ACTA BOT. CROAT. 81 (1), 2022 23 Acta Bot. Croat. 81 (1), 23–31, 2022 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-028 ISSN 0365-0588 eISSN 1847-8476 Micromorphology of endemic Centaurea glaberrima subsp. divergens (Asteraceae) Milan Gavrilović*, Pedja Janaćković University of Belgrade, Faculty of Biology, Department of Morphology and Systematics of Plants, Studentski trg 16, 11000 Belgrade, Serbia Abstract – In this study, the micromorphology of the vegetative and reproductive structures of the endemic Centaurea glaberrima Tausch subsp. divergens (Vis.) Hayek (Asteraceae), using scanning electron microscope (SEM), is pre sented for the first time. Uniseriate whip-like non-glandular and biseriate glandular trichomes are found on the surface of all aboveground parts (stem, leaves, peduncles, involucral bract). On the adaxial leaf epidermis ribbed thickenings ( striation pattern) of outer periclinal cell walls, slightly curved anticlinal cell walls and anomocytic stomata are noticed. Rugose abaxial surface with thorny protuberances of the involucral bract is documented. Corolla is glabrous with longitudinally parallel epidermal cells with distinct straight outline. Isopolar, radially symmetric and tricolporate microechinate pollen grains are seen. Short stylar hairs, without cuticular striations, are present along the outer sides of the style, while the inner sides (abaxial surface) constitute the papillate stigmatic surface. Microcharacters found in cypsela are as follows: slightly ribbed body; rotund base; lateral and concave insertion; short, unicellular curly acute trichomes; smooth epidermis; fine-sulcate ornamentation; rod shaped epidermal cells with short, obtuse end walls and straight anticlinal walls; poorly developed minutely dentate pericarp rim; and dimorphic pappus with bristles of different length and morphology, with pinnules restricted to the margins of the bristles. The results obtained contribute to knowledge about the micromorphology of the studied endemic species and provide features for its better identification. The taxonomic significance of the analyzed characters is discussed. Some well defined microcharacters of the studied species might have taxonomic value. Keywords: cypsela, involucral bract, inflorescence, leaf, SEM, stem, taxonomy Introduction Centaurea L. (Asteraceae, Centaureinae, Cardueae) in- cludes nearly 250 species mainly distributed in Eurasia, es- pecially in the Irano-Turanian and Mediterranean regions. It is formed by annual, biennial or perennial herbs, less of- ten shrubs, with usually unarmed leaves (Susanna and Gar- cia-Jacas 2007). Centaurea is a genus of complex taxonomy. According to Hilpold et al. (2014) the genus can be classified into three well delimited subgenera: Centaurea, Cyanus (Mill.) Cass. ex Hayek and Lopholoma (Cass.) Dobrocz. Sub- gen. Centaurea comprises three clades, partly congruent with their geographic distribution: The Eastern Mediterra- nean Clade (EMC) comprises species often with spiny bract appendages; the Western Mediterranean Clade (WMC) comprises species with pronounced spiny bract appendages and the Circum-Mediterranean Clade (CMC) comprises species that are predominantly not or only slightly spiny in their bract appendages. Within the CMC clade, the most distinctive characters are also bract appendages (Hilpold et al. 2014). Three extreme forms of bract are recognized: membranaceous, long ciliate-fimbriate, and reduced or missing appendages. The CMC is a large group comprising, among others, the section Centaurea, which has the highest species number in the Balkans, with subsections Centaurea, Phalolepis (Cass.) Garcia-Jacas, Hilpold, Susanna & Vilat- ersana and Willkommia (Blanca) Garcia-Jacas, Hilpold, Su- sanna & Vilatersana (Hilpold et al. 2014). Centaurea glaberrima Tausch belongs to the Circum- Mediterranean Clade, sect. Centaurea, subsect. Centaurea. According to the description given by Dostál (1976) and Šilić (1990), it is a biennial or perennial plant, with erect, rigid, furrowed, glabrous stem up to 80 cm, paniculate branched below. Leaves are green, glabrous, dotted. Lower leaves are petiolate, 2-pinnatisect with subulate linear seg- ments not more than 1 mm wide. Capitula are solitary, ob- * Corresponding author e-mail: mgavrilovic@bio.bg.ac.rs GAVRILOVIĆ M., JANAĆKOVIĆ P. 24 ACTA BOT. CROAT. 81 (1), 2022 long-ovate, 10 mm long and 6 mm wide. Involucral bracts are ovoid-conical, glabrous, shiny, with brown appendages, mucronate with a 0.5 mm apex and 3–5 fimbriae 0.5 mm long on each side. Cypselae are 3 mm long, gray-blackish, shiny, slightly hairy, with pappus 1/3 as long as cypsela. Flo- rets are pink. C. glaberrima is endemic to the west Balkan Peninsula. It inhabits fields and waste places, as well as rocky cliffs in Croatia, Bosnia and Herzegovina and Mon- tenegro (Dostál 1976). Two subspecies are recognized with- in the C. glaberrima, subsp. glaberrima and subsp. divergens (Vis.) Hayek, which differ in length and number of fimbriae on the involucral bract appendages (Nikolić 2020). Micromorphological characters of leaves (Adedeji and Jewoola 2008, Gavrilović et al. 2019), involucral bracts (Rob- inson 2009, Gavrilović et al. 2019), flowers (Torres and Galetto 2007, Erbar and Leins 2015, Erbar et al. 2018) and especially those of cypselae (Dittrich 1968, Zhang et al. 2013, Ghimire et al. 2016, Kalmuk et al. 2018, Gavrilović et al. 2019, Ozcan and Akinci 2019) have provided useful in- formation for the taxonomy and phylogeny of particular plant groups in Asteraceae. Centaurea taxa were poorly in- vestigated from a micromorphological point of view. Dit- trich (1968) performed a morphological investigation on fruits of the subtribe Centaureinae and defined characters (the form of the hilum, the mechanisms for detaching the fruits (elaiosomes or simple parenchymatic tissues), the consistence of the pericarp and the hairiness of the fruit) which are important for the definition of the genera. In ad- dition, several studies have shown importance of cypsela microcharacters (e.g. surface pattern, indumentum, pappus structure) in the Centaurea taxonomy (Bona 2014, Candan et al. 2016, Ozcan and Akinci 2019). Wagenitz (1955) exam- ined pollen morphology of the Centaurea taxa and divided the genus into eight subgenera: Serratula, Centaurium, Sca- biosa, Crupina, Jacea, Dealbata, Montana and Cyanus, based on pollen shape, exine ornamentation, internal and external layers of columellae, length of colpus, pore shape and costae. However, there are a few novel studies that eval- uate the pollen morphology of Centaurea species (Özler et al. 2009, Shabestari et al. 2013, Baser et al. 2019). Centaurea glaberrima is micromorphologically com- pletely unexplored. The aims of our present study are to ex- amine: (1) the micromorphology of the vegetative and re- productive structures of C. glaberrima subsp. divergens and (2) particular micromorphological traits that might have possible taxonomic value. Materials and methods Plant material Plant material of C. glaberrima subsp. divergens was col- lected in June 2003, during the flowering period, in a natu- ral habitat: Rose, Montenegro (Fig. 1). Plants were identified according to Dostál (1976) and Nikolić (2020), while no- menclature follows Hilpold et al. (2014). Voucher specimens were deposited in the Herbarium of the University of Bel- grade - Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac” (accession number: BEOU 38399). Standard herbarium acronym follows Thiers (2019) +: Index herbariorum (http://sweetgum.nybg.org/science/ih/). Micromorphological analysis No special pre-treatments were applied in preparation for scanning electron microscopy (SEM). Dry plant parts, both vegetative (stem, leaf, peduncle) and reproductive (in- florescence, involucral bracts, petals, pollen, style and cy- psela), taken from herbarium specimens, were sputter-coat- ed with gold for 180 s at 30 mA (BAL-TEC SCD 005), and observed using a JEOL JSM-6460LV electron microscope at an acceleration voltage of 20 kV. The terminology of the cy- psela microstructure follows Barthlott (1981, 1984), Barth- lott et al. (1998) and Zhang et al. (2013), while pappus de- scription follows Small (1919) and Bean (2001). Results Stem and peduncle micromorphology Whip-like uniseriate non-glandular trichomes consist- ed of a short, upright stalk, composed of several cells, and an elongated, curled terminal cell and they are very sparse- ly distributed on the stem and peduncle (Fig. 2A-B). Large glandular trichomes of the capitate type, seen as glandular dots, were found on the stem surface (Fig. 2A-B), while their distribution was much more dense on the peduncle surface, especially near the inflorescence (Fig. 3B). Stomata with well developed outer cuticular ledge were also seen on the stem and peduncle (Fig. 2B). Leaf micromorphology Both adaxial and abaxial leaf surfaces of C. glaberrima subsp. divergens are sparsely covered with non-glandular tri- chomes, the same type as on the stem and peduncle (Fig. 2C- D). Glandular trichomes of the capitate type, which are sunk- Fig. 1. Centaurea glaberrima Tausch subsp. divergens (Vis.) Hayek at the habitat. MICROMORPHOLOGY OF CENTAUREA GLABERRIMA ACTA BOT. CROAT. 81 (1), 2022 25 en under the surface, are evident on both leaf surfaces (Fig. 2C-F). Glandular trichomes are biseriate (Fig. 2F-H). Surface of the glandular trichome is rugose (Fig. 2G). Cells of the ad- axial epidermis are polygonal in shape (Fig. 2E). Outer peri- clinal cell walls are convex (Fig. 2E) and with strongly ribbed thickenings (striation pattern) (Fig. 2E-F). Anticlinal cell Fig. 2. Stem (A-B) and leaf (C-H) micromorphology of C. glaberrima subsp. divergens. A – general view of the stem and leaf with nu- merous glandular trichomes, B – uniseriate non-glandular trichome of stem, C – adaxial leaf side, D – abaxial leaf side, E-F – sunken biseriate glandular trichomes, striation pattern and anomocytic stomata on adaxial leaf side, G – rugose surface of the glandular tri- chome, H – half of the biseriate glandular trichome. GAVRILOVIĆ M., JANAĆKOVIĆ P. 26 ACTA BOT. CROAT. 81 (1), 2022 walls are slightly curved (Fig. 2E). Anomocytic stomata, with well developed outer cuticular ledge, were observed on the adaxial and abaxial leaf surfaces (Fig. 2C-F). Involucral bract micromorphology The C. glaberrima subsp. divergens inflorescences sub- tended an involucre of overlapping bracts with several fim- Fig. 3. Inflorescence micromorphology of C. glaberrima subsp. divergens. A – general view of the inflorescence, B – peduncle with glandular trichomes, C-D – middle involucral bract with glandular trichomes, E – rugose surface with thorny protuberances of the involucral bract, F – glabrous corolla surface, G – microechinate pollen grains, H – short stylar hairs, without cuticular striations. MICROMORPHOLOGY OF CENTAUREA GLABERRIMA ACTA BOT. CROAT. 81 (1), 2022 27 briae (length more than 1 mm) on each side (Fig. 3A-C). The outermost involucral bracts are very densely covered with glandular trichomes of the biseriate capitate type, seen as glandular dots, as well as with sparsely distributed non- glandular trichomes (Fig. 3A-C). Middle involucral bracts are sparsely covered with both types of trichomes (Fig. 3C- Fig. 4. Cypsela micromorphology of C. glaberrima subsp. divergens. A – general view of the cypsela, B – lateral concave insertion, C-D – short, unicellular curly acute trichomes, D-E – fine-sulcate ornamentation of the cypselae, F – minutely dentate apical pericarp rim and dimorphic barbate-aristate pappus, G – lateral insertion of the pinnules, H – acute apex of the bristle. GAVRILOVIĆ M., JANAĆKOVIĆ P. 28 ACTA BOT. CROAT. 81 (1), 2022 D). Abaxial surface of the involucral bracts (especially seen on the middle bracts) is rugose with thorny protuberances (Fig. 3E). Small protuberances are seen over the entire sur- face (Fig. 3E). Stomata with well developed outer cuticular ledge are also observed (Fig. 3E). Corolla, pollen and style micromorphology Corolla is glabrous. Epidermal cells are longitudinally parallel to the long axis of the corolla. Corolla lacks tri- chomes. The outline of the epidermal cells is distinct (Fig. 3F). Epidermal cells are rod shaped with short, obtuse end walls. The cell boundaries are very thin and the centers of the cells are higher than the boundaries. The anticlinal walls of the epidermal cells are straight (Fig. 3F). Exploring inflorescence micromorphology, we observed isopolar, ra- dially symmetric and tricolporate pollen grains. Surface of the pollen grains is microechinate (Fig. 3G). Style is dis- tinctly bifid (branches obtuse), short-pilose above a collar of long sweeping hairs at the bifurcation point (not shown herein). Acute (expanded at the base and pointed at the up- per part) stylar hairs are present along the outer side of the stigma (Fig. 3H). No stylar cuticular striations were found on the hairs. Thus, the upper part of the style and the outer part of the dichotomous stigma formed a secondary pollen presenter. Only the inner side of the stigma (abaxial surface) was covered with papillate stigmatic surface (Fig. 3H). Cypsela micromorphology The fruit of C. glaberrima subsp. divergens is differenti- ated into cypsela and pappus (Fig. 4A). The cypsela is slight- ly ribbed and rotund at the base. Insertion of the cypsela is lateral and concave (Fig. 4A-B). Carpopodium is absent (Fig. 4A-B). Short, unicellular, curly acute trichomes are sparse- ly present on the cypsela surface (Fig. 4C-D). Cypsela lacks glandular trichomes. The elongated epidermal cells are lon- gitudinally parallel to the long axis of the cypsela (Fig. 4D- E). The cypsela coat is not roughish, the epidermis is smooth. Ornamentation of the cypsela is fine-sulcate (Fig. 4D-E). The outline of the epidermal cells of the cypsela is distinct. Epidermal cells are rod shaped with short, obtuse end walls (Fig. 4E). The cell boundaries are very thin and the centers of the cells are higher than the boundaries (Fig. 4E). The anticlinal walls of the epidermal cells are straight (Fig. 4E). Epicuticular secretion is undeveloped. The apical pericarp rim is minutely dentate, poorly developed, and the pericarp crown is totally absent (Fig. 4F). At the upper por- tion of cypsela, a dimorphic barbate-aristate pappus, con- sisting of free, numerous bristles of different size and shape, shorter than cypsela, is present (Fig. 4A, F). The outer pap- pus is pluriseriate, formed by short triangular bristles and long narrow, subulate, barbellate bristles, while the inner pappus is very reduced, almost vestigial, formed by trian- gular bristles (Fig. 4F). Pinnules (pectines) are restricted to the margins of the bristles. Both the ventral and dorsal bris- tle surfaces are smooth (Fig. 4G). Apex of the bristle is acute (Fig. 4H). Discussion We have shown here that the studied species possesses sparsely distributed uniseriate non-glandular and numerous biseriate glandular trichomes on the aboveground vegetative parts, although the species name of this taxon derived from Latin word glaber (hairless, smooth) which means without indumentum. Non-glandular trichomes in Asteraceae ex- hibit a wide range of structural types. Rahiminejad et al. (2010) noted that the type and density of leaf and stem epi- dermal indumentum were of particular taxonomic value for Centaurea species. Metcalfe and Chalk (1972) described uni- seriate trichomes with a long terminal cell characteristic for Centaurea, which is in accordance with our results. How- ever, two types of non-glandular trichomes, with thick- walled and thin-walled basal cells, were recorded on the leaves of C. sadleriana Janka (Luković et al. 2013), C. rupes- tris L. and C. fritschii Hayek (Rusak et al. 1992) and C. cya- nus L. (Haratym et al. 2020). In addition, Haratym et al. (2020) documented two types of non-glandular multicellular trichomes on the stem surface of C. cyanus. Capitate trichomes are found on stem, peduncle and on both leaf sides of the examined species. According to Rob- inson (2009) the presence of glandular trichomes of the short-stalked capitate type is a widely distributed microchar- acter in Asteraceae. Biseriate trichomes are very common within the family (Gavrilović et al. 2018, 2019). Glandular trichomes are found in other Centaurea species (Rusak et al. 1992, Luković et al. 2013, Haratym et al. 2020). These tri- chomes are the main sites of production and accumulation of sesquiterpene lactones (Gavrilović et al. 2018). Cnicin, a biologically active sesquiterpene lactone, has been reported as a constituent of C. glaberrima (Tešević et al. 2007). We found ribbed thickenings (striations) on the surface of the adaxial leaf epidermis of the studied species which might have taxonomic value as was previously stated by sev- eral authors (Adedeji and Jewoola 2008, Gavrilović et al. 2019). Rugose cuticle was observed on the lamina epidermis of Centaurea sadleriana (Luković et al. 2013). Four types of stomata have been reported in Asteraceae: anomocytic, brachyparacytic, anisocytic and diacytic (Adedeji and Jew- oola 2008). We observed the anomocytic type of stomata in the examined species which was also found in C. sadleriana (Luković et al. 2013), C. rupestris and C. fritschii (Rusak et al. 1992). Slightly curved anticlinal cell walls of dorsal epi- dermis observed in the studied species are found also in C. rupestris (Rusak et al. 1992). Our results showed that the microcharacters of involu- cral bracts (rugose surface with thorny protuberances, pres- ence of glandular and non-glandular trichomes) might have taxonomic value. Hilpold et al. (2014) stated that the main morphological character used for systematics within Cen- taurea is the form of the scarious bract appendages. For the first time, our results provide information about such a sur- face of the C. glaberrima subsp. divergens involucral bracts. Haratym et al. (2020) found numerous pointed unicellular non-glandular trichomes on the edges of each tooth of the MICROMORPHOLOGY OF CENTAUREA GLABERRIMA ACTA BOT. CROAT. 81 (1), 2022 29 bract of C. cyanus. Also on the bract abaxial surface they found two types of non-glandular trichomes as well as bi- seriate glandular trichomes. Glandular trichomes are also found on Xeranthemum cylindraceum Sm., but not on X. annuum L. involucral bracts (Gavrilović et al. 2017). The taxonomic significance of epidermal and cuticular structures of the corolla has been pointed out by Baagøe (1977). Häffner (2000) stated that the patterns of the dorsal epidermis are usually homogeneous within the genera, whereas the ventral epidermis is more variable. We found a glabrous and smooth surface of the dorsal corolla epidermis of the studied species. Haratym et al. (2020) have shown a crested pattern on the epidermal surface of the ligulate flow- ers of C. cyanus. We documented straight anticlinal cell walls of the corolla epidermis in contrast to the undulate cell walls in all Centaureinae members that Häffner (2000) investigated. General morphology of pollen of the studied species is found to be in agreement with previous comprehensive pal- ynological studies of Centaurea taxa (Wagenitz 1955, Özler et al. 2009, Baser et al. 2019). Wagenitz (1955) stated that al- most all Centaurea species belonging to the sect. Acrolophus possess Jacea pollen type with reduced spines which is in accordance with our results. However, further methods (ac- etolysis technique, tansmission electron microscopy (TEM)) and investigation of C. glaberrima subsp. divergens pollen is needed to determine additional features (e.g., shape, exine structure, the presence of single- or double-layer columel- lae) which proved to have value in Centaurea taxonomy (Wagenitz 1955, Shabestari et al. 2013, Özler et al. 2009, Baser et al. 2019). Our findings indicate that the style morphology of C. glaberrima subsp. divergens resembles that of other Cardue- ae members: a ring of sweeping hairs on the shaft below the style bifurcation and internal stigmatic surface is a general character which is shared by all Cardueae species (Susanna and Garcia-Jacas 2009). Torres and Galetto (2007) suggest- ed that style morphological characteristics of Asteraceae are far more diverse and represent a highly complex system. Er- bar and Leins (2015) observed and described five different cuticular patterns on stylar hairs which could be helpful in phylogenetic classification of Asteraceae. However, we did not observe any of the described patterns in the C. glaber- rima subsp. divergens style. Even though a longitudinal cu- ticular pattern was detected on the stylar hairs in C. cyanus (Haratym et al. 2020), many members of the Cardueae have no stylar cuticular striations (Erbar and Leins 2015). In addition, Erbar et al. (2018) examined style morphology in all Carduoideae tribes and determined 10 new style types in view of shape, bifurcation and distribution of stylar hairs and stigmatic tissue. It was shown that all of the Centaurea species, except C. macrocephala Muss. Puschk. ex Willd., possess Centaurea type styles, which is in accordance with our results. Although, cypselae are glabrous in most representatives of the Centaureinae (Herrando-Moraira et al. 2019) we found sparsely distributed uniseriate trichomes on the cy- pselae surface, which is in agreement with Dittrich (1977) who pointed out that Centaurea taxa possess cypselae with monocellular trichomes of different length, which are di- rectly fixed on the pericarp epidermis. In addition, Candan et al. (2016) documented hairy pericarp of cypselae of the investigated species of sect. Acrolophus (Cass.) DC (Centau- rea). Susanna and Garcia-Jacas (2009) stated that majority of genera in Centaureinae possess concave, lateral-adaxial in- sertion areole, often with an elaiosome, which is in accor- dance with our results (although we did not observed elaio- some, it was probably fallen out). Subbasal-lateral insertion was also found by Dittrich (1968) and Ozcan and Akinci (2019) for all investigated Centaurea species. A character that is evident in the subtribe Centaureinae is the presence of a smooth pericarp (Herrando-Moraira et al. 2019, Ozcan and Akinci 2019) which is in accordance with our study. It was shown that the different cellular patterns found in Centaurea pericarp might have diagnostic value (Candan et al. 2016, Ozcan and Akinci 2019). Rod shaped epidermal cells found in examined species are also found in all investigated Cen- taurea taxa (Ozcan and Akinci 2019). Minutely dentate api- cal pericarp rim found in studied species are also present in C. melitensis L. and C. solstitialis L. (Bean 2001). Pappus usu- ally in two structurally different rows (double pappus) are common in Centaureinae (Dittrich 1968, Susanna and Gar- cia-Jacas 2009). In the examined species pappus is dimor- phic: the outer is pluriseriate and the inner very reduced, al- most vestigial, formed by triangular bristles. However, Ozcan and Akinci (2019) have shown that all investigated Centaurea taxa possess the same structures of the pappus bristles, only the bristle lengths in the outer and inner row are different. Candan et al. (2016) documented scabrous or barbellate pappus bristles in Centaurea taxa from the sect. Acrolopus. Häffner (2000) classified genera into three groups based on the bristle cross section and the location of the pin- nules on the bristles. Pappus bristles of the examined species belong to the third type, which is characterized by dorsoven- trally compressed bristles with rows of pinnules (pectines) at the margins, while both surfaces are smooth. However, Bean (2001) found pinnules borne laterally and dorsally in bristles of C. melitensis and C. solstitialis. The present study is the first, and a comprehensive, re- port on the micromorphology of endemic Centaurea gla- berrima subsp. divergens. Micromorphological features of the leaf epidermis (striation pattern, slightly curved anti- clinal cell walls, uniseriate non-glandular and biseriate glandular trichomes), involucral bracts (rugose surface with thorny protuberances, uniseriate non-glandular and biseriate glandular trichomes), corolla (glabrous surface and straight anticlinal cell walls), style (absence of cuticular striations of stylar hairs) and cypsela (minutely dentate api- cal pericarp rim, dimorphic pappus with reduced inner pappus, lateral insertion of the pinnules) might have taxo- nomic value and contribute to better species identification. Micromorphological studies of other related Centaurea taxa should be continued in order to evaluate their phylo- genetic importance. GAVRILOVIĆ M., JANAĆKOVIĆ P. 30 ACTA BOT. CROAT. 81 (1), 2022 Acknowledgments Financial support was provided by the Ministry of Edu- cation, Science and Technological Development of the Re- public of Serbia (451-03-9/2021-14/ 200178). 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