Flower morphology and ontogeny of endemic Crataegus tanacetifolia (Lam.) Pers. ( Rosaceae ) from Turkey

: In this study, flower morphology and ontogeny of Turkish endemic Crataegus tanacetifolia (Lam.) Pers. ( Rosaceae ) were studied using stereo, light and scanning electron microscope. Crataegus tanacetifolia. has corymb type of inflorescence which bears 5-10 hermaphrodite flowers. The first sign in appearance of the flowers is the differentiation of apical meristem as a roundish bulge. It transforms into the floral meristem which gives rise to floral organ primordia in advanced stages. Twenty stamen primordia differentiate as roundish bulge from the sides of floral meristem. Subsequently, the stamen primordia start stretching and differantiating into filaments and anthers respectively. After a short time of stamen initiation, 5 carpel primordia differentiate from the floral meristem. In following stages, the carpel primordia elongate and form an ovary with 5 loculi. During the ovary formation, 5 styles occur on the ovary and then stigmas evolve out of styles tips. ) çiçek morfolojisi ve ontogenisi stereo, ışık ve tarayıcı elektron mikroskobu ile incelenmiştir. Crataegus tanacetifolia 5-10 adet hermafrodit çiçek taşıyan korimbus tipi çiçek durumuna sahiptir. Çiçeklerin ortaya çıkışındaki ilk işaret, apikal meristemin yuvarlak bir çıkıntı olarak farklılaşmasıdır. Apikal meristem ilerleyen evrelerde çiçek organlarını oluşturacak olan floral meristeme dönüşür. Floral meristemin kenarlarından 20 adet stamen taslağı yuvarlak çıkıntılar olarak farklılaşır. Ardından stamen taslakları uzamaya başlar ve sırası ile filamentler ve anterleri oluşturur. Stamen taslaklarının belirmesinden kısa bir süre sonra, floral meristemden 5 adet karpel taslağı farklılaşır. İlerleyen evrelerde, karpel taslakları uzar ve 5 lokuslu bir ovaryum oluşturur. Ovaryum oluşumu esnasında, 5 stilus ovaryumun üzerinde gelişir ve ardından stilusların üzerinde stigmalar geişir. Anahtar Kelimeler: apikal Crataegus tanacetifolia , floral gelişimi, ontogeni


Introduction
The genus Crataegus L. belongs to the subfamily Maloideae of Rosaceae and comprises approximately 200 species worldwide (Campbell et al., 2007;Benli et al. 2008). The homeland of Crataegus is Asian and Mediterranean Countries. According to Davis and Browicz (1972), there are 17 species, a subspecies, two varieties and dozens of hybrids, naturally grown in Turkey. Crataegus is characterised by a corymb which is a specialised determinate inflorescence containing a few to numerous of flowers (Dönmez, 2004). The inflorescence of Crataegus has one type of flower on a single corymb; hermaphrodite. Androecium consist of 5-60 stamens (Rohrer and Robertson, 1994) and gynoecium have 1-5 pistils (Dönmez, 2004). Crataegus species are important medicinal plants. They have been used as multi-effect traditional medicine for prevention and treatment of heart disease, insomnia and hypertension (Vitalini et al., 2009;Chang et al., 2005;Tassell et al., 2010;Dong et al., 2017). Also, they are economically important plants being used in cosmetics and food industry (Lund et al., 2017).
Sex determination in plants is among the most significant subjects that concern biologists due to the importance of reproduction and fertilization for genetical diversity. Also, flower organogenesis is a major area of plant developmental biology, because of its' being one of the main basic topics and the reproductive character and the features of reproductive organs are systematically essential (Kinney et al., 2008). The basic bisexual flower is subdivided into four whorls. Whorl 1 and whorl 2 compose the sterile parts of flower and sex determination takes place in whorls 3 and 4, which contain stamens and pistils respectively (Dellaporta and Calderon-Urrea, 1993). However, the development pattern on the whorls differs between the sexes of flowers; hermaphrodite, pistillate, staminate. The start of flower bud development comprises the conversion of the vegetative meristem into the apical meristem and the transformation of the apical meristem into the floral meristem in the upcoming stage. Floral organs; sepal, petal, stamen and carpel develop from floral meristem, but further development of stamen or pistil is selective, resulting in unisexual flowers (Çetinbaş and Ünal, 2012).
Crataegus tanacetifolia (Lam.) Pers. is an important species, endemic to Turkey. It is a perennial shrub or a small tree, growing up to 10 meters (Sarıbaş and Yaman, 2005). Most of the morphological, anatomical and ontogenic studies conducted in Crataegus have been concentrated on common species (Gyan and Woodell, 1987;Bura et al., 2016;Hamideh et al., 2012). To our knowledge, there are no detailed morphological and ontogenic studies that have been reported for Turkish endemic C. tanacetifolia. Since C. tanacetifolia is endemic to Turkey, there is not sufficient data regarding its morphological and ontogenic characteristics, hence we aimed to give a detailed account of these features. Information on the development of reproductive organs will help advance our understanding of reproductive behaviour and will thus contribute to attempts to solve taxonomic problems in Crataegus, a rather neglected genus in this respect.

Materials and Method
The materials were collected from natural habitats within the borders of Bolu, Lake Abant Nature Park (Turkey) and morphologically analysed under stereomicroscope (Olympus 970931). At least 50 samples were studied for each phase. After fixation process by FAA (Formalinglacial acetic acid-alcohol) solutions, they were embedded in paraffin blocks, sliced at 10 μm by Leica RM2235 rotation microtome, stained with Delafield's hematoxylin, investigated by Olympus BH-2 light microscope. For scanning electron microscope (SEM) analysis, the material was fixed in 2.5% glutaraldehyde in 50 mM cacodylate buffer, pH 7.0 (Platt et al., 1983), dehydrated with an increasing ethanol gradient: from 70% up to 100% and kept in hexamethyldisilazane solution at room temperature (Topcuoglu et al., 2009). After coating with 11 nm of gold by using an automated sputter coater, they were examined with JEOL JMS-59 10LV SEM.

Flower morphology
Crataegus tanacetifolia has corymb type of inflorescence and the corymb contains 5-10 actinomorph, pentameric, hermaphrodite flowers (Fig. 1A). The flowers on the corymb are generally in different developmental stages. Gynoecium contains an ovary with 5 loculi and 5 free stigmas-styles (Fig. 1E). Twenty stamens surround the pistil (Fig. 1B,D). The flowers have 5 green, hairy sepals and 5 free, roundish, white petals. Receptacle is green and hairy (Fig. 1C). Measurements of the mature flower and flower parts of C. tanacetifolia were presented in Table 1.

Flower and flower parts Measurement (mm)
Lenght of the mature flower 15 ± 2 Width of the mature flower 12 ± 3 Lenght of the sepal 9 ± 2 Lenght of the petal 11 ± 3 Lenght of the stamen 11 ± 2 Lenght of the pistil 13 ± 2

Flower ontogeny
Development starts with the differantiation of shoot apical meristem ( Fig. 2A). Tunica, located at the tip of growth cone, consists of a few layers of cells. The diameter of the apical meristem increases as a consequence of cell division. Afterwards, growth cone swells due to the cell division at corpus (Fig. 2B). With the flattening of the growth cone and then swelling up of it, the transformation from bud to flower starts (Fig. 2C). Firstly, the apical flower bud of the corymb and then the lateral flower buds differantiate (Fig. 2D). Afterwards, floral organ primordia are generated at differantiated flower buds.
Floral organ formation starts with the formation of apical meristem. It differantiates as a roundish bulge from the vegetative meristem at growth points (Fig. 2E, Fig. 3A) and consists of well-ordered, thin walled small cells which have abundant cytoplasm and small vacuoli ( While the stamens develop, the roundish shape of them begin to dissappear. Firstly anther and then filament differantiates. During this time, carpel primordia begin to develop and elongate (Fig. 2L). The extending carpel primordia create their own style, the lower part of the pistil becomes swollen and the origin of ovary and ovary loculi are formed. Stigma differantiates on the apex of the style in the advanced stage (Fig. 2M). During the initiation and development, the dimention of the flower bud and reproductive organs increase (Fig. 2N, Fig. 3E,F). Relationship between the flower bud size and flower development stages of C. tanacetifolia were presented in Table 2. Carpel primordia initiation 573 ± 23

Discussions
The corymbs of C. tanacetifolia and C. yaltirikii Dönmez contain 5-10 flowers, the corymb of C. coriifolia Sharifnia & Zarrinkolah (Sharifnia et al., 2016) bears 15 flowers and the corymb of C. ambigua Mey. bears 20 flowers (Tuyakova et al., 2016). The flowers of Crataegus are pentameric and petals are generally white-pink (Dönmez, 2004). Flower sizes of Maloideae are generally 3.4 mm and 4.1 mm. The number of organs is not related to the size of the flowers. For instance, C. suksdorfii Lindl. and C. douglasii Lindl. have larger flowers than C. rivularis Nutl. and C. brockwayae Sarg.. Crataegus rivularis Nutl. has more stamens than C. douglasii Lindl., but has less stamen than C. brockwayae Sarg. (Evans and Dickinson, 1996). The number of stamen differs from species to species in Crataegus (Evans and Dickinson, 1996). The flowers generally contain 10 stamens as in C. transmississippiensis Sarg. and C. galli L. or 20 stamens as in C. tanacetifolia, C. submollis Sarg., C. canadensis Sarg., C. punctata Jacq. (Phipps, 2012). The number of ovary loculi can also differ in Crataegus species. Crataegus tanacetifolia has 1 ovary with 5 loculi, the ovary of C. galli has 1-2 loculi and the ovary of C. punctata has 2-5 loculi. Each of the loculi of these species are linked to the style as in C. tanacetifolia. Most angiosperms produce hermaphrodite flowers with functional male and female sex organs within the same flower (Aryal and Ming, 2014). During the early stages of floral organ development, all floral organ primordia form in male and female flowers. In males, the gynoecium develops as a sterile, undifferentiated rod, while in females, anther development arrests soon after the anther primordia form, then the anthers degenerate (Juarez and Banks, 1998). Therefore, the further development of stamens or pistils is very important in determining the sex of the flower. It is thought that sex is determined by the selective inhibition of gynoecium or androecium development in hermaphrodite flower. Also, the determination process involves the differentiation of gametophyte in pistil or stamen (Dellaporta and Urrera, 1993). For instance, sex determination in pistillate flower of Zea mays arises as a result of programmed cell death of stamen primordia (Cheng et al., 1983). Due to the hermophroditism, there are no atrophy or cell death on sexual organ primorida of C. tanacetifolia.
The developmental stages from the physiological differentiation of flower buds to the blooming of flowers have been studied by many researchers. Durner and Poling (1985) have divided the flowering into 4 stages: induction, initiation, differentiation and development. The floral induction occurs in the leaf upon exposure to a stimulus which ultimately results in the production of a flower bud at a shoot meristem (Durner, 2015). The initiation includes physiological and morphological changes occurring in the meristem upon receipt of stimuli from the leaf. Tal et al. (2017) indicated that enlargement and doming of the shoot apical meristem is a hallmark of the transition from vegetative growth to flowering. According to Evans and Dickinson (1996), the initiation of the flower buds starts with the transformation of apical meristem to the floral meristem. The morphological changes, observed during the conversion of apical meristem to floral meristem, resemble the ones in Prunus avium L. and P. Persica (L.) Batsch; apical meristem flattens and radially extends (Engin and Ünal, 2007). According to Durner and Poling (1985), differentiation is the development of specific floral organs on a single flower or of flowers on a single inflorescence, and development is the macroscopic production of flowers.
As in other plants (Uhl, 2011), all floral organ primordia in C. tanacetifolia develop as a result of periclinal division of floral meristem cells. In C. tanacetifolia, C. suksdorfii Lindl., C. douglasii Lindl. and C. rivularis Nutl., the first differentiated sex organs from floral meristem are stamens. Stamen primordia arise in roundish bulge forms as they do in P. avium and P. persica (Engin and Ünal, 2007). In Maloideae, 20 stamen differantiates in 1-3 rings. However, in C. tanacetifolia, 20 stamen primordia, that are located very close to each other, differantiate as roundish bulges from the outer 1 ring of the floral meristem and they emerge synchronized as they do in Erigeron philadelphicus L. (Harris et al., 1991). Stamen primordia arise out of the sides of floral meristem, as Dadpour et al. (2011) stated, and in stages where stamen primordia start emerging, the flatness of floral meristem starts to become gradually convex.
Female organ development starts with formation of carpel primordium in the center of floral meristem in C. tanacetifolia, as it is a usual manner in flowering plants (Gasser and Beers, 1993). But in Maloideae the time of differantiation of carpel primordia can differ. For instance, carpel primordia differantiates after all stamen primordia have achieved a nearly hemispherical shape in C. suksdorfii (Evans and Dickinson, 1996). But they differantiate shortly after the stamen initiation in C. tanacetifolia. The carpel primordia, which just arise in C. tanacetifolia, are not as roundish as stamen primordia. Uhl (2011) mentioned that various floral organs those differentiate out of floral meristem, might be morphologically different.