Micromorphological Characteristics of Fruit Surfaces of Some Usable Species of the Genus Valeriana (Valerianaceae) Among Ukrainian Flora

emicromorphological features of the fruit surfaces of nine species of Ukrainian flora, namely Valeriana tuberosa L., Valeriana tripteris L., Valeriana rossica P. Smirn., Valeriana stolonifera Czern., Valeriana grossheimiiWorosch, Valeriana sambucifoliaMikan fil., Valeriana officinalis L. s. str., Valeriana wolgensis Kazak. [Valeriana officinalis var. nitida (Kreyer) Rostanski], and Valeriana simplicifolia (Rchb.) Kabath, were examined. Depending on the presence and localization of pubescence on the surface of the fruit, three groups of species were distinguished: those with glabrous fruits, those with fruits pubescent only on the adaxial side, and those with fruits pubescent on both sides. Scanning electron microscopy revealed the additional characteristics of the fruits (microsculpture of the fruit surface, shape of cuticular formations on the surface of the outer periclinal walls of epidermal cells and on the surface of hairs, and structure of the stomatal complex), which were useful for the identification of the species. At the supraspecific level, the revealed features of the fruit surfaces somewhat overlapped and could be used to identify series and sections only as additional features. Based on the studied samples from herbarium material (KW), V. officinalis var. nitida was considered synonymous with V. wolgensis since there were no micromorphological differences between their fruits. e detailed micromorphological characteristics of the fruit surfaces of all the studied species can be used for further comparative morphological investigations of different aerial parts to identify stable features independent of geographic and ecological conditions.


Introduction
ere are more than 200 species of the genus Valeriana among worldwide flora. ey are mainly distributed in the Andes of South America, throughout Eurasia, and in the temperate zone of North America (Takhtajan, 2009). ere are 10 species in the latest nomenclatural checklist for the flora of Ukraine (Mosyakin & Fedoronchuk, 1999). Species of the genus Valeriana L. are known for their medicinal properties; biologically active substances have been found in seven species among the flora of Ukraine (Minarchenko, 2005). To discover reliable characteristics for the identification of such species, it is important to increase the amount of information on the micromorphology of their fruits.
Morphological characteristics of fruits are known to be species-specific for many members of the subfamily Valerianoideae and the order Dipsacales (Al-dabbagh & Saeed, 2020;Tsarenko et al., 2019;Tsarenko, Bulakh, et al., 2020;Tsarenko, Tsymbaliuk, et al., 2020;Tsymbaliuk et al., 2018;Zaĭtseva, 2006). In numerous scientific papers as well as various floristic reports, manuals, etc., the morphological features of the fruits of Valeriana spp. are minimized and only the general characteristics of their shape and morphometric data are provided (Gorbunov, 2002(Gorbunov, , 2014Katina, 1987;Vakulenko et al., 2016). Moreover, the micromorphological structure of the fruit surfaces have not been well studied. It is important to study these features to understand the issues of phylogeny and taxonomy of certain species as well as the family or the order of Dipsacales as a whole. e aim of this work was to conduct a detailed micromorphological characterization of the surfaces of fruits of Valeriana spp., which are important in pharmacology, in order to identify additional features that are relevant for species recognition.

Material and Methods
All fruits were obtained from herbarium specimens of the National Herbarium of M. G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine (KW). To obtain data on the general fruit appearance, they were examined using a light binocular stereoscopic microscope (MBS-9; Micromed). e micromorphological features of the fruits were studied using scanning electron microscopy (SEM) (JSM-6060; JEOL). e samples were sprayed with a layer of gold according to the standard method used. Fruit descriptions were recorded using the terminology generalized in a number of papers (Barthlott, 1981;Plisko, 2000;Ziman et al., 2004).
Photographs of the fruits and the microstructure of their surfaces were taken at ×30 to ×4,000 magnifications. Measurements of the fruit elements were performed using the AxioVision Rel. 4.8 (Carl Zeiss) soware. e sample citation herein is based on the original text of the labels (Table S1).

Results
e fruits of Valeriana spp. are quite similar in appearance. ey are somewhat flattened, especially on the adaxial side, mostly elongated-ovoid or ovoid ( Figure 1A, Figure 2A, Figure 3A, Figure 4A,B, Figure 5A,B, Figure 6A, Figure 7A,B, Figure 8A, Figure 9A,B, Figure 10A,B). e apex of the fruit is elongated, and the base is rounded and slightly expanded, with a barely visible depression in the middle and a basal fruit raphe. e calyx is at the apex of the fruit, which transforms into a white feathery pappus-like structure, with 8-16 long thick leathery awns and numerous white long so hair-like expansions ( Figure 3A, Figure 4C, Figure 6A). ese awns are basically fused together in the form of a membranous crown. In immature fruits, the awns with hairs are wrapped down and unfold only aer ripening. e fruits have a border on the edge on both sides that is composed of parenchymal tissue with a noticeable vascular bundle (lateral ribs).
On the adaxial side there is one longitudinal vein in the center, and on the abaxial side there are three (one central and two lateral) longitudinal veins with vascular bundles, which look like ribs from the outside. Some species have a pubescent fruit surface. e hairs are simple, unbranched, unicellular, and thin-walled. Detailed descriptions of the fruits are provided below. e species were categorized according to the accepted system of the genus (Gorbunov, 2014).  cuticle; and rounded with linear, warty, and striate ornamentation. Some epidermal cells have papillary elongated formations and papillae up to 30-40 μm high, with similar cuticular formations ( Figure 1C,D). e anticlinal walls are straight or wavy. e microsculpture of the fruit surface is of the small tuberculate type ( Figure 1B,C). e fruits are pubescent on both sides (except for the ribs) with simple ribbon-like slightly curved hairs, rounded at the apex and up to 0.4 mm long. e surface has cuticular formations similar to those on the surface of epidermal cells. On the inter-rib zones there are stomata surrounded by five or six radially located lateral cells, the cuticular sculpture of which is mostly of the striate type, and the stomatal  apertures are parallel to the longitudinal axis of the fruits. e cells around the stomata are covered with single unequally divergent wax formations ( Figure 1E). e awns are pubescent, with long ribbon-like, oen twisted, tortuous hairs, and similar cuticular formations ( Figure 1F).  e microsculpture of the fruit surface is of the small tuberculate-crested type ( Figure 3B). e boundaries between the cells are clear. On the ribs, the epidermal cells are located along the longitudinal axis, oblong, and smooth, with convex outer periclinal walls and straight, sometimes slightly curved, anticlinal walls. On the inter-rib zones, they are without a clear orientation, elongated, closer to the border, mostly angular, rounded with convex outer periclinal walls, which in some cells are compressed on the sides and form "ridges" slightly raised above the surface of other cells of epidermis, mainly with striate, and some cells with small tuberculate cuticular sculpture. e stomata are located only on the inter-rib zones, surrounded by four or five radially arranged cells. e shape and cuticular sculpture are similar to those of other epidermal cells, and the stomata are mostly parallel to the longitudinal axis of the fruit ( Figure 3C,D). e awns are pubescent, with long twisted so ribbon-like hairs, and surfaces having cuticular formations similar to those on the surface of epidermal cells.  e anticlinal cell walls are straight or slightly curved. Epicuticular wax is present only in some parts of the surface in the form of uneven irregularly divergent platelets. e fruits are pubescent on both sides (except ribs) with simple rigid tubular narrow-conical or ribbon-shaped slightly curved hairs, rounded at the apex, up to 0.2 mm long, and with rounded and linear warty cuticular ornamentation ( Figure 4F). e stomata are solitary only in the inter-rib zones, surrounded by four or five lateral cells (sometimes the boundaries between the surrounding cells are indistinct), and the stomatal apertures are parallel to the longitudinal axis of the fruits, the shape and cuticular sculpture of which are similar to those of other   e microsculpture of the fruit surface is of the small tuberculate type ( Figure 6B,C). e boundaries between the cells are clear. On the ribs, epidermal cells are arranged in rows along the longitudinal axis, oblong, and almost smooth, with convex outer periclinal walls. On the inter-rib zones, they are without a clear orientation, elongated, or rounded, angular, with convex outer periclinal walls, and with rounded and linear warty or striate cuticular formations ( Figure 6B,C). In some areas the epidermal cells have papillary protrusions of the periclinal cell walls, the surfaces of which are similar to those of cuticular formations. e anticlinal cell walls are straight or slightly curved. Epicuticular wax is present only in some parts of the surface in the form of unequally divergent platelets. e surface of the fruit is pubescent (on the adaxial side between the ribs, and sometimes on the abaxial side), with simple so hairs (up to 0.2 mm long), and rounded at the apex.

Section
On the surface of the fruit (inter-rib zones), the stomata are oriented along the longitudinal axis, surrounded mainly by five (sometimes only one or two) radially located lateral cells, the shape and cuticular sculpture of which are similar to those of other epidermal cells ( Figure 6D). e awns are pubescent, with long twisted so ribbon-like hairs, and rounded and linear warty cuticular formations. Worosch. (Figure 7) e microsculpture of the fruit surface is of the small tuberculate type ( Figure 7C). e boundaries between the cells are clear. On the ribs, the epidermal cells are arranged in rows along the longitudinal axis of the fruit, oblong, and smooth, with convex outer periclinal walls. On the inter-rib zones, they are without clear  orientation, elongated, or rounded, angular, with convex or concave outer periclinal and with clear straight, rounded or slightly curved anticlinal cell walls. Some epidermal cells have papillary protrusions of the outer periclinal walls or are replaced by papillae up to 30-40 μm long, with rounded and linear warty and striate cuticular formations ( Figure 7C). On the surface of the fruit (inter-rib zones) there are occasional stomata, which are surrounded by five lateral cells that are similar to other cells of the epidermal cuticular formations, and the stomatal apertures are parallel to the longitudinal axis of the fruit ( Figure 7D). Epicuticular wax is present only in some parts of the surface in the form of unequally divergent platelets. On the adaxial side, the fruits are pubescent, with hairs rounded at the apex and up to 0.2 mm long. e surface of the hairs is covered with rounded warty cuticular formations. e awns are pubescent, with long twisted so ribbon-like hairs, and similar cuticular formations. (Figure 8) e microsculpture of the fruit surface is of the small tuberculate type ( Figure 8B,C). e boundaries between the cells are clear. On the ribs, the epidermal cells are arranged in rows along the longitudinal axis, oblong, and smooth, with convex outer periclinal and clear, straight or slightly curved anticlinal walls. On the inter-rib zones, the cells are also oriented along the longitudinal axis of the fruits, and some cells are rounded, angular, with convex outer periclinal walls having rounded and linear warty cuticular formations.

Valeriana sambucifolia J. C. Mikan
On the inter-rib zones, the stomata are oriented along the longitudinal axis of the fruit. e stomata are surrounded by two-five lateral cells, located along the longitudinal axis of the fruit, and do not differ in shape and orientation from other  epidermal cells ( Figure 8D). Epicuticular wax is present only in some parts of the surface as unequally divergent platelets of wax. e awns are pubescent, with long twisted so ribbon-like hairs, and rounded and linear warty cuticular formations.
Valeriana stolonifera Czern. (Figure 9) e microsculpture of the fruit surface is of the small tuberculate type ( Figure 9C,E). e boundaries between the cells are clear. On the ribs, the epidermal cells are arranged in rows along the longitudinal axis, oblong, and smooth, with convex outer periclinal walls; on the inter-rib zones, they are mostly without a clear orientation, mostly rounded, angular, or elongated in places, with convex outer periclinal walls, indistinct or in certain places clear anticlinal walls, and in some areas, papillary formations and papillae up to 30-40 μm long having rounded and linear warty cuticular formations ( Figure 9F). Epicuticular wax is present only in some parts of the surface as unequally divergent platelets of wax. On the adaxial and abaxial sides, the fruits are pubescent only between the ribs, with simple rigid narrow-conical or ribbon-like hairs, up to 0.2 mm long, tubular at the base, and rounded at the apex, with similar cuticular formations ( Figure 9D,F). On the inter-rib zones, the stomata are surrounded mainly by five (sometimes two or three) lateral cells, the shape and cuticular sculpture of which are similar to those of other epidermal cells, and the stomatal apertures are parallel to the longitudinal axis of the fruit ( Figure 9E). e awns are pubescent, with long twisted so ribbon-like hairs, and warty cuticles.
Valeriana rossica P. Smirn. (Figure 10) e microsculpture of the fruit surface is of the small tuberculate type ( Figure 10C,D). e boundaries between the cells are clear. On the ribs, Acta Agrobotanica / 2021 / Volume 74 / Article 7414 Publisher: Polish Botanical Society the epidermal cells are oblong, with convex outer periclinal walls, smooth, without warty sculpture, and arranged in rows along the longitudinal axis; on the inter-rib zones, they have different shapes -angular, rounded, or elongated, in certain places with straight or slightly curved anticlinal and convex periclinal walls, and with rounded and linear warty cuticular formations.
On the adaxial side, the surface (except for the ribs and the border) is pubescent, with simple so ribbon-like hairs (0.09-0.3 mm long), tubular at the base, rounded at the top, and with warty cuticular sculptures; on the abaxial side, it is not pubescent. e awns are pubescent, with long ribbon-like hairs. On the inter-rib zones, the stomata, oriented along the longitudinal axis of the fruits, are surrounded mainly by five (sometimes two or three) lateral cells, the shape and cuticular sculpture of which are similar to those of other epidermal cells ( Figure 10D).

Discussion
e morphological features of fruits are important for species identification (Mehebub et al., 2019). Considering the micromorphological features of the fruit surfaces of species of the genus Valeriana, we could distinguish three groups of species (which do not correspond to supraspecific taxa): 1 -those with glabrous fruits (V. tripteris, V. simplicifolia, and V. sambucifolia); 2 -those with fruits pubescent only on the adaxial side (V. grossheimii and V. rossica); and 3 -those with fruits pubescent on both sides (V. tuberosa, V. wolgensis, V. stolonifera, and V. officinalis).
SEM analysis showed that within these groups of species, the microstructure of the fruit surface is similar in terms of the shape of epidermal cells, the presence and shape of cuticular formations on the surface of the outer periclinal walls of epidermal cells and on the surface of hairs, the type and shape of hairs (in pubescent species), and the shape of epicuticular waxy formations, which are present mainly on hairless surfaces; however, there are also some differences between them (Table 1)  vegetative and generative organs; hence, it is understandable that a slight species differentiation manifests at the level of micromorphology on the fruit surface.
However, Karcz (1996), who studied the micromorphological features of the fruit surface of V. officinalis (the author considered the species in a broad sense), found some differences between two varieties (V. officinalis var. officinalis and V. officinalis var. nitida). e micromorphological differences were noted for the structure of cuticular formations on the surface of epidermal cells and the surface of hairs, as well as the presence of epicuticular wax. Karcz (1996) did not rule out that these characteristics are phenotypic, resulting from the conditions prevailing in their habitat. We analyzed samples of V. officinalis fruits that were taken from herbarium material (KW) ( Table S1) and identified as V. officinalis var. nitida, V. officinalis s. str., and V. wolgensis (which we consider synonymous with V. officinalis var. nitida). ere were no differences between the studied samples. Phenotypic variability can probably manifest in the presence and localization of pubescence of fruits of some species. e samples of V. grossheimii studied by us had pubescence of fruits only on the adaxial side; however, Gorbunov (2002) indicated pubescence for this species only on the abaxial side. Fruit samples of V. officinalis s. str. were pubescent on both sides, and according to the literature, the fruits can also be glabrous or pubescent on one side only (Grubov, 1958). However, to identify the species, it is necessary to consider the whole complex of features of the fruit surface microstructure, except the type of wax formation on the epidermal cell surface, which cannot be used as an additional diagnostic feature, because it is similar in all the studied species of the genus. At the supraspecific level, the revealed features of the fruit surface somewhat overlap and can be used to identify series and sections only as additional features.
us, an investigation on the microstructure of the fruit surfaces of Valeriana spp. was conducted, and the diversity in the cuticular sculpture of the outer periclinal cell walls of the epidermis was shown. In turn, the micromorphological features were identified, and these can be used as additional features for the identification of species and supraspecific taxa.
However, our data on the microstructure of the fruit surface for some Valeriana spp., samples of which were collected from the territory of Ukraine, differ slightly from those reported by other scientific sources. is study revealed detailed micromorphological characteristics of the fruit surfaces of all the studied species, and they may be useful for further comparative morphological studies of fruits taken from other aerial parts of the species. ese will help identify stable micromorphological characteristics of fruits that are resistant to geographical and environmental factors.

Supplementary Material
e following supplementary material is available for this article: Table S1. Herbarium specimens of species of the genus Valeriana used for the study of fruits using SEM.