Carpological features of Lonicera L. (Caprifoliaceae Juss.) of the flora of Ukraine

The objective of this study was to carry out macroand micromorphological studies of fruits and seeds of representatives of the genus Lonicera of the flora of Ukraine (both natural and introduced), to clarify their features, to provide additional characteristics and to evaluate the possibility of using carpological features for the diagnosis of taxa of the genus. Material and methods. Materials of the National Herbarium of the M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine (KW) and Herbarium of the M.M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine (KWHA) were used, as well as samples from living plants from the collections of the M.M. Gryshko National Botanical Garden and O.V. Fomin Botanical Garden. The citations are given by the original text of the labels. The fruits and seeds of seven species of the genus (four are introduced, and the other are species of natural flora), which are listed in “Vascular plants of Ukraine. A nomenclatural checklist”, have been studied. Morphological features of fruits and seeds were studied under the light microscope (LM, MBS-9) and scanning electron microscope (SEM, JSM-6060 LA). Samples were sprayed with a layer of gold according to the standard method. The study was conducted at magnification from ×30 to ×3000. Descriptions of fruits and seeds were performed using terminology, summarized in specialized papers. Fruit and seed sizes were measured using Axio Vision Rel. 4.8. Results. The fruits of Lonicera species are free or adnate at the base, surrounded by, for the most part, free bracteoles. A pseudocarp of L. caerulea is formed of the bracteoles of paired flowers concrescent in a tubular cupule, which completely and tightly wraps free paired fruits. The L. nigra has an uneven concrescent of the bracteoles that freely surround the paired fruits, which have concrescent only at the base. The fruits of the species Lonicera are black and dark blue, or red and yellow. The fruits of subgenus Periclymenum (L. caprifolium, L. etrusca, L. periclymenum) are free and spherical. The subgenus Chamaecerasus species’ fruits are mainly spherical, but in some species the fruits are also hemispherical, elliptical and elongated (L. caerulea, L. tatarica). Fruits in this subgenus are free (L. caerulea, L. tatarica) or coalescent at the base (L. nigra, L. xylosteum). All the studied species are characterized by glabrous fruits, except fruits of L. xylosteum that are pubescent with glandular and simple hairs. The seeds are elliptical, ovoid, nearly spherical, flat, concave on one side and convex on the other side, with a recess on the periphery and a crest in the center, at the base they are mainly narrow-rounded. All species of the section Eucaprifolium and one species of the section Coeloxylosteum have truncated seeds. The surface of the seeds is almost similar and pitted; in L. xylosteum it is shallow-pitted; in L. nigra it is tubercular-pitted. In some species, the seed surface is pitted, and also flat cells are present. Conclusions. Based on the results of a critical review of literature materials and analysis of own data, the detailed characteristics of fruits and seeds of the representatives of the genus Lonicera within the flora of Ukraine have been compiled. These characteristics are useful for more accurate identification of fruiting Lonicera plants. The species of the subgenus Periclymenum have exclusively free fruits, while the fruits in the subgenus Chamaecerasus are free or coalescent at the base. The features of color and shape of the fruits https://doi.org/10.46341/PI2020012 UDC 581.471/581.48/582.973


Introduction
The genus Lonicera is represented in the world flora by almost 200 species (Theis et al., 2008;Takhtajan, 2009). The species are distributed in all areas of the Northern Hemisphere (in temperate and subtropical regions of Europe, North and Central America, North Africa, and Asia). South-East Asia is considered to be the primary center of origin for this genus. Some representatives of this genus are decorative plants with beautiful flowers and fruits and were introduced into the culture (Browicz, 1976;Poyarkova, 1958;Zaytsev, 1962). Mosyakin & Fedorochuk (1999) have listed seven Lonicera species for the flora of Ukraine -four are species of natural flora (L. caerulea L., L. etrusca Santi, L. nigra L., L. xylosteum L.), and three species (L. caprifolium L., L. periclymenum L., L. tatarica L.) are introduced from other countries and are widely used in botanical gardens and parks of Ukraine (Barbarych, 1961;Zaytsev, 1962;Glukhov et al., 2002;Lavrinenko, 2016).
According to the APG IV (APG, 2016) and the results of recent phylogenetic studies of Dipsacales Juss. ex Bercht. et J. Presl, the genus Lonicera belongs to the tribe Caprifolieae of the family Caprifoliaceae Juss. (Theis et al., 2008;Takhtajan, 2009;Reveal, 2012). Also, the features of fruits and seeds of Lonicera are well discussed in the literature and listed among the morphological features confirming recent molecular-phylogenetic data on Dipsacales (Bell et al., 2001(Bell et al., , 2005Donoghue, 2003;Jacobs et al., 2008;Theis et al., 2008;Göktürk & Sümbül, 2014).
Carpological characteristics are essential for the identification of these plants in the fruiting stage. The information on the morphological features of seeds is also important for specialists in botanical gardens and arboretums, who directly work with seeds during the seed exchange programs.
It is also important to note, most of the authors of the monographic elaboration of the genus Lonicera in the regional Floras and Identification Keys usually used features of the perianth, a number of flowers on the shoot apex, shoot pubescence, and shape of leaves to delimit taxa. They only briefly focused on the characteristics of fruits (color, size) and seeds as supporting features (Rehder, 1909;Poyarkova, 1958;Barbarych, 1961;Zaytsev, 1962). Despite this, we believe, that carpological features (including micromorphological peculiarities) play an essential role in diagnostics and identification of species in the genus Lonicera, in particular those represented in the flora of Ukraine, during the fruiting stage.
The fruits of Lonicera are inferior syncarpous succulent multi-seeded berries with various colors -yellow, dark-red, blue, and black. They are arranged in pairs, free or coalescent at the base, often one of the paired berries may be underdeveloped. The ovary contains 2-or 3-4 locules with a double-rowed location of numerous anatropous ovules with one massive integument (Plysko, 2000). The species of Lonicera are characterized by different levels of coalescence of adjacent fruits. Free fruits were found in L. syringantha Maxim.; partial fusion was found in L. canadensis Bartram ex Marshall and L. tatarica, and the highest coalescence of adjacent fruits is typical for L. caerulea (Artiushenko & Fedorov, 1986;Plysko, 2000). Malinkina (2001) found that L. tatarica and L. xylosteum have a pair of inferior syncarpous 3-locular ovaries, which unified on a single pedicel with two free bracts. At the base of each ovary, there are two small round bracteoles, which are partially fused with the ovary. Some species have coalescent bracteoles, which form a cupule enclosing the ovaries (Poyarkova, 1958).

Carpological features of Lonicera of the flora of Ukraine
At the beginning of fruit development, 2-3 layers of cells separate from the outer epidermis and together form the exocarp. Between these layers there is a layer of parenchyma cells -mesocarp. The pericarp of the fruit of Lonicera is formed from similar parenchymal cells.
The cells of the exocarp are polygonal in most species in cross-section. The cells of mesocarp are round and thin-walled, containing chromoplasts and drops of oil. The inner part of the mesocarp is rich in calcium oxalate. Endocarp is usually reduced to a single unsclerified layer of cells (Artiushenko & Fedorov, 1986;Plysko, 2000;Jacobs et al., 2009).
The seeds are early separated from the pericarp. Developed seeds have a small (up to 1/6-1/4 length of seed), straight, well-differentiated embryo. The embryo is surrounded by a well-differentiated endosperm and a multilayered spermoderm with lignified exotesta cells (Malinkina, 2001;Jacobs et al., 2009;Lavrinenko, 2012). The microstructure of the seed surface is specified by the features of the walls of exotesta cells. Anticlinal and periclinal walls of exotesta are thickened, lignified, and have pores. The hilum is small, submerged, located at the base of the seed (Jacobs et al., 2009;Lavrinenko, 2012).
Analyzing research articles (Rehder, 1909;Barbarych, 1961;Zaytsev, 1962;Artiushenko & Fedorov, 1986;Zaitseva, 1998Zaitseva, , 1999Zaitseva, , 2006Glukhov et al., 2002;Jacobs et al., 2009;Lavrinenko, 2012Lavrinenko, , 2016, we found that a number of questions regarding carpological characteristics of the genus Lonicera remained outside the attention of researchers (i.e., features of the fruits pubescence, surface microstructure of the seeds, etc.). This fact motivated us to investigate macro-and micromorphology of the fruits and seeds of Lonicera of the flora of Ukraine. These studies were aimed to provide additional characteristics of the fruits and seeds and to evaluate the possibility of application of carpological features for the identification of Lonicera specimens. Morphological features of the seeds and fruits were studied using a light microscope (LM, MBS-9) and scanning electron microscope (SEM, JSM-6060 LA). The study was conducted at magnification from ×30 to ×3000. Descriptions of fruits and seeds were performed using terminology, summarized in a number of relevant specialized papers (Artiushenko & Fedorov, 1986;Plysko, 2000;Ziman et al., 2004Ziman et al., , 2011. Fruit and seed sizes were measured using Axio Vision Rel. 4.8.

Results
Investigated species belong to two subgenera: Chamaecerasus (L.) Rehder (sections Isika (Adanson) Rehder and Coeloxylosteum Rehder) and Periclymenum (L.) Rehder (section Eucaprifolium (Spach) Pojarkova). Based on published materials and obtained results, their characteristics have been compiled. The species are listed below according to the system of the genus Lonicera represented in the "Flora of the USSR" (Poyarkova, 1958). Two fruits are free. A pseudocarp is formed by bracteoles of two paired flowers, which are connate in a common tubular cupule. Cupule completely and tightly wraps free paired fruits. The fruits are dark blue, with bluish cover, fleshy, spherical, hemispherical or elliptical, 8-11 mm in diameter, containing up to 10-20 (-30) seeds.

Genus
The seeds are light-or dark-brown, broadly elliptical or rounded, 1.5-2.3 mm long and 1.4-2.1 mm wide, flat, slightly concave on one side, convex on the other, with a cavity on the periphery and a crest in the center, at the base narrow-rounded, with a small notch. The hilum is small, submerged, located at the base of the seed.
The surface of the seed is mostly pitted. Exotesta cells are polygonal, with concave periclinal and distinctly visible anticlinal walls. The outer periclinal walls have ribs, which become visible at the magnification over ×500. Ribs were without clear orientation. The infructescence consists of two fruits connate at the base. Four bracteoles are irregularly and incompletely connate into a goblet-shaped cupule, which freely wraps the fruit at its base. The fruits are black, with bluish cover, fleshy, spherical, 6-10 mm in diameter, connate in pairs only at the base.
The seeds are light-or dark-brown, elliptical, 2.2-2.5 mm long and 1.8-2.4 mm wide, flat, on one side slightly concave, on the other side convex, with a cavity on the periphery and a crest in the center, at the base narrowly rounded, with a small notch. The hilum is small, submerged, located at the base of the seed.
The surface of the seed is tuberculatepitted. The cells of the exotesta are polygonal, with concave or convex periclinal and distinctly visible anticlinal walls. The outer periclinal walls are ribbed or wrinkled (seen at magnification over ×500). Carpological features of Lonicera of the flora of Ukraine Section Coeloxylosteum Rehder Subsection Tataricae Rehder emend. Nedoluzhko Lonicera tatarica L. (Fig. 3; Tables 1 & 2) Two free fruits, bracteoles are also free and cover the fruits to the middle. The fruits are red or yellow, fleshy, spherical, sometimes elliptical, 6-8 mm in diameter. The seeds are light-or dark-brown, elliptical, 3.0-3.5 mm long and 1.5-2.7 mm wide, somewhat flat, slightly concave on one side, convex on the other, with a cavity on the periphery and a crest in the center, at the base narrowly rounded, somewhat truncated. The hilum is small, submerged, located at the base of the seed.
The surface of the seed is pitted. The cells of the exotesta are polygonal, with concave periclinal and distinctly visible anticlinal walls. The outer periclinal walls with ribs, which are visible at magnification over ×500. Ribs do not have a strict orientation. Subsection Ochranthae Zabel emend. Rehder Lonicera xylosteum L. (Fig. 4; Tables 1

& 2)
The infructescence consists of two fruits, which are connate only at the base. Bracteoles are free. The fruits are dark-red, yellow, fleshy, spherical, 6-9 mm in diameter. The surface of the fruits is sparsely pubescent with two- The seeds are light-or dark-brown, elliptical-rounded or asymmetrical, slightly curved, angular, 2.5-3.0 mm long and 2.0-2.7 mm wide, flat, slightly concave on one side, convex on the other, with a cavity on the periphery and a hardly noticeable crest in the center, narrow-rounded at the base. The hilum is small, submerged, located at the base of the seed.
The surface of the seed is shallowly pitted. The cells of the exotesta are polygonal, with concave periclinal and indistinct anticlinal walls. Outer periclinal walls are covered with a tortuous net visible at the magnification over ×500.
The seeds are light-or dark-brown, broadelliptical, sometimes rounded, often curved, 3-6 mm long and 2-3 mm wide, somewhat flat, slightly concave on one side, convex on the other, with a cavity on the periphery and a crest in the center, somewhat truncated at the base, with a small notch. The hilum is small, submerged, located at the base of the seed. The surface of the seeds is diversiform: in some areas of the seeds, it is pitted. In others -the cells of the exotesta are almost flat, with tortuous anticlinal walls, the walls of the cells are clearly visible. The outer periclinal walls are ribbed and wrinkled (seen at the magnification over ×1000). Lonicera periclymenum L. (Fig. 6; Tables 1 & 2) Two free fruits, bracteoles are free too. The fruits are red, fleshy, spherical, up to 6 mm in diameter.
The seeds are light-brown, elliptical, ovoid, 2.0-3.5 mm long and 1.5-2.0 mm wide, flat, slightly concave on one side, convex on the other, with a cavity on the periphery and a crest in the center, narrow-rounded at the base, somewhat truncated. The hilum is small, submerged, located at the base of the seed.
The surface of the seeds is pitted in some areas of the seeds, and the cells of the exotesta are almost flat, with concave or more or less   (Fig. 7; Tables 1 & 2) Several fruits are free, some of which are much smaller or undeveloped. The bracteoles are free too. The fruits are red, fleshy, spherical, up to 8-10 mm in diameter.

Lonicera etrusca Santi
The seeds are brown, broadly elliptical or ovoid, somewhat angular, 3.0-4.0 mm long and 2.7-3.5 mm wide, flattened-convex, slightly concave on one side, convex on the other, with

Discussion
According to our results, we did not find any characteristics that could be diagnostic at the level of subsections or higher. We have only found that there are some species-level differences in the microstructure of the seed surface. The surface of the seeds of all the species studied is almost identical. It is mostly pitted due to concave external periclinal walls of the exotesta. The surface of L. xylosteum seeds is shallowly pitted, as reported by Jacobs et al. (2009). However, the seed surface in L. nigra is tubercular-pitted. The seed surface in L. caprifolium and L. periclymenum is pitted, but almost flat cells of the exotesta are also present.
Our research confirmed the feasibility of the fruit microcarpological features for more accurate identification of species-level taxa in the genus Lonicera. Only a few publications devoted to microcarpology of the genus Lonicera (Plysko, 2000;Jacobs et al., 2009;Lavrinenko, 2012). Several micromorphological characteristics of Lonicera species have been applied to confirm the molecular phylogeny of the genus (Jacobs et al., 2009). These features also can be successfully applied for resolving controversial questions of taxonomy, what has been shown for Dipsacales (Tsymbalyuk et al., 2018;Tsarenko et al., 2019) and some rare Caryophyllales Juss. ex Bercht. et J. Presl (Amini et al., 2011;Martynyuk et al., 2015aMartynyuk et al., , 2015bMartynyuk et al., , 2016Martynyuk et al., , 2018Shykhaleyeva et al., 2018).

Conclusions
Based on the results of the analysis of literary sources and our own data of macro-and micromorphological studies of the fruits and seeds of representatives of the genus Lonicera, their detailed characteristics were prepared. It should facilitate the more precise identification of the species in the fruiting state. A carpological feature that is specific to representatives of the subgenus Periclymenum is the presence of exclusively free fruits, unlike representatives of the subgenus Chamaecerasus with both free and connate at the base fruits.
At the species level, we suggest using the following features of fruits and seeds: differences in fruits coloration of representatives of the genus; the presence of pseudocarp in the L. caerulea; the difference in shape (i.e., for L. caerulea and L. tatarica with the spherical, elliptical shape of the fruits was also noted); the presence of glandular and simple hairs on fruits of L. xylosteum; the difference in the shape of the seeds (elliptic or ovoid in L. etrusca, L. nigra, and L. tatarica, and broadly elliptic or almost rounded in L. caerulea, L. periclymenum, and L. xylosteum).
There are no characteristics of the microstructure of the surface of the seed that can be diagnostic at the level of subsections or higher. The seeds have only some differences at the species level. In most of the studied species, the seed surface is pitted -seeds of L. nigra are tubercular-pitted and seeds of L. xylosteum with shallow-pitted surfaces, while seeds of L. caprifolium and L. periclymenum have almost flat exotesta cells.