SYSTEMATIC IMPLICATIONS OF ACHENE CHARACTERISTICS IN GENERA CENTAUREA L., CYANUS MILL., PSEPHELLUS CASS. AND RHAPONTICOIDES VAILL. (ASTERACEAE)

This study examines the exomorphic achene characteristics of 23 taxa belonging to Centaurea, Cyanus, Psephellus and Rhaponticoides in Asteraceae using light microscope and scanning electron microscope (SEM). The exomorphic characteristics studied are shape, size, colour and surface pattern of achene, and hair, length and colour of pappus. The results of the present investigation showed that achene size of the studied taxa is 3–7 × 1–4 mm. Pappus length is 1–14 mm, deciduous for three taxa and inner row is differentiated from the outer, 1–3 mm long, for 10 taxa. SEM studies showed eight different types of achene surface patterns for the studied taxa: Glebulate, glebulate-ruminate, reticulate, ribbed, ruminate, smooth, smooth-glebulate and undulate. Achene characteristics are useful for both intrageneric and intraspecific classification of the studied taxa. Introduction Morphological and anatomical studies on fruit and seed structure play an important role in systematics (Kumar et al., 2012). Microstructural details of the seed and fruit coat enable the distinguishing of taxa or the discovery of their affinities. This is especially useful for families in which the identification of particular taxa is complicated (Kumar et al., 2012; Bona, 2013; Piwowarczyk et al., 2014). Furthermore, observations of micromorphological features can also provide us with information about developmental strategies, adaptation to different environmental conditions and evolutionary tendencies within related groups of plants (Kreitschitz and Vallès, 2007; Moazzeni et al., 2010). The family Asteraceae is one of the largest angiosperm families and comprises about 1,300 genera and 2,500 species distributed over three subfamilies and 17 tribes (Ayad et al., 2012). The genus Centaurea L. s.l. is one of the largest genera in the family Asteraceae. Among the challenging taxonomic problems persisting in the Asteraceae is the delimitation of the genus Centaurea (Bancheva and Raimondo, 2013; Ranjbar et al., 2013). Centaurea has recently been divided into four genera, namely Centaurea, Rhaponticoides, Psephellus and Cyanus (Wagenitz and Hellwig, 2000; Greuter, 2003a, b). Cyanus, however, is not widely accepted by different authors (Susanna and Garcia-Jacas, 2007). Though previous studies support the use of achene surface patterns as diagnostic characters at species and subspecies levels for the genus Centaurea s.l., understanding the importance of these characters at generic and subgeneric levels requires further studies (Uysal et al., 2005; Çelik et al., 2005a, b; Aksoy et al., 2010; Okay and Demir, 2010; Shabestari et al., 2013; Bona, 2014; Candan et al., 2015). With this in mind, this study examines the exomorphic achene characters of 23 taxa belonging to the genera Centaurea, Cyanus, Psephellus and Rhaponticoides in Asteraceae by using light microscope (LM) and scanning electron microscope (SEM). Email: mehmetbona@gmail.com


Introduction
Morphological and anatomical studies on fruit and seed structure play an important role in systematics (Kumar et al., 2012).Microstructural details of the seed and fruit coat enable the distinguishing of taxa or the discovery of their affinities.This is especially useful for families in which the identification of particular taxa is complicated (Kumar et al., 2012;Bona, 2013;Piwowarczyk et al., 2014).Furthermore, observations of micromorphological features can also provide us with information about developmental strategies, adaptation to different environmental conditions and evolutionary tendencies within related groups of plants (Kreitschitz and Vallès, 2007;Moazzeni et al., 2010).
The family Asteraceae is one of the largest angiosperm families and comprises about 1,300 genera and 2,500 species distributed over three subfamilies and 17 tribes (Ayad et al., 2012).The genus Centaurea L. s.l. is one of the largest genera in the family Asteraceae.Among the challenging taxonomic problems persisting in the Asteraceae is the delimitation of the genus Centaurea (Bancheva and Raimondo, 2013;Ranjbar et al., 2013).Centaurea has recently been divided into four genera, namely Centaurea, Rhaponticoides, Psephellus and Cyanus (Wagenitz and Hellwig, 2000;Greuter, 2003a, b).Cyanus, however, is not widely accepted by different authors (Susanna and Garcia-Jacas, 2007).
Though previous studies support the use of achene surface patterns as diagnostic characters at species and subspecies levels for the genus Centaurea s.l., understanding the importance of these characters at generic and subgeneric levels requires further studies (Uysal et al., 2005;Çelik et al., 2005a, b;Aksoy et al., 2010;Okay and Demir, 2010;Shabestari et al., 2013;Bona, 2014;Candan et al., 2015).With this in mind, this study examines the exomorphic achene characters of 23 taxa belonging to the genera Centaurea, Cyanus, Psephellus and Rhaponticoides in Asteraceae by using light microscope (LM) and scanning electron microscope (SEM).

Materials and Methods
The materials of this study are the mature achenes of 23 Asteraceae taxa belonging to the genera Centaurea, Cyanus, Psephellus and Rhaponticoides.The collected specimens were kept at the Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Botany Herbarium (ISTE).The exomorphic characteristics of the achene are achene colour, size, shape and surface pattern, and pappus colour, hair, and length (Table 1).All these characteristics are described, illustrated and compared.Up to 50 (at least 10) mature achenes for each taxon were measured and observed under the light microscope.During scanning electron microscopy, two mature achenes were selected and mounted onto stubs with double-sided adhesive tape, and were then coated with gold.The achene surfaces were examined from the lateral sides.For each sample, photographs of testa were taken using the JEOL JSM-5600 at a magnification of 22×-50×, 1000×, and 3000×.The terminology of achene characteristics in this work is based on the descriptions used by Barthlott (1981), Stearn (1992), Koul et al. (2000), and Bojňanský and Fargašová (2007).

Results
The results of the present investigation showed that the achene size of the studied taxa is 3-7 × 1-4 mm.The pappus length is changing from 1 to 14 mm, deciduous for three taxa and the inner row is differentiated (1-3 mm long) for 10 taxa.Testa cells of all the studied taxa appear regularly arranged and elongated-parallel with the seed surface.The results of the studied taxa are distinguished below.
20. P. mucronifer (DC.)Wagenitz: Achene straw-coloured to greyish, narrowly ovate, c. 6 × 3 mm, pappus straw-coloured, scabrous, c. 4 mm long, and its inner row differentiated from the outer row, c. 1 mm long.Seed surface pattern smooth.The cell boundaries are thin and have smooth structure and the centres of the cells are placed at ± equal levels with the boundaries (Fig. 4d-f).

Discussion
Rhaponticoides wagenitziana is the only species of the studied taxa which belongs to genus Rhaponticoides.The achenes are dark brown above, yellow at base and have waved layers which are independent from the cells and cell boundaries.These two characteristics seem specific to R. wagenitziana.More work is required to confirm the use of characteristics for delimitation of the genus Rhaponticoides.
Psephellus coruhensis, P. turcicus and P. psephelloides have reticulate seed surface patterns (Duran and Hamzaoğlu, 2005;Duran et al., 2009).Our study shows that pappus length, colour and achene colour characteristics show differences at the specific level.Additionally, the achene surface pattern can be ribbed, smooth or undulate for the genus Psephellus.Because of the overlap, achene characteristics are not useful for determining generic limits in Psephellus.Despite that, achene characteristics provide strong support in the delimitation of the studied Psephellus taxa at the specific level.P. appendicigerus and P. pulcherrimus were placed into Section Aetheopappus (Wagenitz, 1975).These two taxa are easily separated from other Psephellus taxa by their ribbed achene surface pattern and their pappus, which do not have differentiated inner rows.Other studied Psephellus taxa belong to section Psepheloideae (Wagenitz, 1975) in which their inner pappus row is differentiated.These findings show that achene characteristics could be useful for sectional classification of the genus Psephellus.
There is no specific pappus length, pappus colour, achene size or colour for the genus Cyanus, but all the studied Cyanus taxa have smooth or smooth-glebulate achene surface pattern.
This study supports the use of achene surface patterns as diagnostic characters at both specific and infraspecific levels in the genus Centaurea.There are other achene characteristics, including achene length and colour, pappus length and colour, that might be helpful to distinguish more taxa.However, though achene characteristics provide strong support in the delimitation of the studied taxa at specific level, understanding the importance of these characteristics for intrageneric classification of the genus Centaurea requires further study.Centaurea drabifolia subsp.cappadocica and C. drabifolia subsp.floccosa are separated from each other based on stem length in the Flora of Turkey (Wagenitz, 1975).This study shows that achene characteristics are useful in the separation of these taxa, because pappus length, achene colour and achene surface patterns of these taxa are different from each other.The SEM study of achene surfaces showed that the cell wall in C. kurdica is thicker than that of C. sclerolepis, especially on the margins and could be a useful character in the delimitation of species (Uysal et al., 2005).
In conclusion, the achene characteristics of 23 taxa belonging to the genera Centaurea, Cyanus, Psephellus and Rhaponticoides were evaluated in this study.Results support the use of achene characteristics as diagnostic characters for intrageneric and intraspecific classification of these taxa.Achene characteristics provide strong support for the delimitation of the studied taxa at specific level, but understanding the importance of these characteristics at generic and intrageneric levels needs further studies.Hopefully, this investigation will encourage additional studies about achene morphology of this complex family to further elucidate the complex taxonomy of the Asteraceae.