Botany Letters ISSN: 2381-8107 (Print) 2381-8115 (Online) Journal homepage: http://www.tandfonline.com/loi/tabg21 Pollen morphology of the genus Cota J.Gay (Asteraceae) in Turkey Mehmet Ufuk Özbek, Funda Özbek, Birol Başer, Evren Cabi & Mecit Vural To cite this article: Mehmet Ufuk Özbek, Funda Özbek, Birol Başer, Evren Cabi & Mecit Vural (2016): Pollen morphology of the genus Cota J.Gay (Asteraceae) in Turkey, Botany Letters, DOI: 10.1080/23818107.2016.1225266 To link to this article: http://dx.doi.org/10.1080/23818107.2016.1225266 Published online: 26 Sep 2016. Submit your article to this journal Article views: 20 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tabg21 Download by: [Namik Kemal Universitesi ] Date: 13 October 2016, At: 03:09 Botany Letters, 2016 http://dx.doi.org/10.1080/23818107.2016.1225266 Pollen morphology of the genus Cota J.Gay (Asteraceae) in Turkey Mehmet Ufuk Özbeka, Funda Özbeka, Birol Başerb, Evren Cabic and Mecit Vurala a Faculty of science, Department of Biology, Gazi University, ankara, turkey; bFaculty of arts and science, Department of Biology, Bitlis eren University, Bitlis, turkey; cFaculty of arts and science, Department of Biology, namık Kemal University, tekirdağ, turkey ABSTRACT ARTICLE HISTORY Pollen morphology of 22 Cota taxa naturally distributed in Turkey was investigated to describe their pollen features and to evaluate the diagnostic value of pollen characters for systematic purposes using light microscopy and scanning electron microscopy. Pollen grains of Cota are radially symmetrical and isopolar. Their shape is oblate-spheroidal with the polar axes 21.6– 34.56 μm and the equatorial axes 23.04–33.6 μm. The pollens are trizonocolporate. The outline is elliptic in equatorial view and triangular in polar view; amb intersemiangular. Exine sculpturing is echinate. Inter-spinal region exhibits perforate and microreticulate–perforate ornamentations. Numerical analysis revealed that dimensions of the pollen grains and surface ornamentation are the most reliable characters for delimiting the taxa within the genus. received 6 april 2016 accepted 26 July 2016 Introduction Cota J.Gay, which belongs to tribe Anthemideae (Asteraceae), is represented by 63 taxa in the world and is mainly distributed in Europe (excluding northern Europe), North Africa, Caucasia and Central Asia (Davis and Hedge 1975; Oberprieler, Vogt, and Watson 2007). In Turkey, the genus consists of 22 taxa, nine of which are endemic. Taxa of the genus are mainly distributed and common in the Mediterranean and Irano-Turanian phytogeographic regions of Turkey (Özbek, Vural, and Daşkın 2011; Özbek 2012). Cota was earlier classiied as a section in the genus Anthemis L. in Flora of Turkey (Grierson and Yavin 1975). In this account, Anthemis s.l. was divided into three sections namely Sect. Anthemis (29 species), Sect. Maruta (Cass.) Griseb (six species) and Sect. Cota (J.Gay) Rupr. (15 species). Ater the generic and infrageneric concepts of Anthemis were changed, Anthemis sect. Cota was accepted as a genus, Cota J.Gay (Oberprieler 2001; Greuter, Oberprieler, and Vogt 2003; Lo Presti, Oppolzer, and Oberprieler 2010). he genus Cota morphologically resembles Anthemis s.s., but Cota difers from Anthemis by obconical, dorsiventrally lattened achenes, with prominent lateral ribs that are smooth or with 3–10 ribs on each side. he achenes of Anthemis s.s. are obovoid to obconical, circular or quadrangular in cross-section, smooth or with c. 10 tubercular ribs. Some Cota species have economic importance and are used for various purposes, such as drugs, foods and CONTACT Mehmet Ufuk Özbek © 2016 société botanique de France mufukozbek@gmail.com KEYWORDS asteraceae; Cota; light microscopy; pollen morphology; scanning electron microscopy; turkey dyes (Ghafoor 2010; Öztürk et al. 2013). In addition, the lowers of the genus Cota are used as antiseptic and healing herbs. he main components are natural lavonoids and essential oils, which are widely used as antiinlammatory, antibacterial, antispasmodic and sedative agents (Reddish 1929). Several cytological studies conducted in some Cota species have concentrated on the chromosome count. Reports of chromosome numbers for Anthemis melanoloma Trautv. ssp. trapezuntica Grierson, Anthemis tinctoria L. var. pallida DC., A. tinctoria L. var. tinctoria and Anthemis triumfetti (L.)DC. (İnceer and Hayırlıoğlu Ayaz 2007), Anthemis altissima L. var. altissima (Kuzmanov, Andreev, and Georgieva 1977; Stephanov 1982; Oberprieler 1998), Anthemis coelopoda Boiss. var. bourgaei Boiss. (Chehregani and Mehanfar 2008) and A. tinctoria (Van Loon 1982; Magulaev 1992; Oberprieler 1998) were recorded as 2n = 18. Özbek (2010) also reported and conirmed diploid chromosome numbers as 2n = 18 for all Cota species. Most molecular phylogenetic studies have focused on tribal overviews (Watson, Evans, and Boluarte 2000; Oberprieler, Vogt, and Watson 2007), geographical (Francisco-Ortega et al. 1997; Oberprieler and Vogt 2000; Oberprieler 2004a, 2004b, 2005; Himmelreich et al. 2008) and taxonomic (Oberprieler 2001; Vallés et al. 2003; Guo, Ehrendorfer, and Samuel 2004) subgroups of the tribe. Lo Presti, Oppolzer, and Oberprieler (2010) studied the molecular phylogeny including c. 75% of all known species belonging to 2 M. U. ÖzBEk ET al. the closely related genera Anthemis and Cota based on nucleotide sequences from two plastid regions and one nuclear marker (internal transcribed spacer) with a multivariate analysis of 25 micromorphological and anatomical characters. hey reported that molecular and morphological data both support a sister-group relationship between four perennial species – namely, Anthemis calcarea Sosn., Anthemis fruticulosa M.Bieb., Anthemis marschalliana Willd. and Anthemis trotzkiana Claus – and the main clades of Cota and Anthemis. Pollen morphological properties have been determined as distinctive characters for the family Asteraceae (Wagenitz 1955; Stix 1960; Erdtman 1969; Skvarla and Larson 1965; Blackmore 1982, 1990; Pınar and İnceoğlu 1996; Punt and Hoen 2009). Wodehouse (1926, 1935) led detailed studies of Anthemideae pollen grains on Anthemis cotula L. and Chamaemelum nobile (L.) All. Further palynological studies were carried out in Anthemideae by Čigurjaeva and Tereškova (1983), De Leonardis et al. (1991); Martin et al. (2003) and Vezey et al. (1994). Oberprieler (1998) studied the pollen morphology of 104 samples belonging to 35 North African Anthemis species. He found that the dimensions of pollen grains show variation between species. Benedí i González (1987) reported that annual specimens of Anthemis subgenus Anthemis have smaller pollen grains than the perennial specimens. Exine ornamentation is another diagnostic character for the classiication of the Anthemideae. Stix (1960) described two exine patterns in the Anthemideae: Anthemis type with spines and Artemisia type without spines. here are only a few reports of Cota taxa based on palynology. Dauti et al. (2014) investigated the pollen morphology of the eight species belonging to the genus Anthemis distributed in Albania. he aims of this study are to provide detailed pollen morphological characteristics of 22 taxa belonging to the genus Cota using light microscopy and scanning electron microscopy and to clarify the usefulness of these features in terms of systematic implications. Table 1. the collection data of investigated Cota specimens. C1 C2 Taxa C. tinctoria (L.) J.Gay ex Guss. var. tinctoria C4 C. tinctoria (L.) J.Gay ex Guss. var. pallida (DC.) U.Özbek & Vural C. tinctoria (L.) J.Gay ex Guss. var. discoidea (all.) U.Özbek & Vural C. euxina (Boiss.) U.Özbek & Vural C5 C. virescens (Bornm.) U.Özbek & Vural* C6 C. triumfettii (L.) J.Gay ex Guss. C7 C. melanoloma (trautv.) Holub subsp. melanoloma C8 C9 C. melanoloma (trautv.) Holub subsp. trapezuntica (Grierson) oberpr. & Greuter* C. antitaurica (Grierson) Holub* C10 C. oxylepis Boiss.* C11 C. hamzaoglui U.Özbek & Vural* C12 C. fulvida (Grierson) Holub* C13 C14 C. altissima (L.) J.Gay C. coelopoda (Boiss.) Boiss. var. coelopoda C15 C17 C. coelopoda (Boiss.) Boiss. var. longiloba (Grierson) U.Özbek & Vural C. coelopoda (Boiss.) Boiss. var. bourgaei (Boiss.) U.Özbek & Vural C. austriaca (Jacq.) sch. Bip. C18 C. dipsacea (Bornm.) oberpr. & Greuter* C19 C. palaestina reut. ex Unger & Kotschy C20 C. wiedemanniana (Fisch. & C.a.Mey.) Holub C21 C. halophila (Boiss. & Balansa) oberpr. & Greuter* C22 C. pestalozzae Boiss.* C3 C16 locality C3 Burdur: Gölhisar to altınyayla 12 km, inner valley, rocky slopes, 665 m, 30.06.2005 B3 eskişehir:35 km from Kütahya to eskişehir, Quercus forest, 960 m, 02.06.2007 C5 adana: Pozantı, against Horoz village, rocky and stony slopes, 1290 m, 22.06.2007 a2 İstanbul: Karaburun, against Horoz village, rocky and stony slopes, 0–5 m, 26.08.2007 C6 osmaniye: Hasanbeyli, Gökçedağ road, around tV station, Quercus forest openings, 1280 m, 06.07.2008 a7 Giresun: tamdere to Giresun, aksu village, around Karagöl, subalpine meadows, 2000 m, 27.07.2007 a8: Bayburt: Bayburt to aşkale, Kop Mountain, high mountain meadows, 2332 m, 28.07.2007 a7 trabzon: soğanlı pass to Çaykara 3. km, high mountain meadows, 2275 m, 28.7.2007 B6 adana: saimbeyli to tufanbeyli, above obruk upland, rocky slopes, 1800–1850 m, 05.07.2008 C5 niğde: Ulukışla, Maden village, around Bulgar Maden, stony slopes and screes, 2100 m, 25.07.2007 a2 Bursa: Uludağ, above hotels, between cable cars and near an old volfram mine, subalpine scrubs, 2050–2100 m, 31.07.2009 C3 Isparta: yenişarbademli, above Melikler upland, Pinus nigra forest, 1680–1800 m, 24.08.2012 a1 Çanakkale: truva, roadside, 50 m, 04.06.2007 B1 Manisa: 6 km from salihli to Kula, Pinus plantation area, 98 m, 02.05.2007 C5 İçel: Çamlıyayla (namrun), around namrun castle, roadside, 20.06.2007 B1 Manisa: sarıgöl to Buldan 5. km, roadside, 640 m, 03.06.2007 C5 niğde: between Çiftehan to alihoca village, rocky slopes, 1300 m, 22.06.2007 B1 İzmir: armutlu to Bayramlı, Pinus brutia forest, 410 m, 04.06.2007 C6 antakya: around st Peter (Pierre) Church, 115 m, 08.06.2008 a3 ankara: Beypazarı, sarıyer Dam, meadows, 470 m, 16.05.2005 C6 antakya: İskenderun, arsuz, Kale village, sands and sandy soil near seashore, 0–5 m, 07.05.2007 C4 Konya: 30 km from seydişehir to akseki, alacabel pass, rocky slopes, 1825 m, 20.05.2007 Voucher and specimen code U. Özbek 1898 (GaZI) U. Özbek 2300 (GaZI) U. Özbek 2432 (GaZI) U. Özbek 2695 (GaZI) U. Özbek 2753 (GaZI) U. Özbek 2661 (GaZI) U. Özbek 2666 (GaZI) U.Özbek 2679 (GaZI) U. Özbek 2752 (GaZI) U. Özbek 2655 (GaZI) U. Özbek 2812 & M. Vural (GaZI) U. Özbek 2852 (GaZI) U. Özbek 2332 (GaZI) U. Özbek 2216 (GaZI) U. Özbek 2397 (GaZI) U. Özbek 2311 (GaZI) U. Özbek 2445 (GaZI) U. Özbek 2323 (GaZI) U. Özbek 2715 (GaZI) U. Özbek 1824 (GaZI) U. Özbek 2271 (GaZI) U. Özbek 2290 (GaZI) BoTaNy lETTErs 3 Figure 1. simpson and roe test for the studied taxa of Cota. (a) Polar axes (P). (B) equatorial axes (e). Table 2. eight palynological characters to distinguish the 22 taxa of the genus Cota. Taxa /Characters C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 P/E 0.92 0.92 0.92 0.91 0.94 0.97 0.94 0.95 0.95 0.95 0.93 0.94 0.97 0.94 0.94 0.94 0.93 0.98 0.92 0.92 0.94 0.93 Exine 2.68 2.49 2.57 2.74 2.76 2.89 2.87 2.89 2.82 2.92 2.84 2.81 2.91 2.82 2.83 2.82 2.77 2.62 2.81 2.4 2.73 2.73 Intine 0.82 0.85 0.83 0.86 0.86 0.8 0.83 0.84 0.79 0.74 0.82 0.75 0.78 0.84 0.8 0.81 0.91 0.78 0.78 0.74 0.84 0.77 Colpus length 14.57 14.66 14.74 15.52 13.93 14.48 16.78 16.68 16.8 15.2 14.71 14.43 15.51 16.56 16.14 16.28 15.76 14.91 16.14 15.42 13.85 14.44 Colpus width 4.08 4.36 4.03 4.42 3.88 4.12 3.84 3.82 4.56 4.9 5.19 5.02 3.79 4.12 4.01 4.26 4.03 3.87 3.91 3.55 3.79 3.71 Inter-spinal ornamentation 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 amb 26.19 24.15 26.93 26.57 26.09 27.69 30.81 30.5 30 28.83 28.51 28.69 29.07 29.16 29.24 29.78 28.73 25.59 30.49 26.07 27.12 26.54 t 14 14.05 14.88 14.34 12.89 14.64 15.96 16.12 16.02 15.02 14.97 15.09 15.55 14.88 14.64 14.14 14.94 12.38 15.57 13.73 14.33 13.52 4 Taxa C. tinctoria var. tinctoria C. tinctoria var. pallida C. tinctoria var. discoidea C. euxina C. virescens C. triumfetti C. melanoloma subsp. melanoloma C. melanoloma subsp. trapezuntica C. antitaurica C.oxylepis C. hamzaoglui C. fulvida C. altissima C. coelopoda var. coelopoda C. coelopoda var. longiloba C coleopoda var. bourgaei C. austriaca C. dipsacea C. palaestina C.wiedemanniana C. halophila C.pestalozzae Polar axes (P) Equatorial axes (E) P/E Pollen shape Exine Min 24 Max 28.08 Mean 25.54 ± 1.3 Min 24.4 Max 29.76 Mean 27.46 ± 1.2 0.92 ± 0.07 oblate-spheroidal 2.68 ± 0.09 22.02 26.96 24.92 ± 1.78 23.92 28.8 26.73 ± 1.57 0.92 ± 0.04 oblate-spheroidal 24 29.76 26.37 ± 1.58 27.84 30 28.53 ± 0.76 0.92 ± 0.04 23.04 22.32 25.92 27.84 27.84 26.4 29.76 31.2 25.31 ± 1.12 24.92 ± 1.32 27.61 ± 1.04 29.49 ± 0.99 24.96 23.04 26.88 28.8 30.24 27.84 30.72 33.6 27.58 ± 1.53 26.27 ± 1.66 28.36 ± 0.97 31.12 ± 1.49 28.8 34.56 30.32 ± 1.43 29.52 32.64 26.88 25.92 26.4 25.92 27.84 26.88 31.68 30.72 30.72 28.8 30.72 31.68 29.56 ± 1.32 28.3 ± 1.44 28.38 ± 1.09 26.78 ± 0.76 29.06 ± 1.03 29.53 ± 1.33 28.8 28.08 27.84 25.92 27.84 28.8 24.96 28.56 26.57 ± 1.26 26.88 28.88 24.96 23.04 26.88 21.6 24.96 23.04 28.8 26.88 30.72 28.8 28.8 30.72 Clg, colpus length; Clt, colpus width. Intine Colpus 0.82 ± 0.12 Clg 14.57 ± 0.51 Clt 4.08 ± 0.39 2.49 ± 0.37 0.85 ± 0.11 14.66 ± 0.86 4.36 ± 0.69 oblate-spheroidal 2.57 ± 0.42 0.83 ± 0.12 14.74 ± 0.98 4.03 ± 0.3 0.91 ± 0.03 0.94 ± 0.02 0.97 ± 0.04 0.94 ± 0.04 oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal 2.74 ± 0.17 2.76 ± 0.17 2.89 ± 0.31 2.87 ± 0.19 0.86 ± 0.12 0.86 ± 0.17 0.8 ± 0.15 0.83 ± 0.17 15.52 ± 1.05 13.93 ± 1.03 14.48 ± 0.46 16.78 ± 0.77 4.42 ± 0.58 3.88 ± 0.31 4.12 ± 0.4 3.84 ± 0.41 31.56 ± 0.75 0.95 ± 0.04 oblate-spheroidal 2.89 ± 0.17 0.84 ± 0.12 16.68 ± 1.34 3.82 ± 0.32 33.6 31.68 31.68 31.68 32.64 33.6 30.9 ± 1.32 30.06 ± 1.02 30.32 ± 1.02 28.41 ± 1.57 29.77 ± 1.3 31.18 ± 1.54 0.95 ± 0.02 0.95 ± 0.03 0.93 ± 0.02 0.94 ± 0.04 0.97 ± 0.04 0.94 ± 0.03 oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal 2.82 ± 0.28 2.92 ± 0.24 2.84 ± 0.09 2.81 ± 0.11 2.91 ± 0.22 2.82 ± 0.16 0.79 ± 0.13 0.74 ± 0.17 0.82 ± 0.16 0.75 ± 0.18 0.78 ± 0.1 0.84 ± 0.12 16.8 ± 0.98 15.2 ± 1.22 14.71 ± 0.55 14.43 ± 0.56 15.51 ± 0.78 16.56 ± 1.53 4.56 ± 0.39 4.9 ± 0.18 5.19 ± 0.29 5.02 ± 0.51 3.79 ± 0.35 4.12 ± 0.53 26.2 28.8 27.99 ± 0.68 0.94 ± 0.03 oblate-spheroidal 2.83 ± 0.09 0.80 ± 0.12 16.14 ± 0.28 4.01 ± 0.41 27.6 ± 1.01 27.84 30.72 29.14 ± 1.07 0.94 ± 0.02 oblate-spheroidal 2.82 ± 0.14 0.81 ± 0.13 16.28 ± 0.87 4.26 ± 0.1 26.36 ± 1.19 25.03 ± 1.36 28.94 ± 1.17 26.04 ± 1.77 26.89 ± 1.09 25.93 ± 1.7 24.96 24 28.8 24.96 25.92 25.92 29.76 26.4 33.6 29.76 29.76 30.72 28.06 ± 1.12 25.42 ± 0.65 31.002 ± 1.22 27.9 ± 1.42 28.32 ± 0.96 27.72 ± 1.4 0.93 ± 0.06 0.98 ± 0.03 0.92 ± 0.02 0.92 ± 0.05 0.94 ± 0.04 0.93 ± 0.03 oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal oblate-spheroidal 2.77 ± 0.27 2.62 ± 0.34 2.81 ± 0.21 2.4 ± 0.43 2.73 ± 0.24 2.73 ± 0.19 0.91 ± 0.09 0.78 ± 0.2 0.78 ± 0.19 0.74 ± 0.12 0.84 ± 0.12 0.77 ± 0.1 15.76 ± 1.008 14.91 ± 1.3 16.14 ± 1.07 15.42 ± 0.74 13.85 ± 0.5 14.44 ± 1.23 4.03 ± 0.34 3.87 ± 0.34 3.91 ± 0.12 3.55 ± 0.37 3.79 ± 0.16 3.71 ± 0.45 M. U. ÖzBEk ET al. Table 3. Pollen morphology of Cota (values in μm): part I. Table 4. Pollen morphology of Cota (values in μm): part II. Taxa Pore aperture type Plg 6.43 ± 0.46 Plt 7.16 ± 0.44 6.49 ± 0.88 spine Perforation number at base 5–15 ornamentation ornamentation of interspinal area echinate Perforate 26.19 ± 1.09 amb t tricolporate length 3.24 ± 0.38 Width at base 4.97 ± 0.47 7.11 ± 0.85 tricolporate 3.23 ± 0.26 4.77 ± 0.71 6–16 echinate Perforate 24.15 ± 1.1 14.05 ± 0.89 6.59 ± 0.28 7.19 ± 0.63 tricolporate 3.55 ± 0.4 4.92 ± 0.68 9–17 echinate Perforate 26.93 ± 0.84 14.88 ± 0.66 6.67 ± 0.48 6.25 ± 0.59 6.45 ± 0.82 8.2 ± 0.46 7.54 ± 0.42 6.56 ± 0.83 7.22 ± 0.89 8.32 ± 0.58 tricolporate tricolporate tricolporate tricolporate 3.07 ± 0.34 3.2 ± 0.36 3.82 ± 0.06 3.77 ± 0.3 5.1 ± 0.44 4.83 ± 0.47 5.52 ± 0.37 5.93 ± 0.39 6–10 6–15 8–15 8–13 echinate echinate echinate echinate Perforate Perforate Perforate Perforate 26.57 ± 1.39 26.09 ± 1.19 27.69 ± 1.04 30.81 ± 0.85 14.34 ± 0.93 12.89 ± 1.04 14.64 ± 0.55 15.96 ± 0.7 8.31 ± 0.77 7.98 ± 0.82 tricolporate 3.45 ± 0.38 4.72 ± 0.37 8–12 echinate Perforate 30.5 ± 0.79 16.12 ± 1.07 7.77 ± 0.72 8.34 ± 1.05 7.33 ± 0.55 7.56 ± 0.64 7.53 ± 0.72 7.38 ± 0.74 8.48 ±0.47 8.9 ± 0.71 7.82 ± 0.6 7.94 ± 0.42 7.39 ± 0.8 7.32 ± 0.91 tricolporate tricolporate tricolporate tricolporate tricolporate tricolporate 3.5 ± 0.3 3.2 ± 0.29 3.52 ± 0.51 3.55 ± 0.37 3.66 ± 0.44 3.85 ± 0.48 5.24 ± 0.44 4.9 ± 0.46 5.24 ± 0.61 5.5 ± 0.37 5.28 ± 0.46 4.81 ± 0.05 6–16 10–20 7–15 8–16 8–15 10–15 echinate echinate echinate echinate echinate echinate Perforate Perforate Perforate Perforate Perforate Perforate 30 ± 1.55 28.83 ± 1.26 28.51 ± 0.95 28.69 ± 1.02 29.07 ± 0.99 29.16 ± 1.71 16.02 ± 1.06 15.02 ± 0.98 14.97 ± 1.18 15.09 ± 0.77 15.55 ± 0.82 14.88 ± 1.16 7.24 ± 0.5 7.05 ± 0.46 tricolporate 3.64 ± 0.31 4.68 ± 0.15 9–14 echinate Perforate 29.24 ± 1.13 14.64 ± 1.01 7.33± 0.69 7.38 ± 0.7 tricolporate 3.49 ± 0.41 4.44 ± 0.65 7–13 echinate Perforate 29.78 ± 1.25 14.14 ± 0.95 7.42 ± 0.68 6.3 ± 0.8 7.2 ± 0.66 6.43 ± 0.7 tricolporate tricolporate 3.39 ± 0.47 3.04 ± 0.26 4.81 ± 0.67 4.49 ± 0.34 8–14 7–13 echinate echinate 28.73 ± 1.22 25.59 ± 0.9 14.94 ± 0.74 12.38 ± 1.6 C. palaestina 8.52 ± 0.56 8.51 ±0.46 tricolporate 3.804 ± 0.17 5.26 ± 0.43 9–13 echinate 30.49 ± 1.11 15.57 ± 0.85 C.wiedemanniana 6.89 ± 0.73 7.48 ±0.92 tricolporate 3.05 ± 0.33 4.72 ± 0.21 6–10 echinate 26.07 ± 1.16 13.73 ± 0.83 C. halophila 6.48 ± 0.68 6.7 ±0.51 tricolporate 3.86 ± 0.32 5.63 ± 0.29 8–12 echinate 27.12 ± 1.17 14.33 ± 0.45 C. pestalozzae 7.23 ± 0.82 7.04 ± 0.75 tricolporate 3.74 ± 0.21 5.42 ± 0.4 7–12 echinate Perforate Microreticulate– perforate Microreticulate– perforate Microreticulate– perforate Microreticulate– perforate Microreticulate– perforate 26.54 ± 0.98 13.52 ± 1.19 Plg pore length, Plt pore width. 14 ± 1.11 BoTaNy lETTErs C. tinctoria var. tinctoria C. tinctoria var. pallida C. tinctoria var. discoidea C. euxina C. virescens C. triumfetti C. melanoloma subsp. melanoloma C. melanoloma subsp. trapezuntica C. antitaurica C. oxylepis C. hamzaoglui C. fulvida C. altissima C. coelopoda var. coelopoda C. coelopoda var. longiloba C coleopoda var. bourgaei C. austriaca C. dipsacea 5 6 M. U. ÖzBEk ET al. Figure 2. Pollen morphology of Cota by light microscopy. 1–4 C. tinctoria var. tinctoria; 5–8 C. tinctoria var. pallida; 9–12 C. tinctoria var. discoidea; 13–16 C. euxina; 17–20 C. virescens; 21–24 C. triumfetti. Material and methods Pollen morphological analysis Plant materials Pollen slides were prepared using the Wodehouse (1935) technique. Pollen grains were stained with glycerine-jelly plus basic-fuchsin with slight heating of the slide, then a coverslip was placed on it. hese preparations were investigated and measured under the light microscope. he photographs were taken with the Leica DM750 digital photomicrograph system. Measurements were based on at least 30 pollen grains per specimen for each Flowers of the taxa in the genus were collected from diferent localities of Turkey during the revision of the genus in Turkey. Collectors and localities are given in the specimens investigated list (Table 1). hese specimens are deposited in the Gazi University, Faculty of Science Herbarium (GAZI). he order of the species was adopted from Özbek (2012). BoTaNy lETTErs 7 Figure 3. Pollen morphology of Cota by light microscopy. 1–4 C. melanoloma subsp. melanoloma; 5–8 C. melanoloma subsp. trapezuntica; 9–12 C. antitaurica; 13–16 C. oxylepis; 17–20 C. hamzaoglui; 21–24 C. fulvida. morphological characteristic. For scanning electron microscopy studies, dried pollen grains were transferred onto stubs and then coated with gold. hey were observed and photographed with a JEOL JSM 6060 scanning electron microscope. he terminologies of Faegri, Kaland, and Krzywinski (1989), Faegri and Iversen (1992), Punt et al. (2007), Punt and Hoen (2009), Pınar and İnceoğlu (1996), Pınar and Oybak Dönmez (2000) and Çeter et al. (2013) were followed. he pollen shape class, based on the ratio of polar axis to equatorial axis (P/E), was identiied using Erdtman’s system (Erdtman 1969). Numerical analysis he Simpson and Roe graphical test (Van der Pluym and Hideux 1997) was used for statistical calculations (Figure 1). Pollen characteristics of the taxa and coeficients of correlation were determined, and they were grouped using the clustering analysis method [unweighted pair group method using arithmetic averages (UPGMA), dissimilarity, standardized variables]. Eight palynological characters were selected to distinguish the 22 taxa (operational taxonomic units) of the genus Cota (Table 2). A primary matrix was created using 22 taxa 8 M. U. ÖzBEk ET al. Figure 4. Pollen morphology of Cota by scanning electron microscopy. 1, 2 C. tinctoria var. tinctoria; 3, 4 C. tinctoria var. pallida; 5, 6 C. tinctoria var. discoidea; 7, 8 C. euxina; 9, 10 C. virescens; 11, 12 C. triumfetti; 13, 14 C. melanoloma subsp. melanoloma; 15, 16 C. melanoloma subsp. trapezuntica; 17, 18 C. antitaurica; 19, 20 C. oxylepis; 21, 22 C. hamzaoglui; 23, 24 C. fulvida. (operational taxonomic units) and all eight characters for the multivariate analysis. Since Gower’s Formula (Jabee, Ansari, and Danish Shahab 1971) allows the inclusion of ordinal and nominal variables in the data matrix, it was used to calculate the primary mixed data for dissimilarities. UPGMA was selected because it is the most commonly used method and it has advantages over other methods in accurate relection of the similarity matrix as measured by the co-phenetic correlation coeicient of Sokal and Rohlf (1962), symmetrical hierarchical structure (McNeill 1979), and congruence with classiication derived by traditional methods (Ward 1993). All computations were made using the MVSP 3.22 sotware. Size, symmetry and shape The pollen grains of Cota are radially symmetrical and isopolar. The pollens of the genus are oblatespheroidal with the polar axes 21.6–34.56 μm and the equatorial axes 23.04–33.6 μm (Figure 1). Their dimensions are smaller in C. dipsacea (Bornm.) Oberp. & Greuter and larger in C. melanoloma (Trautv.) Holub var. trapezuntica (Grierson) Oberp. & Greuter. The outline of all pollen grains is elliptic in equatorial view and triangular in polar view; amb intersemiangular (Table 3; Figures 2–7). Apertures Results he main palynological features of Cota taxa examined are summarized in Tables 3 and 4 and they are shown in Figures 2–7. he pollen grains of Cota are trizonocolporate. Colpus is long (13.85–16.8 μm) and narrow or broad (3.55–5.19 μm); margins distinct, regular and ends acute. Colpus membrane is more or less granulate. he porus is BoTaNy lETTErs 9 Figure 5. Pollen morphology of Cota by light microscopy. 1–4 C. altissima; 5–8 C. coelopoda var. coelopoda; 9–12 C. coelopoda var. longiloba; 13–16 C. coelopoda var. bourgaei; 17–20 C. austriaca. 6.25–8.52 μm in length and 6.43–8.9 μm in width. he shape of porus is circular or lalongate (Table 3; Figures 2–7). Exine he exine (without spine length) ranges from 2.4 to 2.92 μm. Ectexine is thicker than endexine without costae and cavea. he intine is 0.74–0.91 μm thick. he spines are generally conical with a broadened base and gradually tapering into pointed tips. he spine length varies between 3.04 and 3.86 μm; the spine width is 4.44–5.93 μm. Perforations at the base of the spines are found to be diferently seriate in investigated taxa. Most of the species have 1–2 seriate perforations, some of them [C. tinctoria (L.) J.Gay var. tinctoria, C. tinctoria (L.) J.Gay var. pallida (DC.) U. Özbek & Vural, C. virescens (Bornm.) U. Özbek & Vural, C. triumfetti (L.) J. Gay] have 1–3 or rarely 2–3 (C. tinctoria (L.) J.Gay var. discoidea (All.) U. Özbek & Vural) and 2–4 (C. oxylepis Boiss.) seriate perforations. he number of perforations is 5–20 in the genus. Exine sculpturing is echinate in all investigated species, but the ornamentation of the inter-spinal area is diferent among the taxa. It is generally perforate (17 taxa); conversely in C. dipsacea, C. palaestina Reut. ex. Unger & Kotschy, C. wiedemanniana (Fisch. & C.A. Mey.) Holub, C. halophila (Boiss. & Balansa) Oberp. & Greuter and C. pestalozzae (Boiss.) Boiss. the ornamentation is microreticulate–perforate (Table 3; Figures 2–7). Numerical analysis of the palynological character A dendrogram of cluster analysis of Cota taxa based on eight palynological character states of 22 taxa has been constructed. his dendrogram shows the similarities that exist among the taxa being studied. Cluster analysis divided the taxa into three main groups with a 68% level of similarity: clusters A, B and C. Cluster A includes three sub-groups, A1, A2 and A3. Cluster A1 includes C. hamzaoglui U.Özbek & Vural, C. fulvida 10 M. U. ÖzBEk ET al. Figure 6. Pollen morphology of Cota by light microscopy. 1–4 C. dipsacea; 5–8 C. palaestina; 9–12 C. wiedemanniana; 13–16 C. halophila; 17–20 C. pestalozzae. (Grierson) Holub and C. oxylepis. Cluster A2 divided into three sub-groups, namely A2a, A2b, A2c. Cluster A2a includes only C. austriaca (Jacq.) Sch. Bip. Cluster A2b comprises C. melanoloma (Trautv.) Holub subsp. melanoloma, C. melanoloma subsp. trapezuntica and C. antitaurica (Grierson) Holub. Cluster A2c includes C. coelopoda (Boiss.) Boiss. var. bourgaei (Boiss.) U. Özbek & Vural, C. coelopoda (Boiss.) Boiss. var. longiloba (Grierson) U. Özbek & Vural and C. coelopoda (Boiss.) Boiss. var. coelopoda. Cluster A3 consists of C. altissima (L.) J. Gay and C. triumfetti. Cluster B includes three sub-groups, namely B1, B2 and B3. Cluster B1 includes C. euxina (Boiss.) U. Özbek & Vural. Cluster B2 includes C. tinctoria var. tinctoria, C. tinctoria var. discoidea and C. tinctoria var. pallida. B3 includes only C. virescens. Cluster C includes three sub-groups, namely C1, C2, C3. Cluster C1 divided into two sub-groups, namely C1a, C1b. Cluster C1a includes C. wiedemanniana. Cluster C1b comprises C. pestalozzae and C. halophila. Cluster C2 includes C. dipsacea and inally in Cluster C3 C. palaestina is present (Figure 8). Discussion he pollen grains of the Anthemis are attributed to the “Achillea type” of Faegri, Kaland and, Krzywinski (1989), Anthemis type of Moore, Webb, and Collinson (1991) and Anthemis arvensis type of Valdés, Díez, and Fernández (1987) and Punt and Hoen (2009). his study showed that the pollen morphology is a taxonomically signiicant character for the studied Cota taxa. Pollen grains have been found to be similar generally in relation to the type of aperture (tricolporate) and ornamentation (echinate). Variations in pollen grains of Cota have been noted mainly in pollen size and inter-spinal area ornamentation besides colpus length and width, Amb, t and perforation number at spine base. Oberprieler (1998) stated that pollen size shows variation between BoTaNy lETTErs 11 Figure 7. Pollen morphology of Cota by scanning electron microscopy. 1, 2 C. altissima; 3, 4 C. coelopoda var. coelopoda; 5, 6 C. coelopoda var. longiloba; 7, 8 C. coelopoda var. bourgaei; 9, 10 C. austriaca; 11, 12 C. dipsacea; 13, 14 C. palaestina; 15, 16 C. wiedemanniana; 17, 18 C. halophila; 19, 20 C. pestalozzae. Figure 8. Dendogram showing dissimilarity distance of the investigated taxa of Cota. their studied Anthemis species. Çeter et al. (2013) found some diferences in inter-spinal area ornamentations between their investigated species of Matricaria L. and Tripleurospermum Sch. Bip. Stix (1960) detected that pollen grains are generally prolate and spheroidal, but they are never more than slightly oblate in Asteraceae. Oberprieler (1998) reported that pollen grains were uniformly spiny, spheroidal and 12 M. U. ÖzBEk ET al. trizonocolporate in all investigated Anthemis species distributed in North Africa. We observed that all investigated taxa were oblate-spheroidal (P/E: 0.91–0.98). he thickness of exine and intine was nearly the same. Çeter et al. (2013) noted that these measurements are similar in the Matricaria and Tripleurospermum taxa. Oberprieler (1998) observed no diferences between pollen sizes of annuals and perennials in their investigated Anthemis species, in contrast to Benedí i González (1987). Our indings are consistent with those of Oberprieler (1998). he pollen grains are homogeneous in size and apertures. he smallest pollen was observed in C. dipsacea, and the largest in C. melanoloma subsp. trapezuntica. he aperture form of this genus is trizonocolporate. he shape of porus was circular in eight Anthemis species studied by Dauti et al. (2014). In our study the pores are not only circular but also lalongate in outline. Stix (1960), Skvarla et al. (1977), Salgado-Labouriau (1982) and Mesin, Crawford, and Smith (1995) declared that subapical perforations at the spine base in Asteraceae are a good taxonomic character for separating taxa. Subapical perforations in the spine base were observed in all investigated Cota specimens. hese perforations are found to be diferent between the species in terms of their arrangement and numbers. Pollen ornamentation, the most signiicant characteristic, can be generally used to distinguish taxa from each other (Pınar, Ekici et al. 2009; Pınar, Duran et al. 2009). Dauti et al. (2014) stated that A. altissima, A. triumfetti, A. tinctoria and the other ive Anthemis species have an echinate exine. Our indings are consistent with this observation; however, we added the inter-spinal area ornamentation. Mesin, Crawford, and Smith (1995) and Çeter et al. (2013) stated that the ornamentations between spines are an important characteristic for Asteraceae. Çeter et al. (2013) reported granulate-perforate, reticulate-perforate or rugulate-perforate ornamentations in the inter-spinal region in the genera Matricaria and Tripleurospermum. In our study, ornamentations between spines were generally perforate, but microreticulate-perforate ornamentations were observed in C. dipsacea, C. palaestina, C. wiedemanniana, C. halophila and C. pestalozzae. Cluster analysis indicated that the Cota specimens were divided into three main groups according to pollen morphological characters. In the dendrogram generated from palynological data, in the irst group of Cluster A the percentage of similarity between C. hamzaoglui and the morphologically related species C. fulvida is > 92%. In the second group, C. melanoloma subsp. melanoloma, C. melanoloma subsp. trapezuntica and C. antitaurica are quite (> 85%) similar. Cota coelopoda var. bourgaei, C. coelopoda var. longiloba and C. coelopoda var. coelopoda are also closely related with > 92% similarity. Cota tinctoria var. tinctoria, C. tinctoria var. discoidea and C. tinctoria var. pallida are closely related with 86% similarity in Cluster B2 group. hese three varieties are also more similar and morphologically resemble C. euxina and the other related species C. virescens. In Cluster C, C. palaestina was separated from the other taxa at irst. In this cluster, C. pestalozzae and C. halophila are more similar (>84%) to each other than the others. hese data support the separation of the taxa on morphological characters. Our numerical analysis showed that the ratio of P/E, colpus length and width and the ornamentation of the inter-spinal area are valuable variables for discriminating the taxa of Cota. his is the irst comprehensive report on the pollen morphology of 22 taxa belonging to the genus Cota examined using light microscopy and scanning electron microscopy. In conclusion, we found correlation between our results and classiication of the taxa in this genus, so pollen features – especially pollen size and inter-spinal area ornamentation – proved to be the most useful characters for the systematics of the taxa. Acknowledgement he authors thank the Scientiic and Technological Research Council of Turkey for inancial support (TUBITAK, TBAG Project No 105T353). Disclosure statement No conlict of interest was declared by the authors. Funding his work was supported by the Scientiic and Technological Research Council of Turkey [grant number TUBITAK, TBAG Project No 105T353]. Notes on contributors Mehmet Ufuk Özbek is doctor at Gazi University, Department of Biology. Contribution: collection and identiication of samples; writing of article. Funda Özbek is assistant at Gazi University, Department of Biology. Contribution: pollen morphological analysis and writing of article. 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