54 ACTA BOT. CROAT. 84 (1), 2025 Acta Bot. Croat. 84 (1), 54–62, 2025 CODEN: ABCRA 25 DOI: 10.37427/botcro-2025-003 ISSN 0365-0588 eISSN 1847-8476 Reproductive biology of Centaurea kilaea (Asteraceae, Cardueae) – an endemic species from Türkiye Ciler Kartal Trakya University, Faculty of Science, Department of Biology, 22030 Edirne, Türkiye Abstract – In this study, the embryology of Centaurea kilaea Boiss., a species endemic to Türkiye, was examined using light microscopy. The anthers of C. kilaea are tetrasporangiate; the anther wall development is dicotyledonous; and the tapetum is amoeboid. The meiotic division of the microspore mother cells is regular, and when the pollen grains are thrown from the anthers, they are three-celled. The ovary of C. kilaea is inferior, bicarpellary, syncarpous, and unilocular, which is characteristic of the Asteraceae family. It carries only a single ovule with basal placentation. The ovule is anatropous, unitegmic, and tenuinucellate. The megaspore mother cell undergoes meiotic division, giving rise to a linear tetrad of megaspores. The chalazal megaspore remains functional, and the other three megaspores degener- ate rapidly. The functional megaspore undergoes three mitotic divisions in succession. As a result, a Polygonum-type embryo sac, with eight nuclei and seven cells, is formed. The antipodal cells persist until the first divisions of the zy- gote. In the mature embryo sac stage, the integument consists of the endothelium, peri-endothelial region, parenchy- matous cells, and outer epidermis, from the inside out. Endosperm development is initially free nuclear, becoming cellular in the globular embryo stage. Embryo development is of the asterad type. The mature seed does not contain endosperm, but the endothelium persists. Keywords: Asteraceae, Centaurea kilaea, endemic, female gametophyte, male gametophyte Introduction The genus Centaurea L., comprising more than 300 spe- cies, is one of the largest genera in the Asteraceae and is widespread across the world (Bremer 1994). Most species of the genus are distributed in the Balkan Peninsula and Tür- kiye (Siljak-Yakovlev et al. 2005). Türkiye is one of the main diversity centers of Centaurea, where it is represented by 204 species, 59% of which are endemic (Sirin et al. 2020). Centaurea kilaea Boiss. is a plant endemic to Türkiye that spreads only in the provinces of Kirklareli, Istanbul, Sa- karya, and Bolu (Wagenitz 1975). The species is classified as endangered (EN) according to the Red Data Book of Turk- ish Plants (Ekim et al. 2000). The chromosome number of the species has been reported as 2n = 4x = 36, and the amount of 2C nuclear DNA as 3.68 pg (Meric et al. 2010). Embryological studies on members of the Asteraceae family date back to earlier years (Desole 1954, Davis 1962, 1964, Renzoni 1970). Recently, the embryology of some spe- cies belonging to the Asteraceae family has been studied; some examples are Centaurea achtarovii, Arnica montana, Pilosella brzovecensis, Ageratum conyzoides, and Ageratum fastigiatum (Franca et al. 2015, Yankova-Tsvetkova et al. 2016, 2018, Bonifacio et al. 2018, Janas et al. 2021). Members of the Asteraceae family have an inferior, bicarpellate, and unilocular ovary. The ovary contains an anatropous, unitegmic, and tenuinusellate ovule with basal placentation (Davis 1966). Although most species contain Polygonum- type embryo sacs, some also include Allium-type, Drusa- type, Adoxa-type, and Fritallaria-type embryo sacs (Davis 1966, Musial et al. 2012). In members of the Asteraceae fam- ily, the anthers are tetrasporangiate. Although there is gen- erally a plasmodial type of tapetum, the existence of a secre- tory tapetum has been proven in some species (Bonifacio et al. 2018, Franca et al. 2015). Following the simultaneous type of meiosis, tetrahedral, decussate, or isobilateral type tetrads are seen. Pollen is thrown from the anther as three- celled (Davis 1966). Scientific data on C. kilaea are also very limited. The chromosome number and nuclear DNA content of the spe- cies were determined by Meric et al. (2010). In addition, an- ti-proliferative compounds of the species were isolated and Corresponding author e-mail: cilermeric@trakya.edu.tr mailto:cilermeric@trakya.edu.tr REPRODUCTIVE BIOLOGY OF CENTAUREA KILAEA ACTA BOT. CROAT. 84 (1), 2025 55 tested against human tumor cell lines (Sen et al. 2015). A literature review shows that there are no studies on the em- bryology of this species. The aim of this study is to reveal the reproductive biology of C. kilaea, which is distributed in a limited area and included in the endangered category according to the Red Data Book of Turkish Plants (Ekim et al. 2000). Materials and methods Materials Capitula containing the buds, blooms, and fruits of C. kilaea were collected from the coast of Igneada (Kirklareli, Türkiye; 41°52'53'' N, 27°59'32'' E) in July. The samples were fixed in an ethyl alcohol/acetic acid mixture (v/v, 3:1) for 24 h, then transferred to 70% ethanol and kept at 4 °C. Like- wise, pollen grains were collected from florets blooming in the same habitat. Histological method For histological observations, various sizes of anthers and ovaries were embedded in historesin according to the manufacturer’s instructions (Leica historesin-embedding kit). Longitudinal and transverse serial sections (4 μm thick) of the embedded blocks were obtained using a rotary microtome with a tungsten carbide blade. The sections were stained in 0.5% (w/v) toluidine blue O solution (in 0.1 M phosphate buffer at pH 6.8) at 60 °C for 2 min. They were washed in distilled water for 30 seconds and then dried in air (O’Brien et al. 1964, modified). Finally, they were mounted with EntellanTM (Merck) for microscopy. Histochemical method Periodic acid–Schiff reaction (PAS) was applied for the determination of insoluble polysaccharides. The 4 µm-thick sections were kept in 1% (w/v) periodic acid solution (in 90% ethyl alcohol) for 30 min and rinsed with distilled water at the end of the period. After staining with Schiff’s reagent (Fisher Chemicals) for 30 min, the slides were placed for 5 min each in 0.5% (w/v) sodium metabisulfite solution. They were washed for 5 min with running water and rinsed with distilled water. Then, the slides were dried in the air and mounted with EntellanTM (Merck). For determination of proteins, the slides were stained with 0.025% (w/v) Coo- massie Brilliant Blue G-250 (CBB) in distilled water/acetic acid/methanol (v/v/v, 87:10:3) solution for 10 min in an oven (60 °C). They were then washed for 2 min in distilled water. Then the slides were dried in air and mounted with EntellanTM (Merck) (Heslop-Harrison et al. 1973). Pollen viability Acetocarmine dye was used to investigate pollen viabil- ity. Ripe pollen grains from newly opened anthers were transferred onto a clean slide, and a few drops of aceto-car- mine were added to the slide. After 20 min, stained pollen grains were considered fertile (viable), and the unstained pollen grains sterile (non-living). A total of 4000 pollen grains were counted, and the percentage of viability was cal- culated. All observations and photography were carried out us- ing an Olympus CX21 light microscope and the KAM- ERAM software program (Argenit, Türkiye). Results Anther wall and male gametophyte development In C. kilaea, the anthers are tetrasporangiate, and the anther wall development is of the dicotyledonous type. The anther wall is thin and consists of four layers, each in a sin- gle layer of cells from the outside to the inside, as follows: the epidermis, endothecium, middle layer, and tapetum. In the early stages, the anther consists of meristematic tissue surrounded by the epidermis. The archesporial cells differ- entiate beneath the epidermis of the microsporangium and divide periclinally, forming the primary parietal cells and the sporogenous cells (Fig. 1A). The primary parietal layer undergoes a periclinal division, giving rise to two second- ary parietal layers (Fig. 1B). The cells of the outer secondary parietal layer undergo periclinal divisions, forming the en- dothecial layer (outer) and middle layer (inner). The cells of the inner secondary parietal layer differentiate directly into the tapetum (Fig. 1C). At the microspore mother cell stage, the volume of tapetum cells increases, and they begin to di- vide before the microspore mother cells (Fig. 1D). At the tetrad stage, the middle layer is flattened, and dark-colored materials appear in the cytoplasm of tapetal cells. However, the transverse inner and side walls of the tapetal cells are still intact (Fig. 1E). At the young microspore stage and the vacuolated microspore stage, the inner tangential and ra- dial walls of the tapetum cells are broken down. The tapetal protoplasmic arms extend into the interior of the locule (Fig. 1F and Fig. 1G, arrowheads). At those stages, the mid- dle layer is flattened or not observed. At the pollen mitosis stage, the tapetal protoplasts move into the locule and fuse to form the multinucleate tapetal periplasmodium. This mass surrounds the pollen grains (Fig. 1H). At the mature pollen stage, the endothecial cells radially elongate and de- velop fibrous thickenings, which arise from their inner tan- gential walls. The tapetal periplasmodium disappears. The mature anther wall consists of the epidermis and endothe- cium with fibrous thickenings (Fig. 1I). The sporogenous cells develop directly into the micro- spore mother cells in C. kilaea. They are observed with large nuclei and obvious nucleoli (Fig. 1D). Microsporocytes un- dergo simultaneous meiotic division, generating tetrahedral tetrads (Fig. 1E). Microspores released from the tetrad have a prominent nucleus in the middle of the cell (Fig. 2A). Then, the nucle- us is pushed to the side due to the vacuole formed inside the cell (Fig. 2B), and it undergoes the first pollen mitosis, re- sulting in the formation of two unequal cells, a large vegeta- KARTAL C. 56 ACTA BOT. CROAT. 84 (1), 2025 tive cell and a small generative cell (Fig. 2C and Fig. 2D). After the first mitotic division, the vacuole becomes invis- ible, and the generative cell divides to form two sperm cells (the second pollen mitosis) (Fig. 2E). Sperm cells are initial- ly round in shape, then take the form of threads (Fig. 2F). Mature pollen grains are yellow, prolate-spheroidal, tricol- porate and echinate, characteristic of the Asteraceae family. In the PAS reaction employed for staining insoluble polysaccharides in the anther wall layers and pollen, all the anther wall layers and sporogenous cells give a negative re- action in terms of polysaccharide content at the beginning of development (Fig. 3A). In the tetrad stage, tapetum cells react weakly to PAS. At this stage, the epidermis and endo- thecium cells contain insoluble polysaccharides. Callose gives a positive PAS reaction at this stage (Fig. 3C). Insoluble polysaccharides are seen in the cytoplasm of the mature pollen grain (Fig. 3E). In protein staining with CBB, there is no considerable difference between the protein contents of the anther wall layers and sporogenous cells at the begin- ning of development (Fig. 3B). However, contrary to the oth- er wall layers, the cytoplasm of tapetum cells becomes rich in protein content starting from the tetrad stage (Fig. 3D). The tapetum layer, which gives a strong positive reaction in the young pollen stage, begins to break down and transfers the protein-structured substances to the maturing pollen grains. Other anther wall layers are poor in protein content at the mature pollen stage (Fig. 3F). Pollen viability After the acetocarmine staining, the pollen viability rate was calculated as 87%, the dyed, large, and properly shaped mature pollen grains being considered fertile, and the un- stained pollen sterile. Ovule and female gametophyte development C. kilaea has an inferior ovary, characteristic of the As- teraceae family. The ovary is syncarpous, unilocular, and carries a single ovule with basal placentation. The ovule is anatropous, tenuinucellate, and unitegmic (Fig. 4A). A sin- gle archespore cell develops under the epidermis, and this archespore cell differentiates directly into a megaspore mother cell (Fig. 4B). The megaspore mother cell undergoes Fig. 1. Anther wall development in Centaurea kilaea: A) arche- spore cells and their divisions, B) outer and inner secondary pa- rietal layers, C) division of the outer secondary parietal layer (ar- rows), D) microspore mother cell stage, E) tetrad stage, F) free microspore stage, G) vacuolated microspore stage (arrowheads indicate tapetal extensions), H) pollen mitosis stage, I) anthesis stage. as – archespore cell, atp – amoeboid tapetum, en – endoth- ecium, ep – epidermis, ft – fibrous thickenings, isp – inner sec- ondary parietal layer, ml – middle layer, mm – microspore moth- er cell, ms – microspore, osp – outer secondary parietal layer, pg – pollen grain, pp – primary parietal cells, sc – sporogenous cells, tp – tapetum, tt – tetrad, v – vacuole. Scale bars = 20 μm. Fig. 2. Male gametophyte development in Centaurea kilaea: A) free-microspore stage, B) vacuolated microspore stage, C) telo- phase stage at the first pollen mitosis (arrows indicate chromosome groups at poles), D) vegetative and generative nuclei, E) second pollen mitosis, F) mature three-celled pollen grain. e – exine, gn – generative nucleus, ms – microspore, p – pore, sn – sperm nucle- us, v – vacuole, vn – vegetative nucleus. Scale bars = 20 μm. REPRODUCTIVE BIOLOGY OF CENTAUREA KILAEA ACTA BOT. CROAT. 84 (1), 2025 57 meiosis to produce a linear megaspore tetrad. The mega- spore at the chalaza side is the functional megaspore; the other three megaspores rapidly degenerate (Fig. 4C). The functional megaspore undergoes three successive mitotic divisions to produce a two-, four-, and then eight-nucleate megagametophyte (Figs. 4D–H). The embryo sac development is of the Polygonum type. The mature embryo sac consists of a three-celled egg appa- ratus, (an egg cell and two synergids) at the micropyle side, three antipodal cells at the chalazal end, and two polar nu- clei in the central part of the sac. Before fertilization, the polar nuclei fuse to form the secondary nucleus (Fig. 4G and Fig. 4H). The synergids degenerate after fertilization. The antipodal cells persist until the first division of the zygote. Obturator cells originating from the funiculus are long, tu- bular shaped, and show prominent nuclei. They are elon- gated toward the synergids. The micropylar canal is com- pletely filled with the obturator cells and the secretion produced by these cells (Fig. 5E and Fig. 5F). In the ovules of C. kilaea, the megaspore mother cell, nucellus, and integument cells react poorly, in terms of in- soluble polysaccharides, with the PAS reaction at the begin- ning of development (Fig. 5A). In the mature embryo sac stage, the part of the integument tapetum facing the embryo sac and the integument cells forming the micropylar open- ing show an intense PAS-positive reaction (Fig. 5C). In ad- dition, at this stage, the obturator cells and their secretion give an intense PAS-positive reaction (Fig. 5E). In the ovules of C. kilaea, the megaspore mother cell, nucellus, and in- tegument cells react moderately in terms of protein content with CBB at the beginning of development (Fig. 5B). In the mature embryo sac stage, the integumentary tapetum cells give an intense positive reaction to CBB (Fig. 5D). At this stage, it is observed that the obturator cells also give a posi- tive reaction, but the protein is not secreted (Fig. 5F). Endosperm and embryo development During fertilization, one of the male gametes fuses with the egg cell, while the other male gamete fuses with the sec- ondary nucleus and forms the primary endosperm nucleus. Fig. 3. Histochemical aspects of the anther wall and pollen devel- opment in Centaurea kilaea: A) insoluble polysaccharide content in the microspore mother cell stage, B) protein content in the microspore mother cell stage, C) insoluble polysaccharide content in the tetrad stage, D) protein content in the tetrad stage, E) in- soluble polysaccharide content in the mature pollen grains, F) protein content in the mature pollen grains. mm – microspore mother cells, pg – pollen grain, tp – tapetum, tt – tetrad. Scale bars = 20 μm. Fig. 4. Ovule and female gametophyte development in Centaurea kilaea: A) ovule, B) megaspore mother cell, C) functional mega- spore and degenerated microspores, D) two-nucleate stage of megagametogenesis, E) metaphase stage of the second mitotic di- vision, F) four-nucleate stage of megagametogenesis (white arrows indicate nucellar cell remnants), G) and H) mature embryo sac (the photos are taken from two consecutive sections of the same em- bryo sac; arrowheads indicate mucilage accumulation). ac – antip- odal cell, ch – chalaza, dm – degenerated megaspores, ec – egg cell, es – embryo sac, fm – functional megaspore, fu – funiculus, in – integument, it – integumentary tapetum, mc – megaspore mother cell, mi – micropyle, sy – synergid cell, se – secondary nucleus, v – vacuole. Scale bars = A, 100 μm; B–H, 20 μm. KARTAL C. 58 ACTA BOT. CROAT. 84 (1), 2025 The primary endosperm nucleus begins to divide before the zygote (Fig. 6A). The endosperm initially develops as free nuclei. The antipodal cells are still intact in this stage (Fig. 6B), and break down after the first zygote division. In the globular embryo stage, wall formation is observed between the endosperm nuclei, starting from the micropylar side (Fig. 6C). The mature seed does not contain endosperm (Fig. 7F). The zygote has a pronounced polarity; the cytoplasm and nucleus are located at the apical end; there is a large vacuole in the basal portion (Fig. 6A). The zygote develops into an embryo by mitotic divisions. The first division is transverse and produces a basal cell toward the micropyle side and an apical cell toward the chalazal side. Embryo de- velopment is of the asterad type. The basal cell undergoes transverse divisions to give the suspensor, comprising 4–6 cells (Figs. 6C–E). The globular embryo is formed by the transverse and longitudinal divisions of cells in the proem- bryo (Fig. 6D). With further development, cells rapidly di- vide and the globular embryo becomes heart-shaped (Fig. 6E). After successive divisions, a mature dicotyledonous embryo forms with an obvious plumule (Fig. 6F). Integument differentiation In C. kilaea, the integument is initially homogeneous in structure (Fig. 4B). In the zygotene stage of meiotic prophase I, the innermost layer cells of the integument begin to elon- gate radially to differentiate into the endothelium (integu- mentary tapetum). The nucellar cells are still intact in this stage. At the end of megasporogenesis, the nucellar cells be- gin to deteriorate (Fig. 4C). During mitotic divisions (mega- gametogenesis), the nucellar cells break down and the rem- nants occur around the embryo sac (Figs. 4D-F). The mature embryo sac is surrounded by single-layered and radially elongated integumentary tapetum (Fig. 4G and Fig. 4H). In this stage, mucilage begins to accumulate rapidly on the walls of the integumentary cells close to the endothelium (Fig. 4G and Fig. 4H, arrowheads). After fertilization, the deposition of mucilage material continues between the pri- mary wall and the plasma membrane on all sides of the cells, and the mucilage pushes the protoplast to the center of the cell (Fig. 7A). Afterwards, the protoplast of the mucilage cells degenerates, and the cell wall breaks down (Fig. 7B). In the globular embryo stage, the peri-endothelial region is com- pletely filled with mucilage (Fig. 7C). Meanwhile, the endo- thelial cells elongate longitudinally to adapt to the growth of the embryo sac (Fig. 7B and Fig. 7D). In the torpedo-shaped embryo stage, the mucilage material appears homogeneous (Fig. 7D). As the embryo grows, the mucilage material is rap- idly consumed, disappearing completely in the mature seed. Endothelium persists in the mature seed (Fig. 7F). The integument has thin-walled parenchyma cells be- tween the peri-endothelial zone and the outer epidermis Fig. 5. Histochemical aspects of ovule and embryo sac develop- ment in Centaurea kilaea: A) insoluble polysaccharide content at the megaspore mother cell (mc) stage, B) protein content at mc stage, C) insoluble polysaccharide content in the mature embryo sac (es) stage, D) protein content at the mature es stage, E) insol- uble polysaccharide content of the obturator cells (oc) and secre- tion (asterisk), F) protein content of the obturator cells. Scale bars = 20 μm. Fig. 6. Endosperm and embryo development in Centaurea kilaea: A) zygote and nuclear endosperm, B) persistent antipodal cells, C) and D) globular embryo with suspensor (arrows indicate the walls of endosperm cells), E) heart-shaped embryo, F) mature embryo. ac – antipodal cell, co – cotyledon, ed – endosperm, em – embryo, it – integumentary tapetum, pl – plumule, ra – radicle, ss – suspensor, v – vacuole, z – zygote. Scale bars = A–D, 20 μm; E, 100 μm; F, 200 μm. REPRODUCTIVE BIOLOGY OF CENTAUREA KILAEA ACTA BOT. CROAT. 84 (1), 2025 59 (Figs. 7B–D). There is no specific material accumulation in, or in the walls of, these cells. After fertilization, in the rest- ing phase of the zygote, the outer epidermis cells of the in- tegument begin to elongate radially starting from the mi- cropylar pole. In the heart-shaped embryo stage, the outer epidermis cells elongate, and thickening develops on their radial walls (Fig. 7E). In the mature seed, the outer epider- mis cells form the seed coat. The testa is represented by a single row of radially elongated, lignified cells (macroscler- eids) (Fig. 7F). Discussion In this study, the embryology of C. kilaea (subfamily: Carduoideae, tribe: Cardueae), a tetraploid endemic species in Türkiye, was investigated histologically. C. kilaea shows the characteristics of the Asteraceae family in terms of an- ther structure (tetrasporangiate) and development (dicoty- ledonous anther wall). The anther wall consists of four lay- ers (epidermis, endothecium, middle layer, tapetum). The outer secondary parietal layer develops an endothecium and a middle layer by periclinal division. The tapetum layer dif- ferentiates from the inner secondary parietal layer directly. These results agree with the pattern previously known for Asteraceae (Davis 1966, Yankova-Tsvetkova et al. 2016, 2018). C. kilaea has an amoeboid type tapetum, which is the most representative of Asteraceae. It has also been observed in other tribes such as the Anthemideae (Li et al. 2010), As- tereae (Davis 1968), Calenduleae (Ao 2007), Gnaphalieae (Davis 1962), Heliantheae (Gotelli et al. 2008), Inuleae ( Pullaiah 1979), and Senecioneae (Pullaiah 1983). Secretory tapetum occurs in Eupatorieae (Franca et al. 2015), Stifftieae and Wunderlichieae (Bonifacio et al. 2018). Yankova- - Tsvetkova et al. (2016, 2018) reported that initially, the tapetum of Arnica montana (Madieae) and Centaurea achtarovii (Cardueae) is secretory, and then it transforms into amoeboid. The persistent epidermis, fibrous endothe- cium, and ephemeral middle layers observed in C. kilaea are common in Asteraceae (Davis 1966). Microspore mother cells of C. kilaea show simultaneous cytokinesis, resulting in tetrahedral type tetrads. These findings have also been reported in previous studies (Franca et al. 2015, Bonifacio et al. 2018); the cytokinesis of Ambrosia artemisiifolia is of the successive type (Liu et al. 2011). In C. achtarovii (Yankova-Tsvetkova et al. 2018), the tetrads are generally tetrahedral and isobilateral. A. montana has usu- ally tetrahedral, occasionally isobilateral, rarely T-shaped, and linear tetrads (Yankova-Tsvetkova et al. 2016). The pol- len grains of C. kilaea are shed with three cells (a vegetative cell and two sperm cells) from the mature anther, as in many Asteraceae members (Davis 1966). In Stifftia chrysantha, Stifftia fruticose, Wunderlichia mirabilis, and Wunderlichia senae, the pollen grains are shed with two cells (a vegetative cell and a generative cell) from the anther (Bonifacio et al. 2018). Mature pollen grains of C. kilaea contain insoluble polysaccharides and proteins. Similar observations have been also reported in Galinsoga quadriradiata (Kolczyk et al. 2015). Although C. kilaea is a tetraploid species (2n = 4x = 36) (Meric et al. 2010), meiosis is quite regular and the pollen fertility percentage is high (87%). The ovary of C. kilaea, characteristically for the Astera- ceae family, is single-chambered with two carpels and con- tains a single ovule with basal placentation. This feature has been reported for many Asteraceae family members (Davis 1966, Ao 2007, Chen et al. 2014, Franca et al. 2015, Bonifacio et al. 2018). The single archespore cell forms just below the nucellus epidermis and differentiates directly into the mega- spore mother cell. The megaspore mother cell produces a linear type megaspore tetrad as a result of meiosis. The megaspore on the chalazal side is the functional megaspore, and the other three megaspores degenerate rapidly. This is characteristic of many Asteraceae family members (Davis 1966). Embryo sac development in C. kilaea is Polygonum- type, as it is in 70% of angiosperms. Monosporic Polygo- Fig. 7. Integument differentiation in Centaurea kilaea: A) deposi- tion of mucilage (mu) in the integument (in) cells, B) degenerated mucilage cells stained with PAS (arrows), C) peri-endothelial re- gion filled with mucilage, D) peri-endothelial region (pr) and integumentary tapetum (it) in the torpedo-shaped embryo (em) stage (asterisks indicate integumentary cells between the peri- endothelial zone and the outer epidermis), E) outer epidermis cells in the zygote resting stage (arrows indicate wall thickening), F) outer epidermis cells in the heart-shaped embryo stage. cl – cell lumen, co – cotyledon, ci – crushed integument cells, ep – epider- mis, ow – ovary wall. Scale bars = A, E, F, 20 μm; B, C, 100 μm; D, 200 μm. KARTAL C. 60 ACTA BOT. CROAT. 84 (1), 2025 num-type embryo sac development is mostly seen in As- teraceae family species, but monosporic Oenothera-, bisporic Allium-, tetrasporic Fritillaria-, Adoxa- and Drusa- type embryo sac developments are also seen (Davis 1966, Li et al. 2009, Musial et al. 2012). Thus, the embryo sac devel- opment of plants in the Asteraceae family is highly diverse. The antipodal cells of C. kilaea persist until the first di- vision of the zygote. They are similarly persistent in Crepis bithynica, C. achtarovii, Wunderlichia spp., Ambrosia spp., and Taraxacum udum (Yurukova-Grancharova and Dimitrova 2006, Musial et al. 2013, Chen et al. 2014, Bonifacio et al. 2018, Yankova-Tsvetkova et al. 2018). However, the antipo- dal cells in Calendula officinalis degenerate prematurely, so they are not found in the mature embryo sac of C. officinalis (Ao 2007). Before fertilization, the polar nuclei fuse to form the secondary nucleus of the embryo sac. Similar observa- tions have been reported in previous studies (Chen et al. 2014, Yankova-Tsvetkova et al. 2016, 2018, Bonifacio et al. 2018). Obturator cells with prominent nuclei and their secre- tions completely filled the micropylar canal in C. kilaea. These cells contain insoluble polysaccharides and proteins. The secretion produced by obturator cells gives an intense PAS-positive reaction; however, there is no protein in the secretion. Similarly, in sunflower and Taraxacum spp., the secretion (extracellular matrix) consists of only insoluble polysaccharides (Yan et al. 1991, Plachno et al. 2015). In C. kilaea, the obturator cells and their secretions play a key role in the growth of pollen tubes toward the micropyle, and further, their nutrition. It is similarly reported that the ob- turator plays roles in both the feeding of pollen tubes and their directing toward the micropyle in the sunflower (Yan et al. 1991). In C. kilaea, the polar nuclei fuse before fertilization to form the secondary nucleus. This feature is common for As- teraceae (Davis 1966). Endosperm development is initially of nuclear type in C. kilaea. In the globular embryo stage, cytokinesis occurs between the free nuclei, starting from the micropylar side, and the endosperm transforms into a cellular type. Similar observations have been also reported in Hieracium spp., A. montana, C. bithynica, and C. achtarovii (Yurukova-Grancharova and Dimitrova 2006, Yurukova- Grancharova et al. 2006, Yankova-Tsvetkova et al. 2016, 2018). The cellular endosperm is observed in Ageratum, Ambrosia, Stifftia, and Wunderlichia (Chen et al. 2014, Franca et al. 2015, Bonifacio et al. 2018). The embryogenesis of C. kilaea is of the asterad type, in which basal and apical cells participate in the formation of the embryo, as previ- ously described for Asteraceae (Davis 1966). The presence of integumentary tapetum (endothelium) has been reported in 65 dicotyledonous families with thin ovules (tenuinucellate and weakly crassinucellate) (Kapil and Tiwari 1978). The integumentary tapetum is a common feature in Asteraceae and forms in different developmental stages in the different representatives of the family (Davis 1966). The integument cells are initially homogeneously structured in C. kilaea. In the zygotene stage, the innermost layer cells of the integument elongate radially, and the en- dothelium forms. Endothelium begins to form in C. achtarovii at the one-nucleate stage, in Hieracium pilosella at the end of meiosis, in Taraxacum udum at the diad stage, and in Ambrosia spp. at the leptotene stage (Koltunow et al. 1998, Musial et al. 2013, Chen et al. 2014, Yankova-Tsvetkova et al. 2018). In the mature female gametophyte stage, mucilage be- gins to accumulate rapidly on the walls of the integument cells close to the endothelium. The mucilage deposited be- tween the primary wall and the plasma membrane pushes the protoplast toward the center of the cell. This region is called the peri-endothelial region (Pandey et al. 1978, Kolczyk et al. 2016, Plachno et al. 2016, 2017). In Taraxacum, the cells of the peri-endothelial region begin to differentiate at the young ovule stage, while in Hieracium, the wall changes of the peri-endothelial zone cells start during the four-nucleate embryo sac stage (Koltunow et al. 1998, Plach- no et al. 2016). After fertilization, during the resting period of the zygote, the protoplast of the mucilage cell degenerates and the cell wall breaks down. Lysigenous cavities filled with mucilage are formed in the peri-endothelial region of C. kilaea. In Hieracium and Pilosella, the formation of ly- sigenous cavities occurs at the mature female gametophyte stage, while in Taraxacum, the peri-endothelial cells retain their individuality in the mature female gametophyte stage, and the formation of these cavities occurs during embryo- genesis (Plachno et al. 2016, 2017, Gawecki et al. 2017). In the globular embryo stage, the peri-endothelial region is completely filled with mucilage. Meanwhile, endothelial cells elongate longitudinally to adapt to the growth of the ovule. In the torpedo-shaped embryo stage, the mucilage material appears homogeneous, and it disappears complete- ly in the mature seed. Endothelium persists in mature seeds in C. kilaea. In A. artemisiifolia, endothelial cells begin to deteriorate during the multicellular proembryo stage. Meanwhile, integument cells adjacent to the endothelium also undergo hydrolysis. In the mature embryo period, the endothelium completely disappears (Chen et al. 2014). It is suggested by the present paper that mucilage might be a car- bohydrate source for the developing embryo. Moreover, dis- ruption of peri-endothelial cells creates the necessary space for the growing embryo. In the heart-shaped embryo stage of C. kilaea, the outer epidermis cells of the integument elongate radially, and their radial walls thicken. Like other members of the Asteraceae, the outer epidermis cells form the seed coat in the mature seed (Pandey et al. 1978; Kolczyk et al. 2016). Conclusion This study details the embryological characteristics and the fruit development of C. kilaea, a member of Asteraceae, a threatened coastal dune species that is facing a very high risk of extinction in the wild due to human activities. This is the first study on the reproductive biology and chemical REPRODUCTIVE BIOLOGY OF CENTAUREA KILAEA ACTA BOT. CROAT. 84 (1), 2025 61 composition of the reproductive structures of the species. Тhе revealed features of consisting structures of anthers, ovules, embryo and seeds show the characteristics of the Asteraceae family: tetrasporangiate anthers, dicotyledon- ous type of anther wall development, amoeboid tapetum, tetrahedral microspore tetrads, three-celled mature pollen grains containing insoluble polysaccharides and proteins, anatropous, unitegmic, and tenuinucellate ovule, Polygo- num-type of embryo sac development, antipodal cells per- sisting until the first divisions of the zygote, obturator cells and their secretions (extracellular matrix) completely filling the micropylar canal, PAS-positive extracellular matrix without protein contains, mature embryo sac surrounded by an integumentary tapetum (endothelium), integument consisting of the integumentary tapetum (endothelium), peri-endothelial region, parenchymatous cells, and outer epidermis, from the inside out, with mucilage accumulation on the walls of the integument cells (peri-endothelial re- gion) close to the endothelium in the mature female game- tophyte stage, mature seed without mucilage, Asterad type of embryo development, endosperm of cellular type, mature seed without endosperm and with endothelium. 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