Acta Botanica 2-2016 - za web.indd ACTA BOT. CROAT. 75 (2), 2016 173 Acta Bot. Croat. 75 (2), 173–178, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0037 ISSN 0365-0588 eISSN 1847-8476 Micromorphological, anatomical and cytogenetical studies in endemic Crepis macropus Boiss. & Heldr. (Asteraceae) from Turkey Huseyin Inceer*, Nursen Aksu Kalmuk, Kemal Vehbi Imamoglu, Ozge Duman, Sema Hayirlioglu-Ayaz, Gokhan Arslan Karadeniz Technical University, Faculty of Sciences, Department of Biology, 61080 Trabzon, Turkey Abstract – In the present study, the micromorphological structure of achene, pappus and style using scanning electron microscope (SEM), stomatal characteristics, anatomy of stem and achene together with chromosome number and nuclear DNA content of the Turkish endemic Crepis macropus Boiss. & Heldr. are provided in order to expand knowledge of its taxonomy. The SEM studies in this species show that dense spiny cells are found on the achene surface, the pappus bristle has 3–5 spikes and the style possesses slender papillae. The stem structure is composed of epidermis, collenchyma, parenchymatous cortex and pith. The species has ano- mocytic stomata in both the upper and the lower surface of the leaves. The pericarp of the achene is mainly composed of several layers of sclerenchymatous cells. In this species, the chromosome number is 2n = 2x = 8, karyotype consists of two submetacentric and six subtelocentric chromosomes and nuclear DNA content (2C- value) is 12.96 pg. These data are presented here for the fi rst time and their taxonomic values are discussed. Keywords: anatomy, chromosome, Crepis, endemic, micromorphology, nuclear DNA content, Turkey. * Corresponding author, e-mail: inceer@ktu.edu.tr Introduction The genus Crepis L. belongs to the tribe Cichorieae of the Asteraceae family and comprises over 200 species (Bremer 1994). Its species are distributed throughout the northern hemisphere with single species occurring in South East Asia. Some species also occur in different regions of Africa, the Canary Islands and Madeira (Enke 2008). It is thought that the origin of the genus Crepis is in the Altai/ Tien Shan region in the Central Asia (Babcock 1947a). The genus presently has its highest species diversity in the cir- cum-Mediterranean area (Enke 2008). In the Turkish fl ora, the genus is represented by 42 taxa and eight taxa of them are endemic to Turkey (Ekim 2012). Crepis is a problematic genus from a taxonomical point of view and it is notorious for its lack of discriminating characters (Enke 2008). Polymorphism is common in the genus and many taxonomic characters vary more within a species than between closely related species, and this often leads to unclear species-specifi c boundaries. Additionally, some species of Crepis are especially similar to Hieracium and Lapsana in their morphological characteristics and are also similar to many other Cichorieae in their habits. There- fore, they have been confused both taxonomically and no- menclaturally with each other and other Cichorieae genera. The achenes of Cichorieae are in many cases indispens- able for the identifi cation of the genera and species and pro- vide the systematically most valuable features on all taxo- nomic levels (Kilian et al. 2008). As studies in the Cichorieae have shown before (Pak and Kawano 1990, Pak 1993, Pak et al. 2001, Zhu et al. 2006, Sennikov and Illarionova 2008), achene anatomy is helpful in the generic delimitation and infrageneric classifi cation of Crepis (Enke 2008, 2009, Jana and Mukherjee 2012). In general the achene surface features are also taxonomically valuable, mainly at species level, and more rarely concur with supraspecifi c delimitation (Kilian et al. 2008). The pappus has always been an important feature for the discrimination of groups on all taxonomic levels in the Cichorieae (Kilian et al. 2008). The style morphology has been an important morphological character in major clade delimitation of the Asteraceae (Bremer 1996). Some taxonomic value of micromorphology of the pappus and style has been reported in Crepis (Enke 2008, 2009). Stem and leaf anatomy are considered as diacritical character in some groups of the tribe Cichorieae (Carlquist 1967), but there are rare data on stem and leaf anatomy of Crepis in the literature (Metcalfe and Chalk 1979). The relevance of cytological and cytogenetical informa- tion to knowledge of the taxonomy and evolution of Crepis INCEER H., AKSU KALMUK N., VEHBI IMAMOGLU K., DUMAN O., HAYIRLIOGLU-AYAZ S., ARSLAN G. 174 ACTA BOT. CROAT. 75 (2), 2016 was noted long ago. Karyotype and genome size similari- ties and differences, especially chromosome number have been used as criteria to infer species relationships in Crepis (Babcock 1947a, b, Siljak-Yakovlev and Cartier 1986, Sil- jak-Yakovlev and Wraber 1988, Kamari et al. 1991, Godelle et al. 1993, Dimitrova and Greilhuber 2000, 2001, Siljak- Yakovlev et al. 2010, Enke et al. 2011, 2015). Most of the micromorphological, anatomical and cyto- genetical studies conducted in Crepis have concentrated on common species, with some work having been interested in endemic species (Kamari et al. 1991, Kamari 1992, Enke 2009, Enke et al. 2011, Siljak-Yakovlev and Peruzzi 2012). To our knowledge, except the chromosome counting of C. dioritica (2n = 8, Davis et al. 1988) in Turkey, no micro- morphological, anatomical and cytogenetic studies have been reported for Turkish endemic species of Crepis. Crepis macropus is an endemic species that belongs to section Berinia (Babcock 1947a, b, Enke 2009). This en- demic species is distributed in central, north and north-west Anatolia. It grows on chalky, dry, stony or rocky slopes, steppe and fi eld sides at altitudes from 300 to 1600 m (Bab- cock 1947b, Lamond 1975). Crepis macropus exhibits clos- er similarities to its Balkan relatives, C. turcica and C. al- banica, than to those of Anatolia, but it is more like C. turcica based on its habit, involucres, fl orets and achenes (Babcock 1947b). According to Babcock (1947a), C. mac- ropus is an “intermediate” type with regard to its phyloge- netic position in the genus Crepis, and there is strong posi- tive correlation between this advanced type and adaptation to low altitude and arid environment, and it is relatively young species. Thus, C. macropus might be neo-endemic and according to the terminology Siljak-Yakovlev and Pe- ruzzi (2012), it can be considered schizoendemic. According to the relevant literature records, except for molecular analysis based on ITS and chloroplast matK se- quences (Enke and Gemeinholzer 2008, Enke 2009), the micromorphological, anatomical and cytogenetical charac- teristics of C. macropus have not yet been studied. We aimed to give a detailed account of the micromorphological structure of achene, pappus and style, leaf epidermal char- acteristics, achene and stem anatomy, karyotype and nucle- ar DNA content for this Turkish endemic species. Material and methods Plant materials Plant samples were collected from natural populations in 2013 and 2014 from Ankara (Beynam Forest, rocky slopes, 1320 m a.s.l., June, 11th, 2013, achene and pappus for micromorphological observations; leaf, stem and achene for anatomical observations; achene for cytogenetic analy- sis: Inceer 1009), Çankırı (near Çakmak Village, rocky slopes, 885 m a.s.l., June, 27th, 2014, leaf for cytogenetic analysis: Aksu 196) and Nevşehir (Ürgüp National Park, 1095 m a.s.l, July, 1st, 2013, capitulum for micromorpho- logical observations of style: Inceer 1015) provinces. Vouchers are deposited in the herbarium at the Karadeniz Technical University, Department of the Biology (KTUB). Scanning electron microscopy Micromorphological structure of achene, pappus and style was analyzed on the scanning electron microscope (SEM). Dry and mature specimens were mounted directly on the stubs using double-sided adhesive tape, and then ob- served in Agilent FESEM 8500 scanning electron micro- scope. The micromorphological characterization was per- formed according to Enke (2008). Anatomical studies Anatomical observations were performed in the leaf, stem and achene. For this purpose, peripheral sections of upper (adaxial) and lower (abaxial) epidermis of the leaves were taken by hand using commercial razor blades from fi xing materials in FAA (5% formaldehyde : 5% glacial ace- tic acid : 70% ethyl alcohol). Semi-permanent slides were mounted in glycerine. Stomatal index was assessed in both the upper and lower epidermis (Meidner and Mansfi eld 1968). Transverse sections from middle parts of the stem were taken by hand using commercial razor blades from fi xing materials in formaldehyde-glacial acetic acid-ethyl alco- hol and were stained with safranin and then mounted in en- tellan. Transverse sections of achene were carried out with the paraffi n method (Algan 1981) and stained with hema- toxylin and then mounted in entellan. Three slides obtained from three individuals were prepared and the anatomical characters were measured using an ocular micrometer un- der the light microscope. The photographs were taken using a Leica DM 4000 microscope and a Leica DFC 490 digital camera. Illustrations were drawn with a lucida camera at- tached to a light microscope. Cytogenetic analyses The root tip meristems obtained directly from germi- nated achenes were used for chromosome analysis. The root tips were pre-treated with 0.05% aqueous colchicine solution for 3–5 h at room temperature and then fi xed in absolute ethanol-glacial acetic acid (3:1) for at least 24 h at 4 °C (Inceer and Hayirlioglu-Ayaz 2007). They were hy- drolyzed in 1 N HCl at 60 °C for 12–15 minutes. Staining was carried out in 1% lacto-propionic orcein for 12–18 h at room temperature and squash preparations were made in 45% acetic acid. The best metaphase plate of each individual was photo- graphed with a Leica DM 4000 microscope with a Leica DFC 490 digital camera. Five well-spread metaphase plates of different fi ve individuals were used for chromosome analysis and idiogram building. Karyotype analysis was performed according to Levan et al. (1964). Idiograms were prepared based on the average measurements of each chro- mosome pair. Chromosomes were classifi ed on the basis of arm ratio in accordance with Stebbins (1971). The intra- chromosomal asymmetry index (A1) was calculated by use of the formula proposed by Romero Zarco (1986), and the interchromosomal asymmetry index (A2) was measured as the ratio of chromosome length/mean chromosome length. In haploid idiograms, chromosomes were arranged accord- ing their length. STUDIES ON CREPIS MACROPUS FROM TURKEY ACTA BOT. CROAT. 75 (2), 2016 175 The young leaves were taken from three specimens in natural population for fl ow cytometric analysis. The leaves of Lycopersicon esculentum cv. ‘Swanson’ (2 pg/2C) potted and grown were used as an internal standard. Nuclear DNA content was assessed by fl ow cytometry as follows. Leaf fragments of the sample plant and the standard plant were chopped using a razor blade in 1 mL of woody plant buffer (Loureiro et al. 2007; 0.2 M Tris HCl, 4 mM MgCl2×6H2O, 2 mM Na2EDTA×2H2O, 86 mM NaCl, 10 mM potassium metabisulphite, 1% PVP-10, 1% (v/v) Triton X-100, pH 7.5) supplemented with 50 μg mL–1 propidium iodide and 50 μg mL–1 DNAse-free RNAse, fi ltered through a 30 μm mesh and stored on ice, in the dark, until measurement. Three independent samples were extracted, fi ltered and measured on the same day. The measurements were made three con- secutive days using BD Accuri™ C6. Usually 10,000 nu- clei per sample were analyzed for nuclear DNA content. Results Micromorphology In this species, all achene surfaces have prominent spiny cells (Figs. 1a, b). The pappus bristles are slender and made up of 3–5 cells in diameter. The pappus bristles have 3–5 spikes per 100 μm, diameter is 21–44 μm (Fig. 1c). The style and the style branches consist of papillae. The papillae are type A and more densely arranged in the style. The style diameter is 102.23 ± 2.91 μm, and the diameter of its arm is 119.43 ± 12.72 μm (Figs. 1d, e). Anatomy In the leaves of C. macropus, there is a single layered isodiametric epidermis with wavy walls on both the adaxial and the abaxial surface. Adaxial epidermal cells are more or less equal to the abaxial ones. Stomata are present on both surfaces of the leaf (amphistomatic) and anomocytic type (stomata without subsidiary cells). They lie more or less at the epidermis level. The guard cells are oval-shaped. The stomata on both the adaxial and abaxial sides are almost the same size. On the adaxial side, the size of stomata is 26.43 ± 0.65 × 37.66 ± 2.17 μm, on the abaxial side, 26.68 ± 0.80 × 37.74 ± 1.37 μm (Figs. 2b, c). However, the stomatal in- dex on the abaxial side is 18.06, whereas it is 14.80 on the adaxial side. The stem of C. macropus is more or less round in the transverse sections. In its stem anatomy, the epidermis con- sists of single layered, roundish-ovate, frequently arranged cells and is surrounded by a more or less thick cuticle layer. Cells dimensions are 15.86 ± 03.02 × 13.42 ± 01.29 μm. Collenchyma is located below the epidermis. It has 2–3 lay- ers at the margins and its thickness is 183.20 ± 10.04 μm. Cortex is composed of 7–8 rows, parenchymatous and oval cells. Its thickness is 183.20 ± 10.04 μm. Vascular bundles are collateral, scattered in a circular order in the ground tis- sue. Phloem and xylem members are clear. The phloem thickness is 95.36 ± 07.81 μm, whereas the xylem thickness is 194.18 ± 15.29 μm. Width of the vascular bundle is 113.66 ± 14.89 μm. The cambium formation is distinguish- able in the vascular bundles. The pith of the stem consists of large, round parenchymatic cells (Fig. 2a). The achene of the species has a rounded outline with 12 ribs consisting of sclerenchymatous cell bundles in the transverse sections. The ribs are prominent without inter rib furrows. Pericarp consists of exocarp, mesocarp and endo- carp. Exocarp is one layered, with a thick outer cell wall, but the cells are collapsed. Mesocarp only consists of scler- enchymatic bundles and cells. Endocarp is two layered and collapsed. The pericarp thickness is 91.09 ± 17.84 μm, and its width is 96.79 ± 19.74 μm. Testa is circular. Its thickness is 2.44 ± 0.14 μm. Endosperm consists of two layered cells. The thickness of the endosperm is 10.30 ± 0.47 μm. Em- bryo is formed by more or less oval or round, parenchy- matic cells. The orientation of cotyledons is at right angles to the axis of achene (Fig. 2d). Cytogenetics The chromosome number of C. macropus is 2n = 2x = 8. The two chromosome types in its karyotype are distin- Fig. 1. Scanning electron microscope micrographs of Crepis mac- ropus: a, b) achene (Inceer 1009), c) pappus (Inceer 1009), d) style (Inceer 1015), e) style arm (Inceer 1015), scale bars: a, d) = 100 μm; b, e) = 10 μm; c) = 40 μm. Fig. 2. Anatomical structure of Crepis macropus (Inceer 1009): a) stem, Cl – collenchyma, Cr – cortex, E – epidermis, Ph – phloem, Xy – xylem, scale bar = 100 μm, b) abaxial surface in the leaves, c) adaxial surface in the leaves, scale bars (b–c) = 50 μm, d) achene, Ct – cotyledons, Es – endosperm, Sc – sclerenchyma, T – testa, scale bar = 50 μm. INCEER H., AKSU KALMUK N., VEHBI IMAMOGLU K., DUMAN O., HAYIRLIOGLU-AYAZ S., ARSLAN G. 176 ACTA BOT. CROAT. 75 (2), 2016 guished: one submetacentric and three subtelocentric chro- mosome pairs. The chromosome length ranges from 5.26 to 7.64 μm. The relative length ranges from 20.25 to 29.42. (Tab. 1, Figs. 3a, b). The classis of karyotype asymmetry is placed in 4A, the index of A1 is 0.69 and the index of A2 is 0.15. The nuclear DNA content (2C-value) of this endemic species is 12.96 ± 0.11 pg. Its monoploid genome size (1C- value) is 6.48 pg (Fig. 4). Discussion The results obtained from micromorphological, anatom- ical and cytogenetical studies on C. macropus are presented in Tab. 1 and Figs. 1–4. Our fi ndings are in agreement with the previous results on the other species of the genus (Bab- cock 1947a, b, Metcalfe and Chalk 1979, Enke and Ge- meinholzer 2008, Enke 2009, Enke et al. 2011, 2015, Yildirim et al. 2011). The additional morphological, ana- tomical and cytogenetical characters supporting systematic delimitation of the genus have been used because the mo- lecular analyses by Enke (2009) could not support the cur- rent taxonomic sections (Babcock 1947b). Enke (2008) pointed that the surface features of achene, pappus and style are taxonomically valuable, mainly at spe- cies level in Crepis. The present morphological analyses show that the achenes of C. macropus have prominent spiny cells on its surface. The pappus is comparatively homoge- nous in the species and it confi rms the general description presented by Enke (2008). Additionally, Enke (2009) re- ported three papillae types as “type A, type B and type C” in style and style branches. The papillae type in the style and style branches of C. macropus is type A in this species. Similar results are reported by Enke (2008) for C. leont- odontoides and C. zacintha. Many anatomical characters of the leaves are useful for systematics, particularly the epidermal cells and stomata (Metcalfe and Chalk 1979, Dickison 2000, Araújo et al. 2010, Inceer and Ozcan 2011). Paradermal sections taken from the leaves show that C. macropus has isodiametric epidermal cells together with anomocytic stomata. The epi- dermal cell walls are also of the same structure, with anti- clinal undulate walls on both the adaxial and abaxial sur- face. These characteristics agree with the results previously reported by Metcalfe and Chalk (1979) and Inceer and Oz- can (2011) for the family Asteraceae. Metcalfe and Chalk (1979) reported that vascular bun- dles are taxonomically important and cambium can occur in the member of Asteraceae family. The stem anatomy of C. macropus is composed of epidermis, cortex, corticular vas- cular bundles with distinguishable cambium formation and parenchymatic pith. The present results are in agreement with previous data of Metcalfe and Chalk (1979). The achene anatomy has proven so far to be of some relevance for classifi cation of the Crepis species. Enke (2009) reported four different achene anatomy types, “type Fig. 3. Somatic metaphase of Crepis macropus (Inceer 1009): a) microphotograph of mitotic chromosome plate (2n = 8), b) hap- loid idiogram, scale bar = 10 μm. Fig. 4. Flow cytometry histograms: A) Peak of standard Lycoper- sicon esculentum cv. ‘Swanson’ (2C = 2.00 pg), B) Peak of Crepis macropus, Aksu 196 (2C = 12.96 pg). Tab. 1. Morphometric data of karyotype of Crepis macropus. Data are arithmetic mean ± standard deviation, n = 5. S – short arm length, L – long arm length, T – total length, L/S – arm ratio, SAT – satellite, CI – centromeric index, RL – relative length, sm – submetacentric, st – subtelocentric. Chromosome pairs S L T L/S SAT CI RL Chromosome type(μm) (μm) (μm) (μm) 1 5.28±0.48 2.36±0.25 7.64±0.53 2.24 – 30.89 29.42 sm 2 5.16±0.14 1.53±0.52 6.69±0.54 3.37 – 22.87 25.76 st 3 4.95±0.26 1.43±0.32 6.38±0.59 3.46 – 22.41 24.57 st 4 4.32±0.10 0.94±0.25 5.26±0.26 4.60 – 17.87 20.25 st STUDIES ON CREPIS MACROPUS FROM TURKEY ACTA BOT. CROAT. 75 (2), 2016 177 I, type II, type III and type IV” in Crepis. We observed that C. macropus possesses type IV in its achene anatomy. Ac- cording to Enke (2009), in type IV, exocarp can be col- lapsed and the costae are very prominent with deep or no intercostal furrows. Crepis is a model group for genetic studies explaining evolution and speciation in higher plants because it has low chromosome numbers. Therefore, many chromosomal studies on Crepis have been published so far (Babcock 1947a, b, Siljak-Yakovlev and Cartier 1982, Dimitrova and Greilhuber 2000, 2001). Different basic chromosome num- bers x = 3, 4, 5 and 6, are present in the genus Crepis (Bab- cock 1947a, b, Kamari 1992, Dimitrova and Greilhuber 2000, 2001, Enke 2008) and karyotypes can be symmetrical or asymmetrical (Babcock 1947b). Crepis macropus is a diploid species with 2n = 2x = 8 chromosomes. This spe- cies has the same chromosome number with the members of the section Berinia such as C. turcica, C. merxmuerlleri, C. sibthorpiana, C. sonchifolia (Nazarova 1984, Kamari et al. 1991, Kamari 1992, Constantinidis et al. 2002, Enke 2008). There is a minor difference in the chromosome mor- phology between C. macropus and C. turcica (Kamari et al. 1991). The chromosome size values in C. macropus range from 5.26 to 7.64 μm, whereas the chromosome size values range from 4.7 to 6.7 μm in C. turcica (Kamari et al. 1991). The karyotypes in both species also consist of long chromo- somes. Crepis macropus has an asymmetrical karyotype. It is widely accepted that the evolutionary trend is toward an increase in karyotype diversity (Stebbins 1971, Liu et al. 2006, Inceer et al. 2012). It was also reported that increas- ing karyotype asymmetry could occur via a shift of the cen- tromere position from median to subterminal or terminal chromosome regions, or through accumulation of differ- ences in relative size between individual chromosomes (Liu et al. 2006, Inceer et al. 2012). Despite the impressive amount of genome size studies seen the last few years (Bennet and Leitch 2005, Enke et al. 2011, Garcia et al. 2013), taxonomic coverage remains very restricted, with C-values assessed for less than 40% of the Crepis species. The nuclear DNA content of C. macropus is presented with a histogram in Fig. 4, and its genome size (1C-value) is 6.48 pg. The genome size (1C-value) in C. turcica is 6.41 pg (Enke 2009), and thus there is slight dif- ference in genome size between C. macropus and C. turci- ca. According to Leitch et al. (1998) and Soltis et al. (2003), genome sizes can be assigned to a series of distinct catego- ries: very small (1C ≤ 1.4 pg), small (1.4 < 1C ≤ 3.5 pg), intermediate 3.5 < 1C < 14 pg), large (14 ≤ 1C < 35 pg) and very large (1C ≥ 35 pg). In these terms, the endemic C. macropus has an intermediate genome. Similar results are reported in other species of Crepis (Godelle et al. 1993, Siljak-Yakovlev et al. 2010, Enke et al. 2011). The similari- ties in the nuclear DNA content as well as karyotype and external morphology of C. macropus and its relative sug- gest that their genomes evolved from a common ancestral genome and underwent some structural differentiation of the genomes. In conclusion, the present results obtained from micro- morphological, anatomical and cytogenetical analyses will increase taxonomic information about C. macropus in the literature. Furthermore, features of achene, pappus, style, karyotype and nuclear DNA value can be used as taxonom- ic characters for this Turkish endemic species. 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