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International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899

Features Of The Reproductive Sphere Of Species Of The Genus Hosta Tratt

Irina S. Kazakova1, Anna I. Repetskaya1, Irina G. Savushkina1, Natalya V. Nevkrytaya 2, Vladimir S. Pashtetsky2, Natalia A. Kashirina2

1 V.I. Vernadsky Crimean Federal University, 4 Prospekt Vernadskogo, Simferopol, Republic of Crimea, 295007, Russia 2 FSBSI “Research Institute of Agriculture of Crimea", 150 Kievskaya str., Simferopol, Republic of Crimea, 295493, Russia

Abstract Hosta (plantain lily) is a widespread ornamental foliage culture, yet its systematics remains incomplete. The study of the reproductive sphere of five species of hostas (Hosta sieboldii (Paxton) Ingram, H. ventricosa Stearn, H. sieboldiana (Hooker) Engler, H. rectifolia Nakai, H. plantaginea Ascherson) when introduced in the Foothill Crimea is aimed at identifying species-specific traits and selecting promising species for breeding. Morphometric, phenological, cytological methods and electron microscopy were used. Vegetation period in the Foothill Crimea is from April to November; H. rectifolia has the longest blooming period (95 days), and H. ventricosa the shortest (13 days). Flower size in the majority of species is 5-6 cm long to 3-4 cm wide; seed length varies from 8 to 13 mm and is significantly different in all species. Potential seed productivity is 191.2-594.7 ovules per plant; real productivity is 61.3-500.5 seeds. Seed setting comprises 32.1-84.9%, laboratory germination is 14.7-77%. The size of pollen grains on the polar axis ranges from 60.0 to 113.5 μm, on the equatorial axis from 29.5 to 67.1 μm; pollen fertility is 63.6-82.0%. The size and color of seeds are species-specific and can be used as systematic criteria. Three reproductive strategies of species were identified. H. sieboldii, H. sieboldiana, H. plantaginea, and H. ventricosa are recommended for breeding as parental forms. Keywords: Hosta, reproductive sphere, flower, seed, pollen grain.

INTRODUCTION Genus Hosta Tratt. comprises more than 40 species (Bakanova, 1988; Kohlein, 1993; Schmid, 1991). At Baker’s initiative in 1870, hostas were assigned to the Hemerocallidaceae family; the researches carried out in the 1930s showed their proximity to such genera as Yucca, Agave, Camassia, Hemerocallis, Hesperocallis, Leucocrinum, and Manfreda. Based on cytological data obtained by a number of researchers (McKelvey, Sax, 1933, Whitaker, 1934, Akemine, 1935, Sato, 1935, 1942, Suto, 1936, Granick, 1944, Cave, 1948, Wunderlich, 1950, Chupov and Kutyavina, 1981), it was suggested the genus should be included in the Agavaceae family, although karyotypic similarities indicated its proximity to the Liliaceae family. In a recent work on the classification of monocots (Dahlgren et al., 1985), daylilies and hostas were separated – Hemerocalllis was placed in the Hemerocallidaceae family, and the genus Hosta, along with Hesperocallis and Leucocrinum, were singled out as the Funkiaceae family in the order of Asparagales. Later, based on Mathew’s research (1987), Leucocrinum was assigned to the Anthericaceae family, and the taxonomically complex and monotypic genus Hesperocallis was assigned to the Hyacinthaceae family. Thus, only the genus Hosta remained in the Funkiaceae family. Mathew suggested renaming the family as Hostaceae (Mathew, 1988); however, the discussion about the systematic position of the genus is not closed. Thus, in a number of topical botanical electronic databases, hosts are placed in the Liliaceae family (Flora of China www.efloras.org) or Asparagaceae (The Plant List http://www.theplantlist.org/; Germplasm Resources Information Network https: //npgsweb.ars -grin.gov/). Questions of the systematics of the genus remain controversial. In particular, some species were described by comparison with cultured specimens, and a number of species clearly are of hybrid origin (Schmid, 1991).

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899

The largest review devoted to hostas belongs to Schmid (Schmid, 1991) – the author analyzed studies from 1797 to 1990 and offered the system of the genus Hosta the authors of the present research chose to follow. To solve the issues of systematics, we need to identify ecologically flexible and stable features of individual plants and to single out species-specific characteristics. In this regard, obtaining morphobiological data on plants growing in different soil and climatic conditions and, above all, outside the natural range of the species, is crucial. The natural habitat of the genus is located in the East Asian region of the Holarctic kingdom (Takhtajan, 1987). Most species are common in Japan, some in Korea, China, on Sakhalin and the Kuril Islands (Schmid, 1991; Germplasm Resources Information Network, 2018). Hosta is one of the most popular and widespread decorative leafy plants, widely used in temperate regions to create garden compositions. In the southern regions, it is not so frequent, although landscape decorators take considerable interest in hostas, since they are good for the shaded areas of landscape architecture objects; the cultivation experience of the Foothill Crimea is a vivid example (Kazakova et al., 2011, 2012; Kazakova, Repetskaya, 2017). Obtaining an assortment adapted to the hot arid climate will facilitate the introduction of this very promising culture into southern ornamental horticulture. For introductive and breeding work, complex morphological study of species is important. The main sign of successful plant introduction is their ability to reproduce in new conditions (Kohno, Kurdyuk, 1994). The study of the generative sphere of introduced species is of particular importance for solving problems of selection and use in mass gardening. Erdtman's study of the pollen grains of the Hemerocallidaea family (Erdtman, 1956) gives preliminary information on the hostas' generative sphere. Chung and Jones (Chung, Jones, 1989) conducted a study of pollen of 22 species and subspecies of hostas and of 9 closely related taxa. It confirmed the logic of dividing the genus into three subgenera, first proposed by Bailey (1930) and of singling hostas out into a separate family. Based on a study of the pollen morphology, Chinese researchers (J-X. Liu, C-H. Zhao, X-R. Liu, Y-Z. Xi and Y-L. Zhang, 2011) noted certain features of hostas’ similarity with the Liliaceae family, agaves and yuccas. A number of works by Russian and Ukrainian researchers (Boyko, 2010; Results of the introduction ..., 2007; Davletbaeva, Reut, 2017; Sedelnikova, 2012; Smolinska, 2009; Pavlyuk, 2014) provide certain information about the vegetative and generative sphere of the genus (bush height, leaf blade size, peduncle length and flower size), as well as data on the growth rhythms and development of plants introduced in various soil-climatic zones. In the conditions of the Bashkir Cis-Urals and the Forest-Steppe Zone of Ukraine, laboratory germination of seeds and seed productivity of some species were assessed (Boyko, 2010; Davletbaeva, Reut, 2017). Previously, we gave a characteristic of the vegetative sphere of 7 species, 6 forms and 18 varieties of hostas (Kazakova et al., 2012) and analyzed the anatomical and morphological features of leaf blade of five species on the light gradient (Kazakova et al. 2011). There is no comprehensive description of the generative structures of representatives of this genus under cultivation conditions. The authors’ purpose is a comprehensive study of the reproductive sphere of species of the genus Hosta introduced in the Foothill Crimea, aimed at identifying species- specific traits and selecting promising species for breeding.

MATERIALS AND METHODS The work was carried out based on the collection of the N.V. Bagrov Botanical Garden of the Tavricheskaya Academy of V.I. Vernadsky Crimean Federal University (hereinafter BG CFU) in Simferopol. The Botanical Garden is located within the Eastern Foothill agroclimatic region of the northern macroslope of the Crimean Mountains (Vazhov, 1977). The climate is semi-arid, warm, with mild winters. Selyaninov’s hydro-thermal coefficient (HTC) is 0.89, Ivanov's moisture ratio (MR) is 0.56. The average annual temperature is +10.6 °С, the average January temperature is +0.2 °С, the average July temperature is +22.5 °С. The average annual rainfall is 536 mm, 329 mm of which fall during the warm period (April-October) (Agroclimatic reference book, 2011). The BG CFU hostas collection includes 7 species, 49 varieties and 1 form. The objects of research were 5 species: Hosta sieboldii (Paxton) Ingram, Hosta ventricosa Stearn, Hosta sieboldiana

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899

(Hooker) Engler, Hosta rectifolia Nakai, Hosta plantaginea Ascherson, introduced in 2007 (Annotated catalog ..., 2014). To identify the possibility of realizing the genetic potential of hostas species in the Foothill Crimea, an assessment of the morphometric parameters of the plant generative organs was made: flower length and width, capsule and seeds size; the sample for each indicator was 100 pieces. Phenological observations were carried out for 5 years according to I.N. Beideman’s method (Beideman, 1974). The following phenological phases were distinguished: the beginning of growth, the beginning of leaf unfolding, the massive unfolding of leaves, budding, the beginning of blooming, the end of blooming, the ripening of fruits, the beginning of leaves withering, the complete leaves withering. When conducting phenological observations, the dates on which the species entered into a particular phenophase were noted. For comparative assessment of phenological observations, we used the results obtained in other soil-climatic zones: in Central Russia (Botanical Garden of Lomonosov Moscow State University, hereinafter BG MSU; Main Botanical Garden named after N.V. Tsitsin of the Russian Academy of Sciences, hereinafter MBG RAS), in Western Siberia (Central Siberian Botanical Garden, hereafter CSBG), in the Far East (Botanical Garden-Institute of the Far Eastern Branch of the RAS), in the Southern Urals (Botanical Garden-Institute of Ufa Scientific Center of the RAS), in the Forest-Steppe Zone of Ukraine (National Botanical Garden named after N.N. Grishko, hereinafter NBG, National Dendrological Park Sofiyivka, hereafter Sofiyivka), in Bukovina (Botanical Garden of Chernivtsi National University named after Yu. Fedkovich, BG CNU). The ability of plants for vegetative reproduction was determined using the vegetative reproduction coefficient in the seventh year after planting when the plants reach maximum sizes (Mitronina, 2008). The authors took into account the number of planted units per one standard planted unit (Methods of State Variety Testing…., 1968). Potential (PSP) and actual (ASP) seed productivity of plants is used to characterize the success of seed reproduction (Vaynagiy, 1974). Seed germination in laboratory conditions was established according to the ‘Methodological guidelines for seed breeding of introduced species’ (1980). To determine germination, 100 seeds were laid in 4-fold repetition. To include species in breeding programs, potential pollen donors were selected as paternal forms. Pollen was collected during the mass blooming period (June-August). The work was carried out with freshly collected material. The anthers extracted from the flowers were kept at room temperature on parchment paper until they were opened. Temporary preparations for measuring pollen grains were made in glycerin. Morphometric parameters (length and width of the pollen grain) were determined in 30-fold repetition using the automatic system for obtaining images Image Xpress Micro XLS. Pollen fertility was assessed by Z. P. Pausheva’s method (1988). Pollen grains were treated with acetocarmine; pollen with a uniform color was attributed to morphologically normal, pollen with unpainted content was attributed to sterile. Counting the number of fertile and sterile pollen grains was performed in 100-fold repetition. To study the ultrasculpture of pollen, individual pollen grains were taken from dry material, placed on metal tables and sprayed with an alloy of platinum and palladium in an ion sputtering unit. Photographic recording of prepared pollen grains was carried out using a Zeiss EVO40XVP scanning electron microscope in the Interdisciplinary Analytical Laboratory of the Federal Research Center of the Southern Scientific Center of the RAS (Rostov-on-Don). The ultrastructure description of the surface of pollen grains was performed according to N.R. Meyer-Melikyan’s method (Meyer- Melikyan, Severova, 1999) using the Erdtman (1956) and Chung and Jones (1989) classifications of the sexine pattern types. Statistical processing of results was carried out according to generally accepted methods (Lakin, 1980; Plokhinsky, 1970).

RESULTS Hostas belong to the decorative foliage cultures, and their decorativeness coincides with the growing season in the Foothill Crimea – from April to November. Blooming lasts from the second decade of June to the third decade of September and increases the attractive quality of the plants. Over the 5-year observation period, once (in 2012) repeated blooming was observed in H. sieboldii and H. rectifolia. In H. sieboldii, the duration of the first blooming was

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899

40 days, after 43 days the second blooming was noted, which lasted 58 days. In H. rectifolia, the first blooming lasted 10 days, after a week the second blooming began, which lasted 38 days. In different species, the peduncle rises above the rosette of leaves (H. sieboldii, H. ventricosa), hangs above it (H. sieboldiana, H. rectifolia) or is practically at the level of foliage (H. plantaginea). Peduncles are mildly leafed or leafless. In H. sieboldii, the peduncle has several small leaves, the inflorescence itself is loose, with evenly spaced flowers. In H. plantaginea, the peduncle is strong; the inflorescence is thick, short, with one or several underdeveloped leaves. H. sieboldiana has a short and dense inflorescence located on a leafless (or single-leaf) peduncle. H. rectifolia and H. ventricosa have long and loose inflorescences. Below are the dimensions of flowers, fruit, seeds and pollen of the studied species, as well as quantitative data characterizing the features of their reproductive biology.

Hosta ventricosa Stearn Blooming occurs the I-II decade of July. Flower is violet with dark violet veins (Fig. 1a). Perianth is 5.6 ± 0.1 cm long, 3.1 ± 0.4 cm in diameter. Number of peduncles is 10.3 ± 1.4 pcs, number of flowers in the inflorescence is 27.4 ± 0.9 pcs. Capsule is 2.9 ± 0.1 cm long to 0.9 ± 0.1 cm wide. Number of seeds in the capsule is 12.7 ± 0.6, number of ovules is 26.9 ± 0.7. Seed setting is 47.2%. PSP of a plant is 350.2, ASP is 165.4. Seeds are 13.77 ± 0.76 mm long to 5.37 ± 0.11 mm wide (Fig. 2d). Laboratory germination is 77%. Pollen grain sizes: polar axis is 97.2 ± 0.1 µm, equatorial axis is 50.8 ± 0.1 µm. Pollen fertility is 81.5 ± 4.7%. Sexine is in the form of papillae (tubercles) by Erdtman (1956) or rugulate by Chung and Jones (1989) (Fig. 4b).

а) b) Fig.1 Flower srtucture: а) H. ventricosa, b) H. sieboldii.

Hosta plantaginea Ascherson. Blooming occurs from the I decade of August to the I decade of September. Flower is white. Perianth is 12.6 ± 0.6 cm long, 5.4 ± 0.2 cm in diameter. Number of peduncles is 6.6 ± 0.6 pcs., number of flowers in the inflorescence is 14.2 ± 0.3 pcs. Capsule is 6.6 ± 0.2 cm long to 0.9 ± 0.1 wide. Number of seeds in the capsule is 22.7 ± 1.3, number of ovules is 70.8 ± 1.2. Seed setting is 32.1%. PSP of a plant is 191.2, ASP is 61.3. Seeds are 11.87 ± 0.20 long to 3.49 ± 0.07 mm wide (Fig. 2a). Laboratory germination is 42.5%. Pollen grain sizes: polar axis is 113.5 ± 0.1 μm, equatorial axis is 67.1 ± 0.1 μm. Pollen fertility is 79.7 ± 3.1%. Sexine is reticulate by Erdtman (1956) and Chung and Jones (1989) (Fig. 4a).

a) b)

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899

c) d)

Fig.2 Seeds: а) Hosta plantaginea b) Hosta sieboldii c) Hosta sieboldiana d) Hosta ventricosa (10x magnification)

Hosta sieboldii (Paxton) Ingram Blooming occurs from the III decade of June to the II decade of July. Flower is lilac with light violet streaks (Fig. 1b). Perianth is 5.6 ± 0.1 cm long, 3.8 ± 0.1 cm in diameter. Number of peduncles is 11.2 ± 3.8 pcs., number of flowers in the inflorescence is 38.9 ± 1.8 pcs. Capsule is 2.6 ± 0.1 cm long and 0.7 ± 0.1 cm wide. Number of seeds in the capsule is 26.1 ± 0.8, number of ovules is 30.7 ± 0.5. Seed setting is 84.9%. PSP of a plant is 589.1, ASP is 500.5. Seeds are 8.08 ± 0.11 mm long to 3.22 ± 0.08 mm wide (Fig. 2b). Laboratory germination is 22.7%. Pollen grain sizes: polar axis is 88.9 ± 0.1 µm, equatorial axis is 40.9 ± 0.1 µm (Fig. 3). Pollen fertility is 82.0 ± 1.8%. Sexine is club- shaped by Erdtman (1956) or rugulate-baculate by Chung and Jones (1989) (Fig. 4c).

Fig. 3 Morphometric parameters of pollen grains of H. sieboldii (400x magnification)

Hosta rectifolia Nakai Blooming occurs from the I decade of August to the III decade of September. Flower is lilac with light violet streaks. Perianth is 4.3 ± 0.1 cm long, 3.3 ± 0.1 cm in diameter. Number of peduncles is 15 ± 3.3 pcs., number of flowers in the inflorescence is 27.8 ± 0.9 pcs. Does not bear fruit if introduced in the Piedmont Crimea. Pollen grain sizes: polar axis is 60.0 ± 0.1 µm, equatorial axis is 29.5 ± 0.1 µm. Pollen fertility is 69.3 ± 1.8%. Sexine is club-shaped according to Erdtman (1956) or rugulate- baculate by Chung and Jones (1989) (Fig. 4d).

Hosta sieboldiana (Hooker) Engler Blooming occurs from the II decade of June to the II decade of July. Flower is lilac with light violet streaks. Perianth is 5.0 ± 0.1 cm long, 3.3 ± 0.5 cm in diameter. Number of peduncles is 9.9 ± 1.2 pcs., number of flowers in the inflorescence is 30.4 ± 0.6. Capsule is 2.6 ± 0.1 cm long to 0.7 ± 0.1 cm wide. Number of seeds in the capsule is 17.2 ± 0.4, number of ovules is 35.4 ± 0.5. Seed setting is 48.6%. PSP of a plant is 594.7, ASP is 288.9. Seeds are 8.41 ± 0.12 mm long to 3.24 ± 0.08 mm wide (Fig. 2c). Laboratory germination is 14.7%. Pollen grain sizes: polar axis is 69.5 ± 0.1 µm,

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899 equatorial axis is 42.2 ± 0.1 µm. Pollen fertility is 63.6 ± 2.5%. Sexine is club-shaped according to Erdtman (1956) or rugulate-baculate according to Chung and Jones (1989) (Fig. 4e).

a) b) c)

d) e)

Fig.4 Micrograph of pollen grains: а) H. sieboldiana (2400x magnification), b) H. sieboldii (1000x magnification), c) H. rectifolia (2400x magnification), d) H. plantaginea (2400x magnification), e) H. ventricosa (2400x magnification).

DISCUSSION In the Foothill Crimea, the hosta blooms throughout the summer until the beginning of autumn. H. sieboldii in the Bagrov Botanical Garden (BG CFU) blooms in the III decade of June, and much later in Forest-Steppe Ukraine (NBG) and Western Siberia (CSBG), namely, in the I and II decade of July (Catalog of Ornamental Plants, 2009; Sidelnikova, 2012). The close timing of the onset of the blooming phase (II decade of June) in the Foothill Crimea (BG CFU), Forest-Steppe Ukraine (Sofiyivka) and central Russia (BS MSU) is observed in H. sieboldiana (Boyko, 2010; Golikov, 2008). According to the present research, H. plantaginea blooms from the I decade of August to the I decade of September. About the same time is indicated by other authors for central Russia (MBG RAS), Forest-Steppe Ukraine (NBG, Sofiyivka), Bukovina (BG CNU), Poland (Warsaw), and even Siberia (CSBG) (Vavilova, 1997; Catalog of ornamental plants, 2009; Marchinkovski, 2008; Smolinskaya, Chervinskaya, 2009; Sidelnikova, 2012). Significant differences are noted in the onset of blossoming of H. rectifolia in the Crimea (August) and the middle zone of Russia, namely, in MBG RAS (June). Previously, we developed a mathematical model that allows us to establish a physiological zero and lower temperature limits necessary for the hostas to move on to the next phenological phase (Kazakova, Repetskaya, 2017). H. sieboldii, H. sieboldiana and H. ventricosa belong to the group of middle-summer species. For the onset of the budding phase, the lower temperature limit should be + 19.8 °C, and the sum of effective temperatures (that must be reached above this level) is 1.6 °C. In

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899 late-summer H. rectifolia and H. plantaginea, the generative stage of annual development occurs a month later after the sum of effective temperatures of 100-110 °C is reached. Thus, it is possible to predict the seasonal dynamics of the development of hostas species and the varieties obtained on their base in different climatic conditions, which allows us to decide on the feasibility of cultivation and introduction into mass planting. Introduced the Foothill Crimea, the longest blooming phase (56 days) was observed in H. rectifolia, the shortest (16 days) in H. ventricosa. Each flower opens only for one day; long-term decorative effect is achieved due to the large number of flowers in the inflorescence. All the studied species were day-blooming except the evening-blooming H. plantaginea which has a pleasant smell reminiscent of the fragrance of acacia, jasmine or lilac. Flowers on short pedicel, slanting or drooping. The perianth is usually funnel-shaped, but may be bell-shaped or arachnid-shaped, six-cut, with a long tube. Six stamens are loose or fused at the bottom of the perianth (Abramova, 1977; Schmid,1991). In four of the five species, the perianth is 5-6 cm long to 3-4 cm wide. The exception is H. plantaginea, the perianth reaching 13 cm long to 5 cm wide. In China, perianth is 10 cm or more for H. plantaginea and 4-6.5 cm for H. ventricosa (Flora of China http: //www.efloras.org/). Schmidt (Schmid, 1991) indicates the capsule size for H. plantaginea as 12.5 cm to 7.5 cm; in other species, flower sizes are 5–5.5 cm and 3–3.5 cm, respectively. Similar flower sizes in plants cultivated in the semiarid climatic conditions of the Crimean peninsula and growing at the natural habitats in a humid monsoon climate indicate the stability of this characteristic, its species specificity and applicability as one of the signs in the descriptors. The flowers color spectrum is wide enough – from white to lilac and violet. The corolla of H. plantaginea is white with almost no visible veins. H. ventricosa has a violet-striped corolla with brightly colored dark purple veins (Fig. 1a). In the rest of the species, the corolla is lilac with barely visible violet streaks (Fig. 1b). The fruit is an elongated leathery three-compartment capsule that opens along the partitions. Quantitative parameters of plant generative structures are more stable than of vegetative ones. The sizes of flowers and fruits are key morphological characteristics in the differentiation of many closely related species, including some hostas species. The ‘6 cm or less than 6 cm’ capsule is one of the criteria for separating H. plantaginea, on the one hand, and H. ensata, H. ventricosa and H. albofarinosa, on the other (Flora of China www.efloras.org). We have found that under cultivation conditions in the Foothill Crimea, the capsule is 2.9 ± 0.1 cm in H. ventricosa, and 6.6 ± 0.2 cm in H. plantaginea. Thus, the fruit size can serve as an objective criterion for separating some species, in particular H. plantaginea and H. ventricosa. To assess the degree of adaptation and viability of introduced species in the new conditions, the signs that allow more or less accurately determination of how the species manifests itself are employed (Lapin, 1974). The most significant of these are the intensity and quality of seed-bearing plants, which determine the possibility of selecting generations resistant to new conditions. All the studied species, except H. rectifolia, annually form viable seeds in the Foothill Crimea. The lack of seed reproduction in H. rectifolia is to some extent offset by a high vegetative reproduction factor (up to 100 plots/plant). Seeds of the other species are numerous, oblong, flat, thin, single-winged (with a lateral wing) (Fig.2). Seed scar is small, ellipsoidal, punctate, basal. Seeds are shiny, brown, grayish-brown or black. The seed surface is bare and lumpy. Sizes vary from 8 to 14 mm long to 3 to 5.5 mm wide (Table 1). Table 1. Morphological characteristics of seeds of the species of the genus Hosta

Seed colour Seed length with Seed width, Seed thickness, Species wing, mm mm mm Hosta 8.41±0.12 3.24±0.08 0.51±0.06 grayish-brown sieboldiana Hosta sieboldii 8.08±0.11 3.22±0.08 0.57±0.03 black

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Hosta black plantaginea 11.87±0.20 3.49±0.07 0.76±0.07 Hosta ventricosa 13.77±0.76 5.37±0.11 0.86±0.05 brown ] – values differ by 95% at the level of significance

A variety of seed morphology among angiosperms with a relatively constant structure in small taxonomic groups allows using features of seeds in systematics (Ezau, 1980). The seeds size is one of the most stable morphometric parameters of plants. However, in the systematics of the genus Hosta, we did not find the use of such criterion. The seed length is significantly different in all the studied species (P <0.05) (Table 1). In this case, the groups H. sieboldiana and H. sieboldii (8 mm) on the one hand and H. plantaginea and H. ventricosa (13 mm) on the other stand out clearly. In seed thickness, there are also significant differences between groups. The seed width of H. ventricosa (5.37 ± 0.11 mm) significantly exceeds (P <0.05) the value of this parameter in other species. Thus, seed size can be used as a criteria for distinguishing species. The second (quality) criterion is seed colour. Black-colored seeds are formed in H. sieboldii and H. plantaginea; brown and grayish-brown in H. sieboldiana and H. ventricosa (Table 1). It is possible to create a dichotomous key for separating species: 1. Seed length with wing – 8-9 mm - 2. – Seed length with wing – 11-13 mm - 3. 2. Black seeds – Hosta sieboldii. – Grayish-brown seeds – Hosta sieboldiana. 3. Brown seed 3 mm wide – Hosta plantaginea. – Black seed 5 mm wide – Hosta ventricosa.

One of the results of the successful adaptation of the species is the self-reproduction in the new conditions. From the applied point of view, the species most adapted to local conditions are of interest for inclusion in the breeding process as parental forms. To analyze the implementation of the reproductive potential of an introduced species, the assessment of seed production and the percentage of seed setting is indicative. It was established that in H. sieboldiana, H. sieboldii and H. ventricosa the average number of ovules in the capsule is about 30, and in H. plantaginea twice as large (Table 2). In three species out of four, the percentage of seed setting does not exceed 50%.

Table 2. Characteristics of the reproductive sphere of species of the genus Hosta Tratt. Species Hosta Hosta Hosta Hosta Characteristics sieboldiana sieboldii plantaginea ventricosa Number of ovules in the capsule, 35.4±0.5 30.7±0.5 70.8±1.2* 26.9±0.7 pcs Number of seeds in the capsule, 17.2±0.4 26.1±0.8 22.7±1.3 12.7±0.6 pcs Seed setting, % 48.6 84.9 32.1 47.2 Number of peduncles, pcs 9.9±1.2 11.2±3.8 6.6±0.6 10.3±1.4 Number of flowers in the 30.4±0.6 38.9±1.8 14.2±0.3 27.4±0.9 inflorescence, pcs Seed productivity PSP 594.7 589.1 191.2 350.2 ASP 288.9 500.5 61.3 165,4 Laboratory germination,% 14.7 22.7 42.5 77 * The bold type shows maximum values of indicators

The level of seed setting gives a partial view of the reproductive strategy of the species. To obtain a more complete picture, we calculated the potential seed productivity (PSP), actual seed productivity (ASP) and laboratory germination of seeds.

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During germination in laboratory conditions, the swelling stage is marked on Day 3, and on Day 7, the germinal root appears. On Day 15, the maximum number of seedlings was recorded. On Day 20, the first pair of leaves was observed (Fig.5); the first true leaf has a clear distinction between an almost colorless petiole and a green leaf plate.

Fig. 5 Hosta plantaginea seedlings on Day 20

A comprehensive analysis of the characteristics of seed reproduction of hostas under cultivation conditions showed that they exhibit a variety of strategies for achieving reproductive success. H. sieboldii produces the largest number of seeds per plant (ASP is 500.5 pcs.) due to the abundance of flowers and peduncles and the highest percentage of seed setting (Table 2). Almost 85% of ovules in capsules of this species develop into seeds. However, laboratory germination of seeds is not high – slightly more than 20%, which reduces the advantage of large seed productivity. H. ventricosa is characterized by the lowest number of seeds in the capsule (12.7 ± 0.6 pcs), with results in relatively low ASP - 165 pcs/plant (3 times less than in the previous species). Yet due to the high germination (77%), H. ventricosa is able to compensate for low seed productivity. Boyko (2010) indicates seed germination of this species at the level of 85%. H. sieboldiana and H. plantaginea show the same tendencies – the first species produces many seeds with low germination (14.7%), the second produces less seeds with higher germination (42.5%). Thus, in semi-arid warm climate of the Foothill Crimea, the five hosta species studied demonstrate three different reproductive strategies. H. sieboldii and H. sieboldiana are species with high reproductive effort, manifested in the mass production of seeds. H. ventricosa and H. plantaginea produce fewer seeds which have a higher germination rate. Vegetative renewal is characteristic of H. rectifolia in the absence of seed reproduction. Using classical ecological terminology (Begone, Harper, Townsend, 1989), one can speak about the elements of r- and k-strategies in the reproductive biology of the first and second pairs of species, respectively. Annual fruiting and a sufficient number of germinating seeds give reason to assess the degree of adaptation of H. sieboldii, H. sieboldiana, H. ventricosa and H. plantaginea to the conditions of the Foothill zone of Crimea as high and recommend them for inclusion in breeding schemes as maternal forms for obtaining local resistant ornamental sorts. Palynological studies were performed to select prospective paternal forms. Pollen grains of all the studied species are isopolar, symmetrical, with well-defined poles (Fig.4). Between the poles lies a distal groove which belongs to the distal-polar simple seedling pores. The ratio of the length of the polar axis to the length of the equatorial diameter allows an objective conclusion about the shape of the pollen grain. According to the Meyer-Melikyan and Severova (1999) classification, the form is elongated-elliptic (1.6:2.1). In accordance with the size classes proposed by Erdtman (1956) based on the length of the major axis, the pollen grains of the studied species are large – from 60 ± 0.06 μm (H. rectifolia) to 97.2 ± 0.09 μm (H. ventricosa) and very large – 113.5 ± 0.04 μm (H. plantaginea). Chung and Jones (1989) identified five types of sexine patterns in members of the genus Hosta. There are three types among the objects of this study: reticulate in H. plantaginea; rugulate-baculate

International Journal of Advanced Science and Technology Vol. 29, No. 7, (2020), pp. 1888-1899 in H. sieboldiana, H. sieboldii and H. rectifolia, and rugulate in H. ventricosa. Evolution of the pollen, presumably, could begin with reticular sexine pattern, as in H. plantaginea, and then consistently turn into more complex patterns (Chung, Jones, 1989). Pollen fertility was determined by acetocarmine staining according to the percentage of morphologically homogeneous pollen grains coloured carmine red and sterile (Fig.6).

Fig. 6 Fertile (left) and sterile pollen grain of H. sieboldiana (100x magnification)

It is established that the pollen fertility of all species is rather high and exceeds 60%. Hosta sieboldii, Hosta ventricosa and Hosta plantaginea reach the highest values of morphologically normal pollen grains – 82%, 81.5% and 79.7%, which suggests a higher fertilizing ability of pollen in these species. The least viable pollen was found in H. rectifolia; this species does not set seeds. Perhaps the absence of seed reproduction is associated with defects in the development of male gametophyte. Thus, the research revealed: 1. Seed characteristics of four species — Hosta sieboldii, Hosta sieboldiana, Hosta plantaginea and Hosta ventricosa are species-specific. Colour and seed size can be used as a systematic criterion. 2. In the semi-arid warm climate of the Foothill Crimea, the five species studied demonstrate three different reproductive strategies. H. sieboldii and H. sieboldiana have high seed productivity with low germination; H. ventricosa and H. plantaginea produce few seeds with high germination. In H. rectifolia, an intensive vegetative renewal is observed in the absence of seed reproduction. 3. Hosta sieboldii, Hosta sieboldiana, Hosta plantaginea, Hosta ventricosa annually set germinating seeds, have fertile pollen and can be recommended for use in breeding as parental forms.

The work is published along the orders of the Ministry of Education and Science of the Russian Federation with state budget financing on the topic No. FZEG-2017-0016 ‘Development of a system for the rational use of decorative phytobiological resources in the Crimea’.

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