıć et al., 2011). The appendages that are decurrent and not ending with mucro are characteristic for Cyanus, and their involucrum and leaf features are effective in species distinction (Wagenitz, 1975). Anatomical studies can provide useful characters that could aid in the identification of problematic species, and also establish their taxonomical relationships (Metcalfe and Chalk, 1957; Janaćković et al., 2019a,b; Janaćković et al., 2019b). Although there have been many studies on the anatomy of Centaurea species (Metcalfe and Chalk, 1950; Esau, 1977; Metcalfe and Chalk, 1979; Uysal, 1991; Bhattacharya and Johri, 1998; Kaya et al., 2000; Celik et al., 2005; Uysal et al., 2005; Celik et al., 2008; Altundağ and Gürdal, 2009; Aydin et al., 2013; Özcan, 2013; Taşar et al., 2018; Aydın et al., 2019), there are very few studies regarding Cyanus species (Çakırlar et al., 2005; Ozcan et al., 2014; Ozcan, 2018). Examining subgenus Cyanus species to identify their anatomical characteristics, clarifying the systematic value of their anatomical characteristics by way of numerical analysis, and contributing to the systematic position of the examined taxa were the main objectives herein. *Corresponding author, E-mail: burcuyilmaz@selcuk.edu.tr https://doi.org/10.3329/bjpt.v28i2.57128 mailto:burcuyilmaz@selcuk.edu.tr 296 ÇITAK et al. Materials and Methods In this study 20 species of subgenus Cyanus in Turkey have been investigated (Table 1). The voucher specimens of each species, collected from at least five individuals, were stored in the Herbarium of Konya (KNYA). At least five fully-grown leaves and the middle parts of the stems were used in the analysis. Only sections of C. lanigera were taken from the herbarium specimen. All procedures were performed at the Plant Anatomy Laboratory, Department of Biology, Selçuk University, Konya, Turkey, with either herbarium material or material fixed in 70%. Cross- sections of the stem, leaf blade and midrib were obtained using a rotary microtome. The paraffin method was applied and the parts of stem and leaf were treated through alcohol and xylene series. The anatomical tissues were stained with safranin-fast green, which was used as an established method (Johansen, 1940). The vascularization patterns of the stems and leaves, parenchyma pattern, and arrangement of the midrib vascular system were analysed. The microphotographs of the anatomical slides were obtained using a Canon EOS450D digital camera (Ota City, Tokyo, Japan) attached to Leica 1000 DM light microscope (Wetzlar, Germany). A total of nine anatomical characters (six quantitative and three qualitative) were measured (µm) or scored using Kameram v.21 analysis system software (Argenit, Istanbul, Turkey) (Tables 2 and 3). A data matrix was set using the recorded qualitative and quantitative characters. Based on the anatomical characters, the coefficients of correlation among the 20 species were determined and these species were grouped using the clustering analysis method (unweighted pair group method with arithmetic mean (UPGMA), dissimilarity, standardized variables). The clustering analysis was based on Gower’s (1971) general coefficient similarity (Sneath and Sokal, 1973), which was used directly with a mixture of character types (binary, qualitative, and quantitative. Untransformed, centered, and unstandardized data were used to create a covariance matrix. MVSP 3.22 software (Kovach Computing Services, Anglesey, Wales) was used for all of the computations. Table 1. Locality information of the investigated taxa of Cyanus subgenus. Acronym Collection number Species Locality C1 **EŞ-574-MŞ Centaurea reuteriana Boiss. var. reuteriana C2 Muğla: Köyceğiz, Sandras mountain, 1763 m, 29.06.2015 C2* **EŞ-554-MŞ C. reuteriana Boiss. var. phrygia Bornm. B3 Afyon: Sultandağları, 1850 m, 21.05.2015 EŞ-582-MY C. reuteriana Boiss. var. phrygia Bornm. A4 Karabük: Keltepe, step, 1800 m, 09.07.2015 EŞ-562-MŞ C. reuteriana Boiss. var. phrygia Bornm. C3 Isparta: Davraz Dağı, stony places, 1800 m, 16.06.2015 C3* **EŞ-659-MŞ C. lanigera DC. B5 Aksaray: Hasan mountain, 1979 m, 29.06.2016 C4 **EŞ-668-MŞ C. nigrofimbria (K. Koch) Sosn. A8 Trabzon: Çaykara, Soğanlı mountain, 2300 m, 12.07.2016 EŞ-595-AG,HG C. nigrofimbria (K. Koch) Sosn. A8 Trabzon: Çaykara: North of Soğanlı Mountain, steppe, 2300 m, 12.07.2016 C5 **EŞ-642-MŞ C. woronowii A9 Artvin: Ardanuç, 2 km to Rabat Church, 1273 m, 11.06.2016 C6* **EŞ-656-MŞ C. eflanensis (Kaya & Bancheva) Şirin & Ertuğrul A4 Karabük: Bartın-Safranbolu road, 1078 m, 15.06.2016 COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 297 Table 1 Contd. Acronym Collection number Species Locality C7 **EŞ-605-MŞ C. thirkei Sch. Bip. B1 Manisa: Spil Mountain, Merdivencik, P. Nigra openings, 951 m, 30.04.2016 C8 EŞ-591-MŞ C. cheiranthifolia Willd. var. cheiranthifolia A9 Ardahan: Çıldır-Aktaş road, 2100 m, 14.08.2015 **EŞ-672-MŞ C. cheiranthifolia Willd. var. cheiranthifolia A9 Ardahan: Hanak, Aydere Village, step, 2326 m, 14.07.2016. C9 **EŞ-643-MŞ C. cheiranthifolia Willd. var. purpurascens (DC.) Wagenitz A9 Ardahan: Değirmenli village, 2287 m, 11.06.2016 C10 **EŞ-622-MŞ C. bourgaei Boiss. C4 İçel: Mut, 1561 m, 15.05.2016 EŞ-572-MŞ C. bourgaei Boiss. C3 Antalya: Elmalı, Kızlar Sivrisi dağcılar şenlik alanı, road sides, 1900 m, 29.06.2015 C11 EŞ-577-MŞ C. pichleri Boiss. subsp. pichleri A5 Amasya: Merzifon, 1502 m, 07.07.2015 EŞ-567-MŞ C. pichleri Boiss. subsp. pichleri C2 Antalya: Korkuteli, Kırkpınar Plateau, 1600 m, 17.06.2015 **EŞ-535-MŞ C. pichleri Boiss. subsp. pichleri C3 Isparta: Davraz Mountain, steppe, 1600 m, 12.05.2015 C12* **EŞ-635-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz C5 Niğde: Demirkazık Mountain, 1849 m, 02.06.2016 EŞ-576-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz B2 Kütahya: Murat Mountain, near summit, Pinus nigra openings, 1850 m, 01.07.2015 EŞ-613-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz B3 Eskişehir: Kütahya - Eskişehir road, steppe, 820 m, 03.05.2016 EŞ-616-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz B5 Kayseri: Yahyalı - Aladağ road, red- brown soils, 1727 m, 05.05.2016 EŞ-568-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz C4 Konya: Konya-Beyşehir road, turnout of Ayışığı At Çiftliği, steppe, 1270 m, 27.06.2015 EŞ-623-MŞ C. pichleri Boiss. subsp. extrarosularis (Hayek & Siehe) Wagenitz C5Niğde: Niğde/Adana; Aladağ, Narpuz Boğazı, steppe, 2224 m, 16.05.2016 C13 **EŞ-583-MŞ C. triumfettii subsp. axillaris (Čelak.) Stef. & T. Georgiev B2 Kütahya: Akdağ, 1610 m, 14.05.2016 EŞ-579-MŞ C. triumfettii subsp. axillaris (Čelak.) Stef. & T. Georgiev A4 Bolu: Mengen, 1 km to Arak Village, P. nigra forest, 812 m, 08.07.2015 EŞ-548-MŞ C. triumfettii subsp. axillaris (Čelak.) Stef. & T. Georgiev A4 Çankırı: Step, 750 m, 20.05.2015 C14 **EŞ-645-MŞ C. huetii Boiss. A9 Ardahan: Çıldır, 1 km to Gökbelen Village, sides of fields, 1991 m, 12.06.2016 EŞ-666-MŞ C. huetii Boiss. B7 Sivas: Divriği, Göl Mountain, summit, steppe, 1926 m, 01.07.2016 C15* **EŞ-618-MŞ C. mathiolifolia Boiss. C2 Denizli: Honaz Mountain, 1829 m, 12.05.2016 EŞ-549-MŞ C. mathiolifolia Boiss. B3 Afyon: Sultandağı, Dereçine-Büyükyayla road, road sides, 1350 m, 21.05.2015 EŞ-599-MŞ C. mathiolifolia Boiss. C2 Burdur: from Tefenni to Korkuteli, stony places, 1351 m, 28.04.2016 298 ÇITAK et al. Table 1 Contd. Acronym Collection number Species Locality EŞ-561a-MŞ C. mathiolifolia Boiss. C2 Isparta: Davraz Mountain, stony places, 1800 m, 16.06.2015 EŞ-564-MŞ C. mathiolifolia Boiss. C2 Isparta: Davraz Mountain, around ski resort, steppe, 1600 m, 16.06.2015 C16* **EŞ-614-MŞ C. germanicopolitana Bornm. A4 Çankırı: Eldivan, before 3 km to Oğlaklı Village, road sides, 854 m, 04.05.2016 C17 **EŞ-547-MŞ C. depressa Bieb. A4 Çankırı: Hacı Ali Dinlenme Tesisi, road sides, 700 m, 20.05.2015 EŞ-637-MŞ C. depressa Bieb. A5 Amasya: Akdağ, near summit, road sides, 1003 m, 09.06.2016 EŞ-545-MŞ C. depressa Bieb. B3 Afyon: Sinanpaşa, Düzağaç Town, road sides, 1150 m, 15.05.2015 EŞ-557-MŞ C. depressa Bieb. B4 Ankara: Hacı Hasan Village, ‘Sevgi çiçeği’ protected area, fallow places, 950 m, 28.05.2015 EŞ-541-MŞ C. depressa Bieb. C2 Denizli: 950 m, 13.05.2015 C18 **EŞ-598-MŞ C. pinardii Boiss. C3 Burdur: İlyas village, 870 m, 28.04.2016 EŞ-603-MŞ C. pinardii Boiss. B2 Uşak: Southwest of Uşak, near Karabol stream, steppe, 580 m, 29.04.2016 EŞ-602-MŞ C. pinardii Boiss. B3 Afyon: Dazkırı, Sarıkavak Village, edge of fields, 864 m, 29.04.2016 C19* **EŞ-560-MŞ C. tchihatcheffii Fisch. & C. A. Mey. B3 Afyon: Dazkırı, 864 m, 15.06.2015 C20 **EŞ-604-MŞ C. cyanus L. B1 Manisa: Spil Mountain, 647 m, 30.04.2016 *Endemic taxa, ** Selected taxa. Table 2. Anatomical traits used in statical analysis of Cyanus subgenus. Vegetative organ Acronyms Definition of anatomical traits Stem A1 Number of vessels A2 Diameter of pith cells A3 Width of collenchyma Leaf A4 Sclerenchyma cells on phloem A5 Mesophyll type A6 Mesophyll thickness A7 Length of palisade cells A8 Width of palisade cells A9 Width of spongy parenchyma/mesophyll A10 Row of palisade tissue A11 Width of spongy parenchyma A12 The shape of midrib (triangular: 0, linear: 1, oval:2, v-shaped:3) COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 299 Table 3. The anatomical measurements and observations of Cyanus subgenus. Species/Anatomical characters A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 C. reuteriana var. reuteriana 18 93.51 146.43 1 0 690.37 67.39 18.45 0.12 3 88.52 2 C. reuteriana var. phrygia 20 82.19 57.74 1 0 230.76 40.85 12.90 0.46 3 108.44 1 C. lanigera 18 38.25 128.43 1 0 152.79 18.56 7.04 0.27 2 42.41 2 C. nigrofimbria 19 80.71 79.80 1 1 203.39 31.80 20.64 0.31 2 63.35 0 C. woronowii 20 61.58 125.14 0 1 105.23 23.35 13.36 0.28 2 29.71 0 C. eflanensis 16 76.53 103.4 1 0 227.13 32.04 16.55 0.28 2 65.81 2 C. thirkei 20 40.58 97.09 0 0 209.63 25.34 11.07 0.21 3 44.63 0 C. cheiranthifolia var. cheiranthifolia 26 114.73 175.63 1 0 202.09 34.55 16.22 0.28 2 58.53 0 C. cheiranthifolia var. purpurascens 24 82.23 88.72 1 0 421.05 60.37 24.16 0.24 2 103.02 1 C. bourgaei 13 50.05 62.46 1 0 262.81 48.92 14.49 0.17 2 45.41 2 C. pichleri subsp. pichleri 19 57.64 116.33 1 0 165.38 26.22 10.82 0.27 3 44.76 0 C. pichleri subsp. extrarosularis 16 65.31 111.99 1 0 231.94 29.23 14.55 0.22 2 52.53 3 C. triumfettii subsp. axillaris x x x x 0 68.86 21.83 7.89 0.50 2 34.71 2 C. huetii 21 73.64 107.3 1 0 269.87 41.89 15.21 0.21 2 58.18 0 C. mathiolifolia 14 55.93 107.9 1 0 257.08 40.01 14.26 0.21 2 54.83 0 C. germanicopolitana 16 95.15 162.33 1 0 268.47 40.42 21.85 0.12 3 33.87 3 C. depressa 16 101.94 130.21 1 0 230.03 39.81 13.55 0.15 2 34.97 0 C. pinardii 17 68.20 82.46 1 0 354.67 70.98 25.33 0.20 2 72.62 0 C. tchihatcheffii 21 50.79 56.45 0 0 236.21 53.20 18.8 0.14 2 34.83 2 C. cyanus L. 16 81.97 145.26 1 0 217.05 47.30 17.80 0.17 2 38.25 3 Results and Discussion Stem anatomy The stem cross-section varied in size among the species (Table 3). In most, it was oval-shaped with leaf blade parts (Figs 1-5). In the examined species, the stems were densely covered by unicellular trichomes (Figs 1-5). Collenchyma was present at protrusion areas in stem cross- sections. Cortex parenchyma had two types of cells; the first was cylindrical-shaped with abundant chlorophyll, the second was oval-shaped with less chlorophyll. Sclerenchyma was placed above the phloem in the cortical parenchyma of most of the species. In all of the species examined, vascular bundles were collateral type, forming either continuous or discontinuous rings, arcs, or rings with arcs inside (Figs 1-5). Sclerenchymatic caps were found around the vascular bundles in the examined species, except in C. tchihatcheffii, C. woronowii, and C. thirkei (Figs 1,3,4). Vascular bundles were observed near the abaxial surface of the stems and varied in number among the species. 300 ÇITAK et al. Fig. 1. The stem anatomical photographs of Cyanus subgenus. (a) general view, (b) close view 1. C. reuteriana var. reuteriana 2. C. reuteriana var. phyrgia, 3. C. lanigera, 4. C. nigrofimbria. ep:epidermis, cl:chlorenchyma, co:collenchyma, sc:sclerenchyma, ph:phloem, x:xylem, pi:pith Fig. 2. The stem anatomical photographs of Cyanus subgenus. (a) general view, (b) close view 1. C. woronowii, 2. C. eflanensis, 3. C. thirkei, 4. C. bourgaei. ep:epidermis, cl:chlorenchyma, co:collenchyma, sc:sclerenchyma, ph:phloem, x:xylem, pi:pith COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 301 Fig. 3. The stem anatomical photographs of Cyanus subgenus. (a) general view, (b) close view. 1. C. cheiranthifolia var. cheiranthifolia, 2. C. cheiranthifolia var. purpurascens 3. C. pichleri subsp. pichleri 4. C. pichleri subsp. extrarosularis. ep:epidermis, cl:chlorenchyma, co:collenchyma, sc:sclerenchyma, ph:phloem, x:xylem, pi: pith Fig. 4. The stem anatomical photographs of Cyanus subgenus. (a) general view, (b) close view. 1. C. triumfetti subsp. axillaris, 2. C. huetii, 3. C. mathiolifolia, 4. C. germanicopolitana. ep:epidermis, cl:chlorenchyma, co:collenchyma, sc:sclerenchyma, ph:phloem, x:xylem, pi:pith 302 ÇITAK et al. Fig. 5. The stem anatomical photographs of Cyanus subgenus. (a) general view, (b) close view. 1. C. depressa, 2. C. pinardii, 3. C. tchihatcheffii, 4. C. cyanus. ep:epidermis, cl:chlorenchyma, co:collenchyma, sc:sclerenchyma, ph:phloem, x:xylem, pi:pith Fig. 6. The leaf anatomical photographs of Cyanus subgenus. (a) general view of midrib, (b) lamina view. 1. C. reuteriana var. reuteriana, 2. C. reuteriana var. phyrgia, 3. C. lanigera, 4. C. nigrofimbria. ue: upper epidermis, le:lower epidermis, pp:palisade parenchyma, sp: spongy parenchyma, vb:vascular bundle COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 303 Leaf blade and Midrib The shape of the epidermal cells from the adaxial face was either rectangular or oval (Figs 6- 10). The epidermis was uniseriate in all of the species. Collenchyma occurred under the midrib epidermis in all of the species. The mesophyll was equifacial in 17 species with well-defined palisade and reduced spongy parenchyma, bifacial in three species: C. woronowii, C. nigrofimbria, and C. reuteriana var. phrygia (Figs 6 and 7). Palisade parenchyma was composed of two to three layers of elongated cylindrical cells (Table 3). Cells of spongy parenchyma were predominantly cuboid, variably compressed according to the species. Only three species were characterized by loose spongy parenchyma, with larger intercellular spaces. Stomata were observed at the level of the epidermal line (Figs 6-10) in the species. Midrib shape in cross-sectional view also varied among the species (Figs 6-10). On the abaxial side, it was convex in all of the species. On the adaxial side, convex (Figs 6,7,9), concave (Figs 6, 8,10), and planar midribs were found (Figs 6,8,9,10). The midrib vascular system was organized into arches that were formed by collateral bundles arranged in groups of 1 or 3. Statistical analysis The dendrogram derived from the cluster analysis using the UPGMA based on the nine anatomical variables of the 20 Cyanus species is presented in Fig. 11, in which the similarities among the examined species were presented. The dendrogram revealed two main groups: Groups A and B. Group A (with 58% similarity) comprised one perennial plant, C. reuteriana var. phrygia. Group B (with 62% similarity) comprised the remaining 19 taxa of the annuals and perennials of Turkish Cyanus. Group B consisted of two main clusters, which were described further as Clusters B1 and B2. Cluster B1 included C. germanicopolitana and C. reuteriana var. reuteriana (with 72% similarity). Group C consisted of only C. triumfetti subsp. axillaris. Cluster D included two main clusters: Clusters D1 and D2. Cluster D1 included two species: C. cheirantifolia var. purpurascens and C. pinardii. D2 contained two main clusters: Groups E and F. Group E consisted of two species C. nigrofimbria and C. woronowii (with 84% similarity). Cluster F included 10 species in two different subclusters: F1 and F2. Sub-cluster F1 comprised two further subclusters: Groups G and H. Group G contained only C. lanigera. Group H included two subclusters: Group H1 and H2. Group H1 contained C. eflanensis, C. pichleri subsp. extrarosularis, and C. cyanus (with 88% similarity) under two different subclusters. Group F2 included two subclusters, namely Subclusters F3 and F4. Subcluster F3 contained only C. cheiranthifolia var. cheiranthifolia. Subcluster F4 comprised C. matthiolifolia and C. huetii and C. depressa under two different smaller subclusters (with 93% similarity). The anatomical findings on the subgenus Cyanus herein supported the results of Sirin et al. (2017), Sirin et al. (2019), and Citak et al. (2021), from the point of view of the karyological, palynomorphological, and achene morphological data. The stem anatomical characters were determined to vary among the species; however, patterns allowing distinctions among species were not detected. Çakırlar et al. (2005) presented differences in the vascularization patterns of the stems of C. tchihatcheffii and C. depressa, including variations in the vascular bundle type, and number and position of accessory bundles (Fig. 5). The shape of the stem cross-sections was rounded, semi-rounded, rectangle, circular, or irregular in the family Asteraceae (Celik et al., 2005, 2008; Aydin et al., 2013; Aydin et al., 2019) and also in the subgenus Cyanus (Çakırlar et al., 2005, Özcan et al., 2014, Özcan, 2018). The position and the number of layers of the collenchyma tissue are important in plant communities (Metcalfe and Chalk, 1950; Özörgücü et al., 1991; Lersten and Curtis, 1997; Makbul et al., 2008, Aydin et al., 2013, Özcan et al., 2014). In current study, it was observed that different rows of collenchymatic tissue (3–10 rows) were located under epidermis. The arrangement of the vascular 304 ÇITAK et al. Fig. 7. The leaf anatomical photographs of Cyanus subgenus. (a) general view of midrib, (b) lamina view. 1. C. woronowii, 2. C. eflanensis, 3. C.thirkei, 4. C. bourgaei. ue: upper epidermis, le:lower epidermis, pp:palisade parenchyma, sp: spongy parenchyma, vb:vascular bundle Fig. 8. The leaf anatomical photographs of Cyanus subgenus. (a) general view of midrib, (b) lamina view. 1. C. cheiranthifolia var. cheiranthifolia, 2. C. cheiranthifolia var. purpurascens, 3. C. pichleri subsp. pichleri, 4. C. pichleri subsp. extrarosularis. ue: upper epidermis, le:lower epidermis, pp:palisade parenchyma, sp: spongy parenchyma, vb:vascular bundle COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 305 Fig. 9. The leaf anatomical photographs of Cyanus subgenus. (a) general view of midrib, (b) lamina view. 1. C. triumfetti subsp. axillaris, 2. C. huetii, 3. C. mathiolifolia, 4. C. germanicopolitana. ue: upper epidermis, le:lower epidermis, pp:palisade parenchyma, sp: spongy parenchyma, vb:vascular bundle. Fig. 10. The leaf anatomical photographs of Cyanus subgenus. (a) general view of midrib, (b) lamina view. 1. C. depressa, 2. C. pinardii, 3. C. tchihatcheffii, 4. C. cyanus. ue: upper epidermis, le:lower epidermis, pp:palisade parenchyma, sp: spongy parenchyma, vb:vascular bundle. 306 ÇITAK et al. Fig. 11. The combined dendrogram according to anatomical characters of Cyanus subgenus bundles was found to be in an arc shape in all of the studied species of the subgenus Cyanus by Çakırlar et al. (2005), Özcan et al. (2014), Özcan (2018) prior to this study. In the current study, this phenomenon was confirmed. The vascular bundles in the stem were generally arranged as two rings, and especially, those found under the collenchymatic area were bigger than the other vascular bundles. Celik et al. (2005, 2008) and Kaya et al. (2010) reported that the vascular bundles were scattered in a circular manner as one ring in the stem of some species of Centaurea. Clustered sclerenchymatic fibers were located on the upper sides of the vascular bundles in the examined taxa, except in C. tchihatcheffii, C. woronowii, and C. thirkei (Figs 2 and 5). Additionally, a chlorenchymatous tissue below the epidermis was observed in the stem cortex in the examined species. This kind of tissue was reported for the genus Centaurea in some previous studies performed by Uysal et al. (2005), Celik et al. (2005, 2008), and Kaya et al. (2010), and the subgenus Cyanus by Çakırlar et al. (2005), Özcan et al. (2014), and Özcan (2018). The stem cortex usually consisted of parenchymatic oval cells with thin walls in all of the examined taxa, but it varied from two to four rows. It was found that the mesophyll of 17 species were equifacially oriented, with well-developed palisade parenchyma and reduced spongy parenchyma, and in contrast three species were dorsiventrally oriented. According to the observations of Özcan et al. (2014), the mesophyll of C. cheiranthifolia var. purpurascens and C. woronowii were equifacial. The present study confirmed that this was true of the mesophyll of C. cheiranthifolia var. purpurascens (Fig. 8); however, it was not true for C. woronowii (Fig. 7). In the present study, dorsiventral mesophyll was only observed in C. woronowii (Fig. 7), C. nigrofimbria (Fig. 6), and C. reuteriana var. phrygia (Fig. 6) which grow in moist areas, while equifacial leaves were observed in the other investigated taxa, which mainly grow in the dry habitats of the Irano-Turanian phytogeographic region of Turkey. According to Yentür (2003), equifacial leaves were generally characteristic of xerophytic plants, which was in accordance with the observations made herein. The varying midrib shape in Cyanus species can really contribute the systematics of this subgenus. Özcan et al. (2014) and Özcan (2018) reported that there were three vascular bundles in the midrib of C. cheiranthifolia var. purpurascens and C. nigrofimbria, and six in C. woronowii, which was in agreement with the results of the current study. COMPARATIVE ANATOMICAL CHARACTERISTICS OF THE SUBGENUS CYANUS 307 The UPGMA dendrogram derived from the anatomical traits of the stem and leaf discriminated the species of the subgenus Cyanus (Fig. 11). The positions of Cyanus species and their similarities reflected in the clusters were found to be partially agreeable with the previous classification of the subgenus based on the morphological data. C. nigrofimbria and C. woronowii were in the same clade according to their mesophyll type. The subspecies and varieties were close to each other in the dendrogram; however, their different positions in the different clusters can be explained by different ecological habitats, such as dry areas or forest ecosystems. In conclusion, the number of vascularization patterns in stems, mesophyll type, and midrib shape were the most valuable variables for distinguishing the species of the subgenus Cyanus. 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