Morphological Features of Fruits and Seeds of Some Species of the Genus Crataegus L. of the Flora of Ukraine

e genus Crataegus L. is one of the largest species in the family Rosaceae Juss. e fruits are used as medicinal raw materials (Crataegus fructus), as they contain a set of biologically active compounds. e genus is voluminous and taxonomically problematic due to significant variability, hybridization, apomixis, and polyploidy. As a result, it is difficult to determine the species of the genus. Species of the genus have a number of diagnostic features, one of which is a distinct fruit with seeds. e aim of our study was to identify and compare the diagnostic features of the fruits of seven species of the genus Crataegus flora of Ukraine, which are most oen used as medicinal raw materials and are the most common in Ukraine – Crataegus laevigata, C. monogyna, C. pentagyna, C. pseudokyrtostyla, C. sanguinea, C. fallacina, and C. rhipidophylla. e study was performed using scanning and digital microscopy, and descriptions were made by standard methods. As a result, we identified features that clearly distinguished the studied species from each other. We identified mature fruits that contain only one pyrene (C. monogyna, C. pseudokyrtostyla, C. fallacina, and C. rhipidophylla), and several pyrenes (C. laevigata, C. pentagyna, and C. sanguinea) among the studied species of the genus. e studied species are characterized by spherical and ellipsoidal fruits. e predominant color is fulvous/brown with different shades, but almost-black fruits of the species C. pentagyna are well distinguished. Fruits of all studied species of the genus are fleshy, except C. sanguinea, which is floury. e species is characterized mainly by yellow flesh fruits, except C. pentagyna, which has ribbed fruits. e fruits of C. monogyna plants differ from those of other studied species by the spinous-tuberous surface relief. Pyrenes of triangular shape are observed only in C. pentagyna. We suggest that these features can be used as additional diagnostic tools to determine the species of the genus Crataegus flora in Ukraine.


Introduction
ere are more than a thousand species of the genus Crataegus L. worldwide. According to the Catalogue of Life (Roskov et al., 2019), there are 1,447 Latin names of hawthorn, which is the common name of the species in the genus. In the Flora of Eastern Europe, Tsvelëv (2001) has listed over 1,500 species for the genus Crataegus. According to various sources, in Ukraine, there are 33 native species and several cultivated species that are becoming wild (Cherepanov, 1995;Fedoronchuk, 2003Fedoronchuk, , 2017Mosyakin & Fedoronchuk, 1999). e genus Crataegus is voluminous and taxonomically problematic because of significant variability, hybridization, apomixis, and polyploidy (Fedoronchuk, 2017;Iukhymenko, 2019;Mezhens′ka & Mezhens′kiĭ, 2013). As a result, it is difficult to accurately determine the species of the genus. Generally, they differ in the occurrence and morphological features of modified shortened shoots (thorns), such as size, shape, degree of dissection, and pubescence of the leaf blade, and in the shape, size, surface type and other features of fruit and number of seeds.
Comprehensive studies of species of the genus Crataegus for the purposes of taxonomy in Ukraine have been caried out over almost a century, but even currently there are a number of controversial issues due to the inherent hybridization of hawthorns and variability of features depending on environmental growth conditions. is complicates the task of identifying clear diagnostic features of hawthorn raw materials for their standardized use in the pharmaceutical industry and medicine. erefore, a comparative analysis of the main diagnostic morphological and anatomical features of the fruits of model species of the genus Crataegus was undertaken in this study, and the results are provided below.
One of the controversial issues regarding the morphological characteristics of the generative organs of the genus Crataegus is in the determination of the type of fruit. For example, according to Krylov (1903) it is a "spherical drupe, " while according to Bobrov et al. (2009) it is a "pyrenarium-like apple. " Fedoronchuk (2017) classified the fruit as an "apple, " in which fully or partially grown leaves (many-locular carpels) grow with the inner wall of the hollow axis of the flower (hypanthium), which subsequently becomes fleshy, whereas Iukhymenko (2019) and Korovkin (2007) classified it as "stone-like apple, " and according to Vanin (1967) and Gubanov et al. (2013), the fruit is an apple. In other literature sources, the name "berry-like fruit" has been incorporated (Alaghawani & Naser 2013;Christensen, 1992). According to one of the main anatomical and morphological features, the type of hawthorn fruit is a "pyrenarium. " It is characterized by the presence of a hard sheath formed from the inner tissues of the pericarp, which surrounds the seeds and arises from the coenocarpic gynoecium (Artiushenko & Fedorov, 1986) with dry sepals that remain on it (Iukhymenko, 2019;Ufimov, 2013). Following Bojňanský and Fargašová (2007), we consider pyrene as a seed covered with a stony layer, and fruits that contain pyrene as stone fruits. A review of the fragmentary literature available on the morphological features of hawthorn suggested that in the studies of fruits, attention was mostly paid to their size, weight, color, number of pyrenes (seeds surrounded by fruit endocarpium), color of flesh, and morphometric characteristics of seeds. We analyzed the available data on morphological and anatomical features of the Crataegus spp. fruits of the following species: Crataegus orientalis Palas ex. M. Bieb. var. orientalis, C. aronia (L.) Bosc. ex DC. var. aronia, C. meyeri Pojark., C. tanacetifolia (Lam.) Pers, C. pontiaca Koch, C. ambigua C. A. Mey, C. pojarkovae Kossych, and C. orientalis Pall. ex M. Bieb. (Fikret et al., 2006) andC. sanguinea, C. douglasii, andC. submollis (Iukhymenko, 2019). We found that complex studies were either absent or fragmentary data were available. In particular, Turkish botanists studied the genotype features of 42 samples of fruits and seeds of the genus; the length, width, weight, and the ratio of length to width of the fruit were taken into account (Fikret et al., 2006). In addition, a comprehensive study of C. ambigua C. A. May was conducted, including studies of the anatomical structure of the fruit (Tuyakova et al., 2016), and taxonomic analysis of Crimean species (C. pojarkovae Kossych and C. orientalis Pall. ex M. Bieb). Based on these results, it was noted that the main diagnostic features were fruits: their variation in color and size (Letukhova et al., 2014).
Fruits of hawthorns (Crataegus fructus) are mostly used as medicinal raw materials. Because of the presence of a set of biologically active compounds, preparations with hawthorn fruits have multiple therapeutic effects, among which the most significant are lipid-lowering, antiatherosclerotic, hypotensive, and cardioprotective effects (Antsyshkina et al., 1990;Evdokimova, 1999;Pengzhan, 2012;Samylina et al., 2010). e State Pharmacopoeia of Ukraine (2014) (SPU) lists 12 species of the genus Crataegus, the raw materials of which are permitted for use in medicine. Five of these species exist naturally in the flora of Ukraine -Crataegus laevigata (Poir.) DC., C. monogyna Jacq., C. pentagyna Waldst. & Kit., C. pseudokyrtostyla Klok. (C. curvisepala Lindm.), and C. sanguinea Pall. (Mezhens′ka & Mezhens′kiĭ, 2013;Mosyakin & Fedoronchuk, 1999;SPU, 2014). is publication is the first in a series focused on the study of macro-and micromorphological features of raw organs of the genus Crataegus in Ukraine. e aim of this study was to identify and compare the diagnostic features of the fruits of seven species of the genus Crataegus (C. laevigata, C. monogyna, C. pentagyna, C. pseudokyrtostyla, C. sanguinea, C. fallacina Klokov, and C. rhipidophylla Gand.) that are most common in Ukraine and are most oen used as raw materials.

Material and Methods
e fruits of seven species of the genus Crataegus of the flora of Ukraine, collected and identified by the authors, and samples from the National Herbarium of Ukraine (KW; http://sweetgum.nybg.org/science/ih/herbarium-details/?irn=125430) were selected for this study (Table 1).
For each species, 30 fruits were used for analysis. All quantitative indicators were processed using the methods of variation statistics using the program Statistica StatSo ver. 10 (USA). We did not perform a statistical comparison of species, because the aim of this work was to identify diagnostic features of the fruits of species of the genus Crataegus, which are most oen used in medicinal raw materials, rather than resolving problems of species taxonomy.
e sculpture of the fruit surface was studied using a scanning electron microscope (SEM) (JSM-6060 LA; JEOL, Japan). Samples for SEM research were processed according to the standard method: dehydrated objects were fixed on brass tables and sprayed with a thin layer of a mixture of gold and platinum in a vacuum chamber. A Superior 10-220x digital microscope (Sigeta, Ukraine) was used for macromorphological studies. e size of the fruits and seeds was determined using a measuring ruler on a millimeter scale. e terminology following Artiushenko and Fedorov (1986), Bojňanský and Fargašová (2007), and Korovkin (2007) were used for morphological and anatomical feature descriptions.
Crataegus laevigata. e fruits are elliptical to short-ellipsoidal in shape ( Figure 1A). e color is brownish-red, brown, or black, flesh yellow and juicy in consistency. e length of the fruit varies from 5 to 9 mm [mean = 7.6 ± 0.9 mm, CV (coefficient of variation) = 5%], and width varies from 4 to 9 mm (mean = 6.6 ± 0.75 mm, CV = 8%). e relief of the surface of fruits is collicular (Figure 2A), and there are epicuticular waxes, which are represented by wax plates. e number of pyrenes is two (three), and they have two or three sutures on the dorsal and ventral sides ( Figure 1H). ey are irregularly shaped, convex, ribbed on the dorsal side, flat, or grooved on the ventral side. e sculpture of the surface of pyrenes is reticular-collicular ( Figure 1H, Figure 3A).
Crataegus monogyna. e shapes of the fruits vary from widely-short-ellipsoidal or ovoid to rounded ( Figure 1E). e color is dark red, brownish-red, and flesh is yellow and juicy. Fruit length varies from 5 to 10 mm (mean = 7.6 ± 0.95 mm, CV = 9%) and width varies from 4 to 6 mm (mean = 5.7 ± 0.7 mm, CV = 10%). Epicuticular wax is well developed and is represented by wax crusts and crystals ( Figure 2B). ere is one pyrene (Figure 1I), the seam is visible on the lateral side, and there are two or three vague grooves on the dorsal side. Pyrene is characterized by a reticular relief ( Figure 2I, Figure 3C).  Crataegus pentagyna. e fruits are almost spherical, round, or short-ellipsoidal ( Figure 1F). e color is almost black or purple-black with a glaucinus coating at the base. e flesh is underdeveloped and reddish-brown. Fruit length varies from 6 to 9(12) mm (mean = 8.9 ± 1.5 mm, CV = 17%), and width varies from 6 to 8 mm (mean = 7.2 ± 0.5 mm, CV = 6%). e relief of the surface of the fruits is indistinctly collicular ( Figure 2C). Epicuticular wax is well developed and is represented by wax  crusts. e number of pyrenes varies from two to several (three-four) ( Figure 1J). e ventral side of the pyrene is a dilated pterygoid with a longitudinal crest, and the apex is narrow, rounded on the dorsal side, with a longitudinal groove. Pyrenes are characterized by a reticular relief ( Figure 2J, Figure 3J).
Crataegus pseudokyrtostyla. e fruits are spherical, oblong-ellipsoidal, or cylindrical in shape ( Figure 1D). e color is dark red, oen with green spots,  reddish-brown, and the flesh color is yellow-orange. e relief of the surface of fruits is collicular, and epicuticular wax is represented by well-developed waxy irregular platelets ( Figure 2D). Fruit length varies from 9 to 11 mm (mean = 10.1 ± 0.45 mm, CV = 7%), and width varies from 5 to 6 mm (mean = 5.45 ± 0.35 mm, CV = 8%). ere is one pyrene (Figure 1K), characterized by pitted relief ( Figure 2K, Figure 3D).
Crataegus sanguinea. e fruits are almost spherical or short-ellipsoidal in shape ( Figure 1B). eir color is blood-red (brownish-red) and rarely orange-yellow.  e flesh is yellow with floury consistency. Fruit length varies from 7 to 10 mm (mean = 8.6 ± 0.9 mm, CV = 10%), and width varies from 7 to 9 mm (mean = 8.1 ± 0.7 mm, CV = 11%). e relief is collicular-pitted, and epicuticular wax is well developed, represented by wax crusts ( Figure 2E). ere are 2(3)-4(5) pyrenes ( Figure 1L). Pyrenes are elliptic to obovoid, with sutures on the ventral side and one or three keels on the dorsal side. eir relief is collicular-pitted ( Figure 2L, Figure 3E). Crataegus fallacina. Fruits are spherical, short-elliptical, ellipsoidal, or ovoid ellipsoidal ( Figure 1C). e color of the fruit is yellowish-brown or red. e flesh is yellow and juicy. Fruit length varies from 9 to 12 mm (mean = 8.6 ± 0.9 mm, CV = 10%), and width varies from 5 to 9 mm (mean = 8.1 ± 0.7 mm, CV = 11%). e relief of the fruit surface is collicular, with well-developed epicuticular wax ( Figure 2F), which is represented by wax crusts and granules. Papillae are present on the fruit surface. eir area is approximately 0.39-0.41 mm 2 . ere is one pyrene ( Figure 1M). Its dorsal side is convex with four grooves, and the ventral side is flat with one furrow and a prominent nose. e relief is reticulated-pitted ( Figure 2M, Figure 3F).
Crataegus rhipidophylla. e fruits are elongated-ellipsoidal, ellipsoidal, or cylindrical in shape ( Figure 1G). e color is bright or dark red, yellow, burgundy, or black. e flesh is yellow and juicy. Fruit length varies from 8 to 15 mm (mean 12.6 ± 2.74 mm, CV = 18%), and the width is 1.3-2 times greater than the length. e relief of the fruit surface is collicular with well-developed epicuticular wax ( Figure 2G). Wax is represented by wax films and platelets. ere is one pyrene ( Figure 1N). e dorsal side is convex, and the ventral side is flat. e surface relief of the pyrene is reticulate ( Figure 2N, Figure 3G).

Discussion
Morphological features of fruits are important for species identification (e.g., Alaghawani & Naser, 2013;Mehebub et al., 2019). A comprehensive study of the fruits and pyrenes of selected species of the flora of Ukraine was conducted for the first time. In the present study, it was found that the species of the genus Crataegus native to the flora of Ukraine are characterized by spherical and ellipsoidal fruits. e species differ in fruit size, with lengths varying from 5-9 mm (C. laevigata) to 8-15 mm (C. rhipidophylla) ( Figure 4) and widths varying from 4-6 mm (C. monogyna) to 7-9 mm (C. sanguinea) ( Figure 5). e predominant color is fulvous/brown with different shades, but almost black fruits of the species C. pentagyna are well distinguished.
Fruits of all studied species of the genus are fleshy, except C. sanguinea, which is floury. e species are characterized mainly by yellow fruits. e exception is C. pentagyna, which is reddish-fulvous. e fruits are usually topped with nondrooping, triangular, and pointed sepals that are bent outward. e surface of nonfixed fruits (fresh) in the studied species is smooth and shiny, except for the species C. monogyna, which has grooved fruits and the species C. pentagyna, which has ribbed fruits. e fruits of the studied species had very sparse pubescence at the base. e exception is the fruits of C. pentagyna, which have dense glaucinus coating pubescence, present both at the base and at the apex of the fruit. In all studied species, the fruits are covered with a solid layer of wax, and the studied species differ in the type of epicuticular wax. us, wax platelets are characteristic of C. pseudokyrtostyla, C. laevigata, and C. rhipidophylla, wax spathella are characteristic of C. pentagyna and C. pseudokyrtostyla, and wax peels are characteristic of C. fallacina, C. pentagyna, and C. sanguinea. In the vast majority of the studied species, the relief of the fruit surface is collicular (C. laevigata, C. pentagyna, C. pseudokyrtostyla, C. fallacina, and C. rhipidophylla), collicular-pitted (C. sanguinea), and awned-undulating (C. monogyna). We identified mature fruits that contain only one pyrene (C. monogyna, C. pseudokyrtostyla, C. fallacina, and C. rhipidophylla), and several pyrenes (C. laevigata, C. pentagyna, and C. sanguinea) among the studied species of the genus.
We found that, in general, the morphological features of pyrenes in the studied species are similar. e size of pyrenes of different species of the genus Crataegus varies from 5 to 15 mm in length and 4 to 15 mm in width.
us, we identified features that clearly distinguished the studied species from each other. e almost black color is characteristic of the fruits of C. pentagyna. e largest fruits are observed in C. rhipidophylla. e fruits of C. monogyna plants differ from those of other studied species in the spinous-tuberous surface relief. Pyrenes of triangular shape are observed only in C. pentagyna. In our opinion, these features can be used as additional diagnostic tools to determine the species of the genus Crategus flora of Ukraine. Our findings can be included in the relevant chapter of the European Pharmacopoeia, including the State Pharmacopoeia of Ukraine, to the chapter on medicinal herbal raw materials and herbal medicines.