Morphological features of the leaves in clematises as a reflection of their ecological peculiarities

Quantitative morpho-anatomical features of leaves of nine Clematis taxa (C. alpina ‘Pamela Jackman’, C. macropetala ‘Maidwell Hall’, C. integrifolia ‘Aljonushka’, C. ispahanica ‘Zvezdograd’, C. fargesii ‘Paul Farges’, C. texensis ‘Princess Diana’, C. tibetana, C. viticella, and C. heracleifolia) were determined with the aim to analyze their adaptation to the environmental conditions. Among investigated clematises, there were plants with hypostomatic (C. viticella, C. fargesii ‘Paul Farges’, C. heracleifolia, C. texensis ‘Princess Diana’, C. macropetala ‘Maidwell Hall’, and C. alpina ‘Pamela Jackman’), and amphistomatic leaves (C. ispahanica ‘Zvezdograd’ and C. tibetana). In C. integrifolia ‘Aljonushka’ leaves were hypostomatic, but few solitary stomata were also present on the adaxial surface. In the leaves of investigated taxa, the palisade coefficient ranged from 27.3% (C. alpina ‘Pamela Jackman’) to 49.9% (C. tibetana). The leaves also differed significantly in size. In particular, leaves of C. integrifolia ‘Aljonushka’ were almost ten times smaller than such of C. heracleifolia. As a result of UPGMA clustering, the plants that can survive in severe windy weather in open rocky areas, Clematis tibetana and C. ispahanica ‘Zvezdograd’, were joined in a separate cluster. The second cluster combined C. alpina ‘Pamela Jackman’ and C. macropetala ‘Maidwell Hall’ – cultivars blooming in the spring, during a period of significant difference in daily temperatures. A relatively small leaf area in plants from these two clusters may indicate an adaptation by reducing the transpiration area and general windage. The third cluster united the rest of investigated taxa, mostly – the mesophytic plants with a relatively large leaf area. However, due to similar morpho-anatomical structure of the leaf, the third cluster also comprised C. integrifolia ‘Aljonushka’ with the smallest leaves.


Introduction
The genus Clematis L. (Ranunculaceae Juss.) unites valuable ornamental and medicinal plants of various life forms, which grow on all continents except Antarctica (Sneddon, 1975;Takhtajan, 1978;Tamura, 1993;Moon et al., 2013;Zhang et al., 2013). Due to the long history of introduction and selection, many clematises are available for planting in a moderate climatic zone and are successfully cultivated in Ukrainian botanical gardens (Rijekstynja & Rijekstynysh, 1990;Tamura, 1993;Vachnovskaya, 2007). Taking into account the wide range of decorative application, clematises are good plants for urban gardening.
Clematises differ in the rhythms of their development and can be deciduous or evergreen. They are mainly entomophilous, monoecious or dioecious plants with opposite pinnate leaves. Morpho-anatomical diversity of their leaves is often determined by the ecological conditions of their habitats (they often occupy forest edges, rocky slopes, rocky river banks, and also steppes and desert landscapes) in different climatic zones (Rijekstynja & Rijekstynysh, 1990;Tamura, 1993;Buisson & Lee, 1993;Bååth & Anderson, 2003;Ahmad et al., 2016;Arimy et al., 2017).
The mechanisms of such adaptation may differ (e.g., by changing the area and thickness of the leaf blade, mesophyll or epidermal cell sizes, number of stomata, or developing the pubescence), but some genetically determined traits remain intact (e.g., the type of leaf blade, vascularization, stomatal apparatus, and trichomes). Therefore, the anatomical structure of the leaves often serves as a valuable source of taxonomic features (Isnard et al., 2003;Jinghua & Liangqian, 2003;Javed et al., 2012). Taking into account these two aspects, we analyzed morpho-anatomical features of the leaves of nine Clematis taxa to evaluate their adaptive potential to urban conditions.

Material and methods
Morphological peculiarities were analyzed for nine Clematis taxa (Table 1), which were chosen due to their high ornamental features combined with the possibility of growing in public urban plantations. Selected plants vary phenologically and morphologically, so they can be applied for the creation of specific landscape compositions. They also differ by origin, which causes some ecological preferences and peculiarities.
For the morphological analysis, for each taxon five mature leaves of the brunch middle part were selected from the western side of plant habitus. Middle parts of leaflets from selected leaves were used for cuts and preparation of the permanent slides. Imprints of abaxial and adaxial sides of mature leaves were applied to determine the structure of the epidermis (Evert, 2005). The anatomical structure of the leaves was studied using permanent (10 μm thick) and native slides. Differentiated staining of the leaf tissues was performed using safranin (stains lignified elements) and water blue (stains unlignified elements) dyes (Furst, 1979).
The palisade coefficient indicates the percentage of palisade mesophyll layer from total mesophyll layer in the leaf blade and was calculated by the formula: K = 100 * M P / M P+S , where K -the palisade coefficient, in %, M P -the height of the palisade mesophyll cells layer on the cross-section of the leaf, in µm M P+S -the total height of the mesophyll, in µm.
Cluster analysis was performed using UPGMA method. UPGMA is effective when objects actually form different "groves"; however, it works equally well in cases of extended (chain type) clusters (Systat Software, 2010;Addinsoft, 2017). Simultaneously, Principle Component Analysis (PCA) was applied as one of the most frequently used multivariate data analysis methods suitable for our purposes (Addinsoft, 2017).
The anatomical structure of the leaves was investigated using a microscope Nikon Eclipse E-200 (Japan). Measurements of linear dimensions and areas were performed using Image-Pro Premier 9.1 software. Statistica 6.0 and SigmaPlot softwares were used for the statistical processing of obtained data (Box et al., 2005;Systat Software, 2010;Everitt et al., 2011).

Results and discussion
Ecological conditions of the habitat influence affect morphology and anatomy of plants in different ways. In case of studied clematises, altitude and insolation play principal role. The common feature of high-altitudinal plants is relatively thick leaf blades (Kogami et al., 2001;Zarinkamar et al., 2011;Ahmad et al., 2016;Arimy et al., 2017). It was also found that such plants, if compare with the plants from lower altitudes, have lower stomata density (Zarinkamar et al., 2011;Arimy et al., 2017), intensive sclerification around the vascular bundles (Ahmad et al., 2016), thick walls of epidermal cells, and, usually, amphistomatic leaves (Ornellas et al., 2019). From the other hand, in case of sufficient insolation, neutral and filtered shades cause the decrease of leaf thickness and area, leaf weight, stomatal density, palisade cell length, mesophyll thickness, and increase in chlorophyll content, degree of air spaces, adaxial width of palisade cell, and perimeter to area ratio (Buisson & Lee, 1993;Hanba et al., 2002).
In general, the leaves of the studied plants differed significantly in shape and area (Fig. 2). The leaf surface area of the studied plants ranged from 1838 mm 2 in C. integrifolia 'Aljonushka' to 18095 mm 2 in C. heracleifolia.
The leaf blades of the studied clematises demonstrated dorsoventral structure. There were plants with hypostomatic (C. viticella, C. fargesii 'Paul Farges' , C. heracleifolia, C. texensis 'Princess Diana' , C. macropetala 'Maidwell Hall' , and C. alpina 'Pamela Jackman') and amphistomatic leaves (C. ispahanica 'Zvezdograd' and C. tibetana). In C. integrifolia 'Aljonushka' , beside the numerous stomata on the abaxial surface of the leaves, there were also observed solitary stomata on their adaxial side. In all studied plants, the stomata of the anomocytic type were found. The maximal stomatal area (C. macropetala 'Maidwell Hall') almost twice exceeded the minimal discovered one (C. texensis 'Princess Diana'). Ordinary epidermal cells varied in shape and size and differed between the experimental plants and even within the leaf surfaces of the same taxon. The height of mesophyll and the ratio of palisade and spongy layers differed among studied plants too (Fig. 3).
Palisade mesophyll was represented by two layers of cells in C. ispahanica 'Zvezdograd' , C. integrifolia 'Aljonushka' , and C. tibetana, and by a single layer in other studied taxa. Loose spongy mesophyll with large intercellular spaces was found in C. texensis 'Princess Diana' , C. heracleifolia, C. fargesii 'Paul Farges' , and C. tibetana. In other studied plants, spongy mesophyll was denser with clear boundaries between the layers. In C. integrifolia 'Aljonushka' it consisted of three or four cell layers, while in C. viticella and C. alpina 'Pamela Jackman' -of three layers (Fig. 4).
Leaf morphological and anatomical features were analysed by PCA. We used nine clematis taxa as observations and 37 characters and indexes of the leaf blade as variables (Fig. 5). From the list of investigated characters, we selected 22 variables, which made the most substantial contribution to PCA dispersion. Selected features in complex allowed comparing light intensity preferences and drought-resistance of plants and their ability to accumulate and retain water ( Table 2).
The PCA showed that C. texensis 'Princess Diana' differs from other studied plants by the thickness of outer cell walls of upper epidermis, while C. macropetala 'Maidwell Hall' differs by the size of cells of spongy mesophyll and lower epidermis. Clematises with largest (C. heracleifolia and C. fargesii 'Paul Farges') and smallest leaves (C. integrifolia 'Aljonushka'), sure thing, strongly differed from other studied plants by the leaf area. Besides this, C. integrifolia 'Aljonushka' and C. fargesii 'Paul Farges' differed by a high      The third and the largest cluster included C. viticella, C. fargesii 'Paul Farges' , C. integrifolia 'Aljonushka' , C. heracleifolia, and C. texensis 'Princess Diana' with hypostomatic leaves thickness of which varied from 109.91 to 184.90 μm (Fig. 6).
Despite the difference in origin and habitus, among studied plants, C. heracleifolia and C. texensis 'Princess Diana' were found to be the most similar. The leaves of these plants have the largest area and comparatively thin epidermis, which indicates their mesophytic origin. Another subcluster was formed by the cultivars C. fargesii 'Paul Farges' and C. integrifolia 'Aljonushka' . Such species common for Ukraine as C. vitalba and C. integrifolia were used to create these cultivars. Hence, these cultivars can occupy the niche of parental species in the plantations of deciduous woody plants with low canopy density. In urban landscapes, they can be used for growing in containers, to decorate walls and fences.
The natural range of distribution of C. viticella is located in southern Europe, where it grows in the forest ecotons.  I. Kovalyshyn, A. Pinchuk, A. Likhanov This explains the shade tolerance peculiarities in the structure of its leaves. Therefore, this species, as a bright summer accent, fits the woody plantations with low canopy density. Cultivars C. alpina 'Pamela Jackman' and C. macropetala 'Maidwell Hall' are traditionally attributed to the Atragene group, which combines clematises from the Northern Hemisphere forests. These plants grow in partial shade, and bloom in spring during rapid fluctuations of the daily temperature under a sparse canopy of the trees in windy forest biocenoses. Small leaves and their dissection can be a result of adaptation of these plants to mentioned growing conditions. Hence, the best places for their growth in the city are on the edges and along the paths in the parks and squares composed by deciduous trees. In such conditions they will get enough light during the flowering period and enough shade during the hot summer time.

Morphological features of the leaves in clematises and their ecological peculiarities
Stony steppe slopes with high insolation are the natural habitats of C. tibetana and C. ispahanica. Clematis tibetana and C. ispahanica Boiss. 'Zvezdograd' have the following leaf structure features: amphistomatic leaves, high palisade coefficient, and relatively significant total leaf blade thickness. Such features are crucial for the plants growing on mountain slopes with high insolation and fluctuations in daily temperatures. The small area of leaves allows these plants to avoid damage by ground particles during the winds. While the relatively thick cuticle reduces transpiration and refracts sunlight. These traits are genetically determined and persist even in plants growing in urban areas. Therefore, C. tibetana and C. ispahanica 'Zvezdograd' are appropriate for growing on windy, rocky mounds.

Conclusions
Adaptive features of the studied clematises are different. Complex analysis of their leaf morpho-anatomical structure allowed assessing the ecological preferences of these plants. In particular, among the main characters of heliophilous clematises were determined amphistomatic leaves, a high palisade coefficient, and a considerably thick cuticle. The small diameter of vascular bundles and the thin cuticle layer are signs of shade-tolerant clematises. While the mesophytic clematises combine a thin cuticle layer and large size of stomata, mesophilic and epidermal cells. Small-celled leaf tissues, in general, indicate drought-resistant clematises.