Centaurea, Asteraceae) Via Scanning Electron Microscopy

Turkish Journal of Botany Turk J Bot (2016) 40: 147-163 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1503-51

The examinations of achene ultrastructural features of section Acrolophus (Centaurea, Asteraceae) via scanning electron microscopy

1 2, 2 2,3 2 2 Feyza CANDAN , Tuna UYSAL *, Osman TUGAY , Meryem BOZKURT , Kuddisi ERTUĞRUL , Hakkı DEMİRELMA 1 Department of Biology, Faculty of Science and Art, Celal Bayar University, Manisa, Turkey 2 Department of Biology, Faculty of Science, Selçuk University, Konya, Turkey 3 Advanced Technology Research and Application Center, Selçuk University, Konya, Turkey

Received: 30.03.2015 Accepted/Published Online: 29.06.2015 Final Version: 09.02.2016

Abstract: In this study, the achene surfaces of 31 taxa belonging to the genus Centaurea L. were examined in detail by means of electron microscopy. The main aim of this study was to characterize the microsculpture of the achene surface of the Turkish species of Centaurea sect. Acrolophus (Cass.) DC. (Asteraceae). Detailed descriptions of the achene surface were given for each taxon and a fruitful dendrogram was established by numerical analyses derived from the determined micromorphological features. The relationships among the taxa studied were briefly assessed using this generated dendrogram. The results indicated that the examined taxa had very high variations regarding their achene surfaces and these variations have great importance in determining the taxonomic relationships of the discussed taxa. According to the results, pericarp texture and color could be used for taxonomical diagnosis of the Acrolophus species because they contain differences over a broad spectrum. The fruit coat was usually roguish and its ornamentation was fairly variable; therefore, this taxonomical microcharacter might also be useful in distinguishing closely related taxa. The hairiness of the surface of the pericarp was characteristic in all Acrolophus species and it would be evaluated as a decisive micromorphological character of the section within the genus.

Key words: Taxonomy, Centaurea, Acrolophus, Asteraceae, fruit surface, endemic, micromorphology, Turkey

1. Introduction identification (Kaya et al., 2011). The seed coat morphology An important and relatively large genus in the flora of and microsculpturing were investigated in certain genera Turkey, Centaurea L. comprises ca. 250 species, currently of Brassicaceae and provided evidence for the close distributed over Eurasia, especially in Irano-Turanian and relationships among various genera (Koul et al., 2000; Mediterranean regions (Anderberg et al., 2007). Centaurea Moazzeni et al., 2007). These kinds of peculiarities were has one of the highest rates of endemism in Turkey, with used in the identification of families such as Brassicaceae 112 endemics among 181 total species, 18 endemics among (Cruciferae) Burnett and Scrophulariaceae Juss. (Murley, 32 subspecies, and 16 of the 28 varieties (Uysal, 2012). The 1951; Juan et al., 2000). genus Centaurea has been subjected to morphological, Plant species are traditionally examined to perform palynological, and cytotaxonomic studies for a very identification or characterization by stereomicroscopy, long time by several experts (Routsi and Georgiadis, but in many cases, when taxonomists disagree on the 1998; Villodre and Garcia-Jacas, 2000; Çelik et al., 2008; identity of taxa, the micromorphological characters of Gömürgen et al., 2010), but only a limited number of leaves, pollens, fruit, and seeds are examined via scanning studies were performed on the achene morphologies of electron microscopy (SEM) to illustrate diagnostic Centaurea in terms of shedding light on the taxonomy of differences (Shehata, 2006). Seeds display a great deal of the genus (Dittrich, 1968). variation and high morphological diversity, including Morphological and anatomical studies on the fruit valuable taxonomic information. Their shapes, colors, and and seed structures play an important role in systematics. sizes can be of high systematic significance (Chuang and Microstructural details of the fruit coat and seed make Ornduff, 1992). possible the distinguishing of the taxa (Kaya et al., 2011). The seed and fruit are both distinctive features in taxon These features, important characters for taxonomic and diagnosis of the family Asteraceae. In regards to this, there evolutionary studies, were used as parameters for species are many reports providing information for different * Correspondence: [email protected] 147 CANDAN et al. / Turk J Bot taxonomical levels and groups (Wagenitz et al., 1998; Tan cell walls not distinct. Pappus 2.6–3.8 mm. Scales present et al., 2007; Kreitschitz and Valles, 2007). The general (Figures 1a–1c). morphologies of the seeds of Acrolophus (Cass.) DC. taxa Centaurea inermis Velen.: Achenes 3.1–3.9 mm, were described simply by Wagenitz (1975) and Davis et al. ovoid-oblong; swollen, pericarp wholly cream; sometimes (1988). Although some studies were reported on the fruit- grayish lines on cream pericarp, strigose, pitted. Rotund coat surface structure of Centaurea L. species (Celik et al., at the base. Fruit coat roughish, ornamentation irregular 2005a, 2005b; Uysal et al., 2005; Aksoy et al., 2010; Okay sulcate. Cells sometimes one on another. Cells and cell and Demir, 2010; Shabestari et al., 2013; Bona, 2014), none walls distinct. Pappus 0.7–1.5 mm. Scales present (Figures of them gave comprehensive and whole information on 1d–1f). achene surface characteristics of the section Acrolophus. Centaurea kilaea Boiss.: Achenes 3.6–4.3 mm, The present study was mainly undertaken to illustrate lanceolate, swollen, pericarp wholly grayish green; the external morphology of mature seeds using SEM sometimes creamy lines on brown pericarp, strigose, and to obtain information on the achene ultrastructural not pitted. Obtuse at the base. Fruit coat roughish, construction of Acrolophus (Centaurea) taxa of Turkey. ornamentation irregular sulcate. Cells sometimes one on another. Cells and cell walls distinct. Pappus 2.3–3.0 mm. 2. Materials and methods Scales present (Figures 1g–1i). Between 2004 and 2008, many materials belonging to Centaurea cuneifolia Sm.: Achenes 3.0–4.1 mm, section Acrolophus were collected from several localities lanceolate, swollen, cream lines on grayish or brown of Turkey by our research team and the samples collected pericarp, strigose, not pitted. Obtuse at the base. Fruit were deposited in the KNYA herbarium (Table 1). coat roughish, ornamentation irregular sulcate. Cells are For SEM observation, at least 10 achenes for each taxon sometimes one on another. Cells distinct, cell walls not were dehydrated in an acetone series, critical-point-dried distinct. Pappus 0.7–1.5 mm. Scales present (Figures 1j– using carbon dioxide, and mounted on stubs using double- 1l). sided adhesive tape. Achenes were coated with gold under Centaurea zeybekii Wagenitz: Achenes 2.8–4.2 mm, JEOL JSM-6060 model SEM low-vacuum mode at about lanceolate, swollen, cream lines on brown pericarp, 20 nm in thickness for observing their surface structures puberulent, not pitted. Obtuse at the base. Fruit coat (Candan et al., 2009). SEM photographs of all taxa were roughish, ornamentation irregular sulcate. Cells usually taken at 30× size for the achenes’ general view, 1000× for one on another. Cells and cell walls distinct. Pappus 1.3– the general view of the achenes’ surface, and 2000× for 2.5 mm. Scales present (Figures 2a–2c). close views of achenes’ microstructural features. Achene Centaurea tuzgoluensis Aytaç & H. Duman: Achenes surface structural terminology follows Stearn (1983). 3.8–4.3 mm, obovoid, swollen, pericarp wholly dark Fifteen characters were investigated and used in this brown; sometimes creamy-yellow spots on brown analysis. Characters and character states were determined pericarp; sometimes brown lines on creamy white through examination of herbarium specimens stored at pericarp, puberulent, not pitted. Rotund at the base. Fruit the KNYA herbarium. Both qualitative and quantitative coat not roughish, ornamentation irregular sulcate. Cells characters were scored for numerical taxonomic studies and cell walls distinct. Pappus 1.5–2.6 mm. Scales present and constructed as a data matrix (Tables 2 and 3). A (Figures 2d–2f). similarity index indicating the relationship among taxa Centaurea olympica (DC.) K.Koch: Achenes 2.0–3.0 was generated based on the data matrix. Cluster analysis mm, obovoid, swollen, pericarp wholly brown; sometimes was performed using UPGMA methods and a dendrogram creamy lines on brown pericarp, pilose, pitted. Obtuse at based on similarity index was constructed with NTSYS PC the base. Fruit coat roughish, ornamentation irregular v.2.2 (Rohlf, 1997). sulcate. Cells and cell walls not distinct. Pappus absent. Scales absent (Figures 2g–2i). 3. Results Centaurea ulrichiorum Wagenitz, F.H.Hellw. & Parolly: In this study, the seeds of 31 taxa belonging to the genus Achenes 3.3–4.2 mm, lanceolate, compressed, pericarp Centaurea were examined micromorphologically via SEM. wholly grayish white, strigose, pitted. Obtuse at the base. Very detailed information about the structure of the seed Fruit coat not roughish, ornamentation irregular sulcate. surface taxa is given in Tables 2 and 3. Cells distinct, cell walls not distinct. Pappus 1.2–2.2 mm. Centaurea arenaria M.Bieb. ex Willd.: Achenes 2.6–3.6 Scales present (Figures 2j–2l). mm, oblong-rectangular, compressed, pericarp wholly Centaurea polyclada DC.: Achenes 2.0–3.1 mm, creamy or grayish; sometimes creamy lines on brown or obovoid, swollen, pericarp wholly grayish or blackish; grayish pericarp, sericeous, not pitted. Truncate at the sometimes creamy lines on brown pericarp, pilose, pitted. base. Fruit coat roughish, ornamentation striate. Cells and Obtuse at the base. Fruit coat roughish, ornamentation

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Table 1. Collection data of the studied taxa

Collection Taxa Locality Endemic number

Centaurea arenaria [A1] Kırklareli: İğneada, rough paths, 5 m, 15.08.2006, O.Tugay 4486 et al. OT-4486

C. inermis [A2] İstanbul, Yakacık, forest openings, 320 m, 12.08.2006, O.Tugay 4431 et al. OT-4431

C. kilaea [A1] Kırklareli: between İğneada-Beğendik, 50 m, 15.08.2006, O.Tugay 4480 et al. OT-4480 +

C. cuneifolia [B1] Balıkesir: Kaz Dağ, Cızlak Hills, 980 m, 31.07.2006, O.Tugay 4332 and T.Uysal OT-4332-TU

C. zeybekii [B1] İzmir: Nif Dağ, 870 m, 30.07.2006, O.Tugay 4304 and T.Uysal OT-4304-TU +

[C6] Konya: Cihanbeyli, between Gülyazı-Eskil towns, 920 m, 17.07.2008, O.Tugay C. tuzgoluensis OT-5491-TU + 5491 and T.Uysal

C. olympica [A2] Bursa: Uludağ, 970 m, 03.08.2006, O.Tugay 4356 and T.Uysal OT-4356-TU +

[C4] Antalya: Alanya, Demirtaş, Beldibi Village, 990 m, 26.07.2008, O.Tugay 5550 and C. ulrichiorum OT-5550-TU + T.Uysal

C. polyclada [B1] Balıkesir: Zeytinli, Akçay, 11 m, 31.07.2006, O.Tugay 4330 and T.Uysal OT-4330-TU +

C. sipylea [B1] Manisa: Spil Dağ, 1130 m, 30.07.2006, O.Tugay 4322 and T.Uysal OT-4322-TU +

[A3] Bilecik, between Bilecik-Vezirhan, above limestones, 440 m 04.08.2006, O.Tugay C. wiedemanniana OT-4391-TU + 4391 and T.Uysal

[B3] Eskişehir: between Eskişehir-Sarıkaya, stony places, 260 m, 14.07.2005, O.Tugay C. yozgatensis OT-3652-TU + 3652 and T.Uysal

[A2] Bursa: between Bursa-Orhaneli, stony places, 600 m, 13.08.2008, O.Tugay 5628 C. ertugruliana OT-5628-TU + and T.Uysal

[C3] Antalya: between Finike-Elmalı, stony places, 1120 m, 25.07.2006, O.Tugay 4230 C. cariensis subsp. cariensis OT-4230-TU and T.Uysal

[C3] Isparta: between Yenişerbademli-Aksu towns, stony places, 1320 m, 12.07.2004, C. cariensis subsp. maculiceps OT-3569-TU O.Tugay 3569 and T.Uysal

C. cariensis subsp. niveotomentosa [C2] Muğla: Marmaris, Balan Dağ, 560 m, 27.07.2006. O.Tugay 4270 and T.Uysal OT-4270-TU

C. cariensis subsp. microlepis [C3] Antalya: between Elmalı-Fethiye, 1190 m, 25.07.2006, O.Tugay 4240 and T.Uysal OT-4240-TU

C. cariensis subsp. longipapposa [B3] Konya: between Akşehir-Isparta, 1415 m, 16.07.2007, O.Tugay 4981 and T.Uysal OT-4981-TU +

[B6] Kahramanmaraş: between Elbistan-Malatya, 1620 m, 03.08.2007, O.Tugay 5032 C. cariensiformis OT-5032-TU + and T.Uysal

C. werneri [C3] Antalya: between Finike-Elmalı, 420 m, 19.07.2007, O.Tugay 5000 and K.Ertuğrul OT-5000-KE +

C. austroanatolica [C2] Muğla: Marmaris, Balan Dağ, 110 m, 24.07.2006, O.Tugay 4267 and T.Uysal OT-4267-TU +

C. dichroa [C3] Antalya: Kemer, Çıralı, 20 m, 24.07.2006, O.Tugay 4215 and T.Uysal OT-4215-TU +

C. consanguinea [A5] Amasya: Northern slopes, 410 m, 19.08.2006, O.Tugay 4555 and T.Uysal OT-4555 +

C. aggregata subsp. aggregata [B7] Erzincan: Between Refahiye-Sivas, 1790 m, 04.08.2007, O.Tugay 5059 and T.Uysal OT-5059-TU

C. aggregata subsp. albida [B7] Erzincan: between Tercan-Aşkale, 1880 m, 05.08.2007, O.Tugay 5069 and T.Uysal OT-5069-TU +

C .anthemifolia [C4] Konya: Ereğli, Aydos Dağ, 1620 m, 22.07.2008, O.Tugay 5520 and T.Uysal OT-5520-TU +

C. pinetorum [C4] Mersin: Mut, 355 m, 14.07.2007, O.Tugay 4957 and T.Uysal OT-4957 +

C. virgata [C4] Konya: Beyşehir, 1145 m, 15.07.2007, O.Tugay 4965 OT-4965

C. sivasica [B6] Sivas: between Gürün-Kangal, 1810 m, 04.08.2007, O.Tugay 5048 and T.Uysal OT-5048 +

C. diffusa [A1] Çanakkale: Kurşunlu, 35 m, 01.08.2006, O.Tugay 4460 and T.Uysal OT-4460-TU

[B1] Balıkesir: Ayvalık, Altınova, sandy coasts, 1 m, 31.07.2006, O.Tugay 4323 and C. spinosa var. spinosa OT4323-TU T.Uysal

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Table 2. The ultrastructural features of seed surface belonging to Acrolophus taxa (Centaurea).

Taxa Length Compressed Hair status Pitted or Basis Shape (mm) or swollen at pericarp not shape

Centaurea arenaria 2.6–3.6 Oblong-rectangular Compressed Sericeous Not pitted Truncate

C. inermis 3.1–3.9 Ovoid-oblong Swollen Strigose Pitted Rotund

C. kilaea 3.6–4.3 Lanceolate Swollen Strigose Not pitted Obtuse

C. cuneifolia 3.0–4.1 Lanceolate Swollen Strigose Not pitted Obtuse

C. zeybekii 2.8–4.2 Lanceolate Swollen Puberulent Not pitted Obtuse

C. tuzgoluensis 3.8–4.3 Obovoid Swollen Puberulent Not pitted Rotund

C. olympica 2.0–3.0 Obovoid Swollen Pilose Pitted Obtuse

C. ulrichiorum 3.3–4.2 Lanceolate Compressed Strigose Pitted Obtuse

C. polyclada 2.0–3.1 Obovoid Swollen Pilose Pitted Obtuse

C. sipylea 3.2–4.0 Ovoid-oblong Swollen Sericeous Not pitted Rotund

2.3–3.1 C. wiedemanniana Ovoid-oblong Compressed Strigose Not pitted Rotund

C. yozgatensis 1.6–2.5 Ovoid-oblong Compressed Puberulent Pitted Rotund

C. ertugruliana 2.8–3.1 Ovoid-oblong Compressed Strigose Not pitted Rotund

C. cariensis subsp. cariensis 2.7–3.6 Ovoid-oblong Swollen Strigose Not pitted Obtuse

C. cariensis subsp. maculiceps 2.8–3.8 Oblong-rectangular Compressed Strigose Not pitted Obtuse

C. cariensis subsp. niveotomentosa 2.7–3.4 Oblong-rectangular Compressed Sericeous Not pitted Obtuse

C. cariensis subsp. microlepis 2.9–3.5 Oblong-rectangular Compressed Strigose Pitted Obtuse

C. cariensis subsp. longipapposa 3.6–4.3 Oblong-rectangular Compressed Puberulent Pitted Obtuse

C. cariensiformis 3.4–4.2 Lanceolate Swollen Sericeous Not pitted Rotund

C. werneri 2.6–3.7 Ovoid-oblong Swollen Puberulent Not pitted Rotund

C. austro-anatolica 3.0–4.3 Lanceolate Swollen Sericeous Not pitted Obtuse

C. dichroa 3.7–4.3 Obovoid Swollen Strigose Pitted Truncate

C. consanguinea 2.8–4.0 Obovoid Swollen Strigose Not pitted Obtuse

C. aggregata. subsp. aggregata 2.3–3.9 Ovoid-oblong Swollen Strigose Not pitted Rotund

C. aggregata. subsp. albida 2.7–3.2 Oblong-rectangular Swollen Strigose Not pitted Rotund

C. anthemifolia 3.5–5.0 Lanceolate Swollen Puberulent Not pitted Truncate

C. pinetorum 2.4–3.2 Lanceolate Swollen Strigose Pitted Rotund

C. virgata 3.4–4.1 Ovoid-oblong Swollen Puberulent Pitted Obtuse

C. sivasica 2.7–3.2 Ovoid-oblong Swollen Puberulent Pitted Obtuse

C. diffusa 2.1–3.0 Ovoid-oblong Swollen Strigose Pitted Rotund

C. spinosa var. spinosa 2.9–3.6 Ovoid-oblong Swollen Puberulent Not pitted Obtuse

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Table 3. The structural features of seed surfaces.

Achene Pappus Scales

Fruit coat Cell walls Pappus shorter, Present Taxa Cells Present or Length roughish Ornamentation distinct or longer than fruit or distinct or not absent (mm) or not not and with same length absent

Centaurea arenaria Roughish Striate Not distinct Not distinct Present 2.6–3.8 Same length Present

C. inermis Roughish Irregular sulcate Distinct Distinct Present 0.7–1.5 Short Present

C. kilaea Roughish Irregular sulcate Distinct Distinct Present 2.3–3.0 Short Present

C. cuneifolia Roughish Irregular sulcate Distinct Not distinct Present 0.7–1.5 Short Present

C. zeybekii Roughish Irregular sulcate Distinct Distinct Present 1.3–2.5 Short Present

C. tuzgoluensis Not roughish Irregular sulcate Distinct Distinct Present 1.5–2.6 Short Present

C. olympica Roughish Irregular sulcate Not distinct Not distinct Absent - - Absent

C. ulrichiorum Not roughish Irregular sulcate Distinct Not distinct Present 1.2–2.2 Short Present

C. polyclada Roughish Irregular sulcate Distinct Not distinct Present 0.2–0.3 Short Present

C. sipylea Roughish Irregular sulcate Distinct Not distinct Present 2.4–3.3 Short Present

C. wiedemanniana Not roughish Regularly sulcate Distinct Distinct Present 2.6–3.8 Long Present

C. yozgatensis Not roughish Fine sulcate Distinct Not distinct Present 2.5–4.2 Long Present

C. ertugruliana Not roughish Fine sulcate Distinct Distinct Present 1.8–3.5 Same length Present

C. cariensis subsp. Not roughish Fine sulcate Distinct Not distinct Present 1.8–2.5 Short Present cariensis C. cariensis subsp. Roughish Striate Not distinct Not distinct Present 1.3–2.1 Short Present maculiceps C. cariensis subsp. Not roughish Fine sulcate Distinct Not distinct Present 2.8–3.6 Same length Present niveotomentosa C. cariensis subsp. Not roughish Fine sulcate Distinct Not distinct Present 2.0–3.7 Same length Present microlepis C. cariensis Not roughish Fine sulcate Distinct Not distinct Present 4.0–4.6 Long Present subsp. longipapposa

C. cariensiformis Roughish Fine sulcate Not distinct Not distinct Present 1.8–2.6 Short Present

C. werneri Not roughish Fine sulcate Distinct Not distinct Present 3.0–4.2 Long Present

C. austro-anatolica Roughish Irregular sulcate Not distinct Not distinct Present 1.0–2.2 Short Present

C. dichroa Roughish Irregular sulcate Not distinct Not distinct Present 1.0–1.6 Short Present

C. consanguinea Not roughish Regularly sulcate Distinct Distinct Present 0.2–0.3 Short Present

C. aggregata. subsp. Not roughish Regularly sulcate Distinct Not distinct Present 1.9–3.2 Short Present aggregata C. aggregata. subsp. Roughish Striate Not distinct Not distinct Present 2.9–3.6 Long Present albida

C. anthemifolia Not roughish Fine sulcate Distinct Distinct Present 1.0–2.1 Short Present

C. pinetorum Not roughish Fine sulcate Distinct Distinct Present 2.8–3.6 Long Present

C. virgata Roughish Irregular sulcate Not distinct Not distinct Present 0.4–0.5 Short Present

C. sivasica Roughish Irregular sulcate Not distinct Not distinct Absent - - Present

C. diffusa Roughish Regularly sulcate Distinct Distinct Absent - - Absent

C. spinosa Not roughish Fine sulcate Distinct Not distinct Absent - - Absent var. spinosa

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Figure 1. Scanning electron micrographs of the investigated Centaurea taxa: C. arenaria (a–c), C. inermis (d–f), C. kilaea (g–i), C. cuneifolia (j–l). irregular sulcate. Cells distinct, cell walls not distinct. pericarp, strigose, not pitted. Rotund at the base. Fruit coat Pappus 0.2–0.3 mm. Scales present (Figures 3a–3c). not roughish, ornamentation regularly sulcate. Cells and Centaurea sipylea Wagenitz: Achenes 3.2–4.0 mm, cell walls distinct. Pappus 2.6–3.8 mm, bristles in rows. ovoid-oblong, swollen, pericarp wholly creamy or Scales present (Figures 3g–3i). grayish; sometimes creamy lines on brown or grayish Centaurea yozgatensis Wagenitz: Achenes 1.6–2.5 mm, pericarp, sericeous, not pitted. Rotund at the base. Fruit ovoid-oblong, compressed, compressed, pericarp wholly coat roughish, ornamentation irregular sulcate. Cells light brown; sometimes brownish lines on creamy white, sometimes one on another. Cells distinct, cell walls not puberulent, pitted. Rotund at the base. Fruit coat not distinct. Pappus 2.4–3.3 mm. Scales present (Figures 3d– roughish, ornamentation fine sulcate. Cells distinct, cell 3f). walls not distinct. Pappus 2.5–4.2 mm, bristles in rows. Centaurea wiedemanniana Fisch. and C.A.Mey.: Scales present (Figures 3j–3l). Achenes 2.3–3.1 mm, ovoid-oblong, compressed, pericarp Centaurea ertugruliana Uysal: Achenes 2.8–3.1 mm, wholly creamy white; sometimes creamy lines on brown ovoid-oblong, compressed, pericarp wholly creamy white

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Figure 2. Scanning electron micrographs of the investigated Centaurea taxa: C. zeybekii (a–c), C. tuzgoluensis (d–f), C. olympica (g–i), C. ulrichiorum (j–l). or grayish, strigose, not pitted. Rotund at the base. Fruit Centaurea cariensis Boiss. subsp. maculiceps coat not roughish, ornamentation fine sulcate. Cells and (O.Schwarz) Wagenitz: Achenes 2.8–3.8 mm, oblong- cell walls distinct. Pappus 1.8–3.5 mm. Scales present rectangular, compressed, pericarp wholly brownish or (Figures 3m–3o). yellowish, strigose, not pitted. Obtuse at the base. Fruit Centaurea cariensis Boiss. subsp. cariensis: Achenes coat roughish, ornamentation striate. Cells and cell walls 2.7–3.6 mm, ovoid-oblong, swollen, pericarp wholly not distinct. Pappus 1.3–2.1 mm. Scales present (Figures creamy white or creamy yellow, strigose, not pitted. Obtuse 4d–4f). at the base. Fruit coat not roughish, ornamentation fine Centaurea cariensis Boiss. subsp. niveotomentosa (Hub.- sulcate. Cells distinct, cell walls not distinct. Pappus 1.8– Mor.) Wagenitz: Achenes 2.7–3.4 mm, oblong-rectangular, 2.5 mm. Scales present (Figures 4a–4c). compressed, pericarp wholly yellow; sometimes brown

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Figure 3. Scanning electron micrographs of the investigated Centaurea taxa: C. polyclada (a–c), C. sipylea (d–f), C. wiedemanniana (g–i), C. yozgatensis (j–l), C. ertugruliana (m–o).

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Figure 4. Scanning electron micrographs of the investigated Centaurea taxa: C. cariensis subsp. cariensis (a–c), C. cariensis subsp. maculiceps (d–f), C. cariensis subsp. niveotomentosa (g–i), C. cariensis subsp. microlepis (j–l), C. cariensis subsp. longipapposa (m–o). lines on creamy-white pericarp, sericeous, not pitted. compressed, brown lines on creamy-white pericarp, Obtuse at the base. Fruit coat not roughish, ornamentation puberulent, pitted. Obtuse at the base. Fruit coat not fine sulcate. Cells distinct, cell walls not distinct. Pappus roughish, ornamentation fine sulcate. Cells distinct, cell 2.8–3.6 mm. Scales present (Figures 4g–4i). walls not distinct. Pappus 4.0–4.6 mm. Scales present Centaurea cariensis Boiss. subsp. microlepis (Boiss.) (Figures 4m–4o). Wagenitz: Achenes 2.9–3.5 mm, oblong-rectangular, Centaurea cariensiformis Hub.-Mor.: Achenes 3.4– compressed, pericarp wholly creamy or grayish; sometimes 4.2 mm, lanceolate, swollen, pericarp wholly creamy; creamy lines on brown or grayish pericarp, strigose, pitted. sometimes creamy lines on brown or yellow pericarp, Obtuse at the base. Fruit coat not roughish, ornamentation sericeous, not pitted. Rotund at the base. Fruit coat fine sulcate. Cells distinct, cell walls not distinct. Pappus roughish, ornamentation fine sulcate. Cells and cell walls 2.0–3.7 mm. Scales present (Figures 4j–4l). not distinct. Pappus 1.8–2.6 mm. Scales present (Figures Centaurea cariensis Boiss. subsp. longipapposa 5a–5c). Wagenitz: Achenes 3.6–4.3 mm, oblong-rectangular,

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Figure 5. Scanning electron micrographs of the investigated Centaurea taxa: C. cariensiformis (a–c), C. werneri (d–f), C. austro- anatolica (g–i), C. dichroa (j–l), C. consanguinea (m–o).

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Centaurea werneri Wagenitz, F.H.Hellw. & Parolly: Centaurea virgata Lam.: Achenes 3.4–4.1 mm, ovoid- Achenes 2.6–3.7 mm, ovoid-oblong, swollen, creamy oblong, swollen, creamy lines or creamy spots on brown yellow lines on brown pericarp, puberulent, not pitted. pericarp, puberulent, pitted. Obtuse at the base. Fruit coat Rotund at the base. Fruit coat not roughish, ornamentation roughish, ornamentation irregular sulcate. Cells and cell fine sulcate. Cells distinct, cell walls not distinct. Pappus walls not distinct. Pappus 0.4–0.5 mm. Scales present 3.0–4.2 mm. Scales present (Figures 5d–5f). (Figures 7a–7c). Centaurea austro-anatolica Hub.-Mor.: Achenes 3.0– Centaurea sivasica Wagenitz: Achenes 2.7–3.2 mm, 4.3 mm, lanceolate, swollen, creamy lines on gray or brown ovoid-oblong, swollen, pericarp wholly creamy-white, pericarp, sericeous, not pitted. Obtuse at the base. Fruit puberulent, pitted. Obtuse at the base. Fruit coat roughish, coat roughish, ornamentation irregular sulcate. Cells and ornamentation irregular sulcate. Cells and cell walls not cell walls not distinct. Pappus 1.0–2.2 mm. Scales present distinct. Pappus absent. Scales present (Figures 7d–7f). (Figures 5g–5i). Centaurea diffusa Lam.: Achenes 2.1–3.0 mm, ovoid- Centaurea dichroa Boiss. and Heldr.: Achenes 3.7– oblong, swollen, creamy lines or creamy yellow spots on 4.3 mm, obovoid, swollen, pericarp wholly creamy, grayish white or brown pericarp; rarely wholly creamy, grayish; sometimes brown lines on cream pericarp or strigose, pitted. Rotund at the base. Fruit coat roughish, sometimes creamy lines on grayish or brownish pericarp, ornamentation regularly sulcate. Cells and cell walls strigose, pitted. Truncate at the base. Fruit coat roughish, distinct. Pappus absent. Scales absent (Figures 7g–7i). ornamentation irregular sulcate. Cells and cell walls not Centaurea spinosa L. var. spinosa: Achenes 2.9–3.6 mm, distinct. Pappus 1.0–1.6 mm. Scales present (Figures 5j– ovoid-oblong, swollen, yellow lines on brown pericarp, 5l). puberulent, not pitted. Obtuse at the base. Fruit coat not Centaurea consanguinea DC.: Achenes 2.8–4.0 roughish, ornamentation fine sulcate. Cells distinct, cell mm, obovoid, swollen, brown lines on yellow pericarp, walls not distinct. Pappus absent. Scales absent (Figures strigose, not pitted. Obtuse at the base. Fruit coat not 7j–7l). roughish, ornamentation regularly sulcate. Cells and Generalized findings: Achene lengths change between cell walls distinct. Pappus 0.2–0.3 mm. Scales present 1.6 and 5.0 mm, and their shapes can be in outline, e.g., (Figures 5m–5o). oblong-rectangular, ovoid-oblong, lanceolate, or obovoid, Centaurea aggregata Fisch. & C.A.Mey. subsp. respectively. Achenes are usually compressed longitudinally aggregata: Achenes 2.3–3.9 mm, ovoid-oblong, swollen, and can sometimes be swollen or pitted superficially. The yellow, brown or gray lines on creamy or grayish pericarp; achene surface is variable and spotted in some species. The sometimes creamy yellow spots on grayish pericarp, apex of the achene is invariable and it is truncate in all of strigose, not pitted. Rotund at the base. Fruit coat not the taxa. The base of the achene can be obtuse, rotund, roughish, ornamentation regularly sulcate. Cells distinct, or truncate. The fruit coat is usually roguish. Pericarp cell walls not distinct. Pappus 1.9–3.2 mm. Scales present cells take part in the long axis of the achene and they (Figures 6a–6c). vary greatly in shape (short or long, oblong-rectangular, Centaurea aggregata Fisch. & Mey. ex DC. subsp. albida (K.Koch) Bornm.: Achenes 2.7–3.2 mm, oblong- rectangular, finger-shaped, rarely V-shaped). Cell walls rectangular, swollen, pericarp wholly brown; sometimes are usually not distinct. The indumentum varies from creamy spots on brown pericarp; strigose, not pitted. puberulent to pilose or strigose, or even sericeous. The Rotund at the base. Fruit coat roughish, ornamentation length of the pappus is between 0.2 and 4.6 mm and its striate. Cells and cell walls not distinct. Pappus 2.9–3.6 color changes from creamy to creamy white. The pappus mm. Scales present (Figures 6d–6f). consists of 1–5 rows. Bristles are caduceus. Short bristles Centaurea anthemifolia Hub.-Mor.: Achenes 3.5–5 can be in the inner or outer rows. Scales are about 0.5 mm, mm, lanceolate, swollen, pericarp wholly creamy white; and in 1–3 rows. sometimes creamy white lines on yellow pericarp, In the dendrogram, the analyzed species are clustered puberulent, not pitted. Truncate at the base. Fruit coat according to the micromorphological surface features not roughish, ornamentation fine sulcate. Cells and obtained from SEM (Figure 8). Based on the dendrogram, cell walls distinct. Pappus 1.0–2.1 mm. Scales present we can clearly say that the species show large-scale (Figures 6g–6i). variations in terms of their achene surfaces and, therefore, Centaurea pinetorum Hub.-Mor.: Achenes 2.4–3.2 mm, while species with similar achene surfaces occur together lanceolate, swollen, pericarp wholly creamy; sometimes in the same clade, other relatives with different surface creamy lines on brown pericarp, strigose, pitted. Rotund characteristics are located separately in another clade by a at the base. Fruit coat not roughish, ornamentation fine certain percentage or less. A similarity rate of 37% is a fairly sulcate. Cells and cell walls distinct. Pappus 2.8–3.6 mm. low value among the species localized in these main clades Scales present (Figures 6j–6l). and this is a sign of great variation in the achene surface

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Figure 6. Scanning electron micrographs of the investigated Centaurea taxa: C. aggregata subsp. aggregata (a–c), C. aggregata subsp. albida (d–f), C. anthemifolia (g–i), C. pinetorum (j–l). micromorphology of species. When we examine all of distinguished from other relatives by a specific striate the micromorphological characters related to the achene ornamentation of the achene surface and the similarity rate surface, we see that some characters, like ornamentation, among them is only about 64%. Moreover, these species cell walls, and the presence of pits on surface and fruit coat, share common characters, consisting of fruit coat and cell are very effective in distinguishing the species positioned walls, respectively. However, they can be distinguished in different clades or clusters. easily according to the length and basic shape of the When we look at the dendrogram, the species are achenes and the indumentum of the pericarps. In contrast mainly separated according to 2 main clades. The first to our expectations, specifically, taxa like C. cariensis subsp. main clade includes only 3 taxa. A crowded second main maculiceps and C. aggregata subsp. albida, belonging to clade consists of 28 taxa, mainly located in 2 subclades. different species, occur far from their sisters in the same The 3 primary species of the first main clade are basically cluster, which leads one to question the naturalness of the

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Figure 7. Scanning electron micrographs of the investigated Centaurea taxa: C. virgata (a–c), C. sivasica (d–f), C. diffusa (g–i), C. spinosa var. spinosa (j–l). previous classification based on morphological studies. neighbor, with a similarity rate of 62%. In the sister cluster, The taxa positioned within the second main clade can be 8 species share the same cell shape, described by distinct divided into 2 main subclades by a rate of 38%. In the first cells. The species could be assessed within 2 subclusters. subclade, the taxa can be evaluated in 2 clusters separated In the first cluster, 3 endemic species, C. ertugruliana, C. by a similarity rate of 50%. Outside of this first subclade, wiedemanniana, and C. pinetorum, occur together, with a 3 taxa occur together. Taxa (C. cariensis subsp. cariensis similarity rate of 66%. Taxonomically, 2 very close species, and C. aggregata subsp. aggregata) belonging to 2 different C. wiedemanniana and C. ertugruliana, show a high species occur together, with a similarity rate of 85%, similarity (77%) in terms of achene micromorphology. and have a high proportion of common achene surface There are very limited micromorphological differences characteristics, which is quite surprising. In this cluster, C. between them, resulting from differences in their pappus spinosa var. spinosa is also positioned near these 2 taxa as a length and ornamentation. In the second cluster, 5 species

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Figure 8. A dendrogram illustrating taxonomic relationships of the Acrolophus taxa according to seed surface micromorphologies. occur together, with a similarity rate of 59%. Among sivasica, are devoid of a pappus and they are closely related these species, C. yozgatensis and C. cariensis subsp. species, with a similarity rate of 85%. On the other hand, longipapposa are the most similar species with regards to the remaining 10 species occur within a sister cluster their micromorphologies, with a similarity rate of 77%. and they show a relatively lower similarity rate of around C. werneri occurs as a neighbor to these 2 species, with a 57%. C. sipylea and C. tuzgoluensis are the most different similarity rate of 70%. C. yozgatensis and C. werneri, living species of this cluster and the similarity rate between them on rocks, are taxonomically 2 very close local endemic is around 69%. Compared to other relatives occurring in species and are fairly similar in terms of the general view the same cluster, these species are distinguished by their of their appendage shapes as well as their achene surface achene having irregular ornamentation and the presence features. In addition, 2 subtaxa of C. cariensis occur in of a pappus. The most similar species of this cluster are C. the same cluster, with a similarity rate of 70%. It is very zeybekii and C. cuneifolia, with a similarity rate of 85%. This strange that the subtaxa of C. cariensis are positioned in similarity based on the achene surface of these species is different subclades or clusters, and this raises the question highly correlated with their general morphologies. C. kilaea of whether new taxonomic regulations for subranks of is closely related to these 2 species and its similarity is these taxa are required or not. around 77%. As neighbors to these, C. inermis, and, finally, In the sister subclade, 17 species occur in 2 clusters describing a species in the flora of Turkey, C. ulrichiorum, with a similarity rate of 43%. In the first cluster, 7 species occur together near the former cluster, with similarity are positioned together, with a similarity rate of 45%, and rate of 62%, and the relationship between these species they share similar achene characteristics like a swollen can be considered relatively low compared to the previous pericarp and fair reducing. Outside of this cluster, 2 species information. The remaining 2 species of this cluster, C. with distinct spines, C. diffusa and C. cariensiformis, occur anthemifolia and C. consanguinea, take positions relatively together, with a similarity rate of 45%. Of these, C. diffusa is far from the others, with a similarity rate of 61%. a common species and it is easily recognized by its whitish cream flowers. C. dichroa, located near these species, is 4. Discussion separated by a similarity rate of 57%. This species, with Achene micromorphological characters have been found quite remarkable sulfur-yellow flowers, is also fairly distinct useful in systematics of the family Asteraceae (Dittrich, morphologically. Located in the inner part of this cluster are 1985; Abid and Qaiser, 2007, 2009; Garg and Sharma, 7 species that are quite characteristic with reduced pappus 2007; Akcin and Akcin, 2010, 2014). or lack of a pappus. Of these species, morphologically The genusCentaurea was previously revised by speaking, 2 very different species, C. olympica and C. Wagenitz (1975) for the flora of Turkey. However, this

160 CANDAN et al. / Turk J Bot study gave limited information about the descriptive Pulicaria angustifolia (Paria and Chinya, 1998; Abid and achene macromorphological features as regards the section Qaiser, 2007, 2009; Abid and Zehra, 2007; Swelankomo Acrolophus of Centaurea (Wagenitz, 1975). Our work is the et al., 2007). The loss or significant reduction of the first important study that explains in detail the external pappus is not only important for taxonomy of the section, morphology and the ultrastructural formation of mature but it is also a good example of adaptation to changing achenes for Acrolophus (Centaurea) taxa by using SEM. As environmental factors and habitats. reported in previous studies (Celik et al., 2005a; Aksoy et The members of the family Asteraceae (Achillea, al., 2010; Bancheva and Gorgorov, 2010; Shabestari et al., Anthemis, Artemisia, Chrysanthemum) include several 2013; Bona, 2014), Centaurea species have great variation pappus types (Grubert, 1974; Huang et al. 2000; in terms of achene micromorphology, which has great Kreitschitz and Valles, 2007; Akcin and Akcin, 2010, 2014; importance in identifying the species taxonomically. Kreitschitz, 2012). C. virgata and its relatives have fairly Unlike some genera of Asteraceae, Centaurea species have reduced pappus as a result of an adaptation to arid areas. smooth and ribbed achene surfaces and different types of On the other hand, achenes are epappose in C. diffusa, C. pappus that provide dispersal of seeds by wind. Despite all olympica, C. spinosa, and C. sivasica as reported before Acrolophus species having smooth achenes superficially, for Acanthospermum hispidum, Caesulia axillaries, and it is known that ribbed achenes are found in Centaurea Spilanthes acmella (Paria and Chinya, 1998; Abid and akamantis (Georgiadis and Chatzykirakou, 1993) and C. Qaiser, 2007, 2009; Abid and Zehra, 2007; Swelankomo et benedicta (Dittrich, 1968). Similarly, achenes are ribbed al., 2007). The presence of the pappus is evaluated also as a in Amberboa ramosa and Conyza aegyptiaca as reported structural adaptation for effective dispersal by wind, which by Anderberg (1991). Apart from these, Acanthospermum plays important roles worldwide (Kulkarni, 2013), and this hispidum has a spiny achene surface, which shows is a general accepted trend for Asteraceae. Additionally, it structural adaptation for effective dispersal by animals is known as regards the pappus that its manifold forms (Kulkarni, 2013). have a protective function and may also act as dispersal The achene characters of taxonomic value are shape, mechanisms (Susanna and Garcia-Jacas, 2007). size, color, surface, and pappus (Kynclova, 1970; Ritter Slime cells are widespread in higher plants, especially and Miotto, 2006; Abid and Zehra, 2007). In this scope, in fruits and/or seeds in different families like Brassicaceae, Acrolophus species displayed a great deal of variation Euphorbiaceae, Plantaginaceae, Linaceae, Malvaceae, and related to achene characteristics including differences Lamiaceae (Western et al., 2000). According to the literature over a broad spectrum, so that they could be used for about the family Asteraceae, slime cells have been reported taxonomical diagnosis of the species within the section. in Achillea (Kreitschitz and Valles, 2007; Akcin and Akcin, Our data reveal that the ornamentation was fairly variable 2010, 2014), Anthemis L., Chrysanthemum L. (Grubert, among the studied taxa. Some authors stated that seed 1974), and Artemisia L. (Huang et al. 2000; Kreitschitz, ornamentation would be evaluated as a useful tool for 2012). As is known, slime cells play an important role in fruit taxonomic consideration. In this case, we concluded or seed dispersal and in controlling germination (Huang et that this taxonomical microcharacter could be useful al., 2000; Kreitschitz and Valles, 2007). According to our in distinguishing closely related taxa. Kreitschitz and study, there are no slime cells or mucilage observed in the Valles (2007) studied achene morphology of some taxa achenes of Acrolophus taxa. As a result, we hypothesize of Artemisia and Neopallasia and they reported that the that Acrolophus taxa do not need slime cells for their seed surface ornamentation was rugose sulcate. This feature dispersal because of having a pappus. Likewise, they do was seen on most of the taxa of this study. On the other not need mucilage for germination. This could be useful hand, some taxa had striate ornamentation. Another information for their cultivation. character with taxonomic significance, hairiness, is seen The UPGMA dendrogram based on the in many Acrolophus species as reported before for C. micromorphological characters of seed surface (Figure consanguinea and C. polyclada (Celik et al., 2005b; Uysal 8) differentiated the Acrolophus (Centaurea) species et al., 2005). As pointed out for several genera of subtribe from each other. The positions and relationships of the Centaureaineae (Amberboa Vaill., Zoegae L., Russowia Acrolophus species in the cluster were found to be suitable C.Winkl., Karvandarina Rech.f.) and some subgroups like with the classification madeearlier ​​ on a large scale. sect. Acrocentron Cass. and subgen. Cyanus Mill. (Dittrich, Systematic treatments of Acrolophus were conducted in 1968; Susanna and Garcia-Jacas, 2007), indumentums light of several approaches, using DNA sequence data could be evaluated as a general micromorphological (Garcia Jacas et al., 2006; Suárez-Santiago et al., 2007), feature within the section. Pappus is highly variable in the isozymes (Bancheva et al., 2011), and SDS-PAGE (Uysal Acrolophus species and it is of unequal length and varying et al., 2010). Phylogenetic studies of the Acrolophus color as in Amberboa ramosa, Conyza aegyptiaca, and group reported on the absence of congruence between

161 CANDAN et al. / Turk J Bot morphology and molecular data. They claimed that some for characterization at species level within the section morphological features (appendages and leaves) could not Acrolophus. The discussed micromorphological characters be used taxonomically as reliable characters with respect can be used as effective diagnostic characters to separate to the true relationships, due to an adaptive response to the close relative species in the key of the genus. More similar ecological conditions. According to these papers, importantly, the different achene and pappus morphs some morphological traits were developed convergently among the species or even subranks of the same species in several conditions and this was associated with a high could be related to their different dispersal strategies as plasticity of morphological characters. However, SDS- well as different dormancy and germination requirements, PAGE analyses revealed that there was a high congruence so that this may have occurred as a result of an evolutionary between the classical taxonomy of the genus and the total adaptation independently to different habitat types and seed protein profiles (Uysal et al., 2010). Supportively, many microclimatic conditions specific to Turkey, and in by the findings from SEM, we declare that there was a particularly endemics. meaningful correlation between the previous taxonomical classification and achene ultrastructural features of the Acknowledgments species within the section Acrolophus. We are grateful to Selçuk University (BAP-Project No: In conclusion, we can argue that the micromorphological 06401023) and Celal Bayar University (BAP-Project No: features of achene surfaces are of great importance 2008-129) for financial support of this study.

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