Pericarp anatomy and surface micromorphology of some orchids in the Black Sea Region

In this study, we have investigated the anatomy and ultrastructure of the pericarp to determine important characters of the fruits belonging to some Turkish orchidoid species, and to determine which features are related to ecological or habitat preferences. For the purpose, the samples belonging to 19 orchid taxon were collected in the Black Sea Region. SEM and light microscopy photographs were taken with the standard techniques. Variations among taxa were evaluated using various statistical methods such as correlation and discrimination analysis. Among the investigated characteristics, fruit surface ornamentation is related to habitat preferences of the species while morphometric properties of epidermal cells and structural features such as the type of crystal inclusions are important characters at the genus level.


Introduction
Orchidaceae is one of the largest families in terms of biodiversity and is flowering plants with wide area of distribution excluding extreme habitats (Dressler, 1993). With more than 800 genera and nearly 27.000 species, new members are added to the orchid family every year (Chase et al., 2015;The Plant List, 2018). In Turkey, it is possible to encounter over 200 taxa from sea level to over 2000 m elevation (Davis and Davis, 1982;Güner et al., 2012). Additionally, the economic value of these plants is increased through widespread use in food, cosmetic and pharmaceutical industries (Sezik, 1984).
Structural studies of orchids have focused on flower parts generally due to the morphological variety and unrivaled pollination strategies of flowers (Stpiczyńska, 2003;Cozzolino and Widmer, 2005;Schiestl, 2005;Bell et al., 2009;Vereecken, 2009;Anton et al., 2012;Nunes et al., 2015). Furthermore, studies about the internal and surface structure of seeds rather than fruit, symbiotic-asymbiotic germination trials or orchid-fungus relationships are seen to be more notable (Gamarra et al., 2007;Chemisquy et al., 2009;Chen et al., 2012;Gamarra et al., 2012;Tesitelová et al., 2012;Bektaş et al., 2013). Though seeds are very small, they show high rates of variety in testa structure. The physical properties of seeds and the functional outcomes of these properties were revealed in detail by Arditti and Ghani (2000). Also, some studies have revealed the developmental features of seed coats (Molvray and Chase, 1999;Lee et al., 2005Lee et al., , 2007. Many micromorphological studies have stated that seed shape, surface patterning, anticlinal and periclinal wall structures or shape of testa cells are characteristic at different systematic categories and are reliable character to contribute to solving systematic problems in the family (Gamarra et al., 2012;Barthlott et al., 2014;Akbulut and Şenel, 2016;Şeker and Şenel, 2017;Gamarra et al., 2018).
The most accepted opinion related to developmental features of orchid fruit is that they comprise three carpels and the midvein of the carpel is located immediately below the stigma lobes. Orchids have marginal placentation (Brown, 1831;Payer, 1857;Van Tieghem, 1871). Additionally, according to the split-carpel model, the fruit is thought to occur by joining of other flower parts like sepal and petals. In this model, the sepals divide the carpels into two equal pieces and each petal joins to two separate carpel sections to form the fruit (Rasmussen and Johansen, 2006). In spite of detailed investigation of the internal structure of the carpel in these studies, the pericarpic surface morphology or morphometric features have been ignored compared to the seed.
Due to excessive number of species, high rate of variation in flowers or hybridization capacity, revealing characters rapidly and reliably for solutions to systematic problems carries great importance for orchids (Arditti, 1977;Dressler, 1993). Many problems have not been solved due to identification based on flower morphology. With the more common use of molecular markers in recent years, systematic categories of many species have changed (Bateman et al., 2003).
However, the main disadvantages of these methods are high cost and requirement for additional approaches to differentiate closely-related taxa. In this study, the aim was to determine distinguishing features for the pericarp of closely-related orchid species, identification of whether these features are useful for classification and research of the relationships with habitat and ecological preferences of taxa.

Materials and Method
Samples were collected from different localities in the Black Sea Region from 2012-2016 (Table 1). Species identification was completed using Flora of Turkey and Türkiye Bitkileri Listesi (Davis and Davis, 1982;Güner et al., 2012). Fruits were fixated in formalin-acetic acidalcohol (FAA) and then stored in 70% ethyl alcohol. To determine the anatomical and morphometrical characters, cross-section and surface sections were taken from fruit samples from at least three different individuals in each taxon. Anatomical features were investigated using a light microscope (Zeiss AxioLab A1 microscope and the Zeiss Axiocam 105 imaging system) and morphometric features were assessed with at least 30 measurements for each character. Furthermore, pericarp surface micromorphology was identified with scanning electron microscope studies. Samples were passed though alcohol series (30%, 50%, 70% and 95%) to remove excess water from tissues and dried with a critical point dryer. After coating with 15 mm gold-palladium (SEM coating system, SC7620), surface investigations were

Results
The fruit structure of 19 studied orchid taxa were investigated in terms of a variety of features like epidermal cell size, cell shape, surface ornamentation, presence of hairs or papillary structures, and inclusion of crystal or starch grains. On the valves of S. spiralis species, secretory hairs of a variety of sizes were identified. However, hairs were not encountered in the other taxa.
Additionally, the epidermal cell margins were unclear in S. spiralis and there was no surface ornamentation. In other taxa, ornamentation was longitudinal, transversal or in both directions, with striate or undulate. However, SEM photographs of the fruit surface of N. tridentata show reticulate pattern and accumulations similar to secretions on the surface. Similar accumulations were present in S. satyrioides (Figure 1, Table 3).
According to correlation analysis results applied with the aim of identifying the correlation between fruit structural character and ecological needs or habitat preferences of taxa, there was a positive correlation between fruit surface ornamentation and habitat preferences (Table 2). .696 .528 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed). Epidermal cells are isodiametric or elongated, and are pentagonal or hexagonal. In the cross-sections of A. laxiflora, elongated papillary structures is notable between the epidermal cells. Also, starch grains or crystals are clear with density on the mesocarp varying from species to species. While fruits are commonly observed to have raphide bundles, prismatic crystals found in species like S. orientalis and C. viride. Vascular bundles are collateral. In all taxa, the placenta is characterized by a single row cells with large nuclei and dense cytoplasm ( Figure 2, Table 2).
Circular graphs were used with the aim of comparing the mean epidermal cell sizes between taxa (Figure 3). Accordingly, among the three features, the largest epidermal cells were found in O. mascula.
However, the cell length for D. euxina, cell width for C.
viride and cell height for G. conopsea had smallest values measured.
Canonical discrimination analysis was used with the aim of determining the degree of accuracy of groupings according to fruit structural features and which feature was more effective in this grouping. Figure 4 shows the distribution of taxa based on the first two components. Accordingly, at genus level, taxa were accurately grouped with 94.4% success rate.
The most effective characters in canonical discrimination analysis were the morphometric features of epidermal cells like length, width and height. Additionally, the presence and variety of crystal in the tissue or habitat preferences of taxa came to the fore during taxon grouping (Table 4).

Discussions
In the literature, hair with a variety of sizes and structures were reported on the vegetative and generative organs of orchids. Mainly researched for floral structures, these secretory hairs are emphasized to have functions in attracting pollinators acting as osmophore or nectarium due to secretions like mucilage, lipophilic compounds, pectic acid or phenolic compounds (Stpiczyńska and Davies, 2009;Nunes et al., 2014Nunes et al., , 2015. In the literature, endocarpic hairs are mentioned in epiphytic species and it was emphasized that this type of trichome was only found on epiphytes (Beer, 1857;Horowitz, 1901Horowitz, -1902. As seen in our anatomical investigations, the lack of these types of structure in terrestrial species supports this view. Correlation analysis results mean that in habitats with higher humidity rates, the cuticle layer or secondary wall thickness reduces on the fruit surface and indicates that the surface ornamentation may be related to ecological adaptation rather than being a differentiating character for species. As emphasized in previous research, though orchid fruit display homogeneous structure in terms of basic structural features, there is variety in terms of features like the shape, size and content of pericarp cells (Mayer et al., 2011). Though studies about orchid fruit have noted different features for identification of taxa, micromorphological and morphometric features have been ignored. For example, Prillieux (1857) identified 7 different fruit according to dehiscence type and stated that this variety may be useful to characterize species according to some criteria like apically fused vs. free, recurving valves, and valve number. Horowitz (1901Horowitz ( -1902 in a study of many orchids classified taxa according to number and distribution of vascular bundles. In morphometrical investigation, the cell sizes for D. euxina, C. viride and G. conopsea had smallest values. A study using molecular markers like ITS proved the close phylogenetic relationships between these three taxa (Shipunov et al., 2004). Moreover, Bateman et al. (1997) included the C. viride (D. viridis) species in the Dactylorhiza species and this new classification has been adopted by many researchers. In our study, this common morphometric feature in the pericarp epidermal cells of the three taxa support this view.
In this study, though structural character has low differentiation power for Ophrys taxa, discrimination analysis was found that fruit morphometric features have high rates of success for differentiation of other taxa. According to this finding, the pericarp features of orchidoid species may be effective for classification of genus and upper categories.