Seed micromorphology of Orchis Tourn. ex L. (Orchidaceae) and allied genera growing in Edirne province, Turkey

Abstract In this study, the seed micromorphologies of eight taxa of Anacamptis, Neotinea and Orchis growing around Edirne province (Turkey) were investigated using light microscopy and scanning electron microscopy (SEM). Slides prepared with glycerin jelly were used for measurements under the light microscope and fine details of seed testae characteristics were observed with SEM. Seeds of the investigated orchid taxa are fusiform shaped and of different shades of brown. Their lengths and widths are different among the taxa and range between 0.263–0.640 mm and 0.118–0.208 mm, respectively. Testa surfaces of Orchis mascula subsp. mascula, Orchis purpurea subsp. purpurea and Orchis simia subsp. simia, are smooth while those of Anacamptis coriophora, Anacamptis laxiflora subsp. laxiflora, Anacamptis morio subsp. morio, Anacamptis papilionacea and Neotinea tridentata subsp. tridentata are reticulate. An identification key based on seed morphologies and sizes is suggested for the first time, including testae structures of orchids growing in Edirne province. The overall results of the study showed that morphological structures of orchid’s seeds could be used as diagnostic characters in identification.


Introduction
Orchidaceae are one of the most diversifi ed and evolved families in the fl owering plants (Cribb and Govaerts 2005). According to a recent survey (Govaerts et al. 2016) the number of the accepted species currently amounts to 24.000 but might reach 30.000, in view of the ever accelerating rate of new species descriptions every year (Tyteca and Klein 2008). Th e systematics have undergone many changes along the last few decades (Gamarra et al. 2010). Th e latter taxonomic proposals were published by Dressler (1993) and Szlachetko (1995). In the subfamily Orchidoideae, Dressler (1993) divided the tribe Orchideae into two subtribes: Orchidinae with 34 genera and 370 species, and Habenariinae with 23 genera and 930 species (Gamarra et al. 2010). Th e genus Orchis Tourn. ex L. and allied genera Anacamptis Rich. and Neotinea Rchb.f. are some of the most controversial groups belonging to the tribe Orchideae (Orchidaceae). Th e original genus Orchis s.l. used to include more than 1,300 taxa and in its broad concept, had a complex taxonomic history (Vermeulen 1972, Klein 1989, 2004, 2003, Buttler 2001, Szlachetko 2002, Baumann and Lorenz 2006, Kretzschmar et al. 2007, Tyteca and Klein 2008, Delforge 2009). Since Orchis has been proven to be polyphyletic, several species were separated into distinct genera . Also, in many guides and fl oras (see Tutin et al. 1980, Sezik 1984, Renz and Taubenheim 1984, Buttler 1986, Kreutz 1998, Delforge 2006, Buttler 2007) the number of Orchis taxa varies considerably, including species that previously belong to other genera, such as Aceras R.Br., Anacamptis Rich, Dactylorhiza Neck. ex Nevski, Neotinea Rchb.f. and Vermeulenia Á.Löve & D.Löve (Gamarra et al. 2012). Recently, molecular analyses have changed the taxonomy of several species in the genus Orchis , 2003. Th e genera Anacamptis and Neotinea were traditionally considered each as a monotypic genus, represented by A. pyramidalis (L.) Rich. and N. maculata (Desf.) Stearn respectively. Afterwards, the molecular analyses published by Pridgeon et al. (1997) and Bateman et al. (1997) confi rmed the polyphyletic status of Orchis s.l., and many species were placed into the expanded genera Anacamptis and Neotinea (Gamarra et al. 2012 Bateman et al. (1997) and Pridgeon et al. (1997), some Orchis species were nested in Anacamptis and Neotinea. However, based on either morphological or molecular data, the (old) genus Orchis has been split into three genera: Herorchis D. Tyteca Tyteca and Klein 2008). According to these authors, Neotinea and Anacamptis returned to their former monotypic position with the species N. maculata and A. pyramidalis respectively. Th e genera Herorchis and Odontorchis included the rest of the species of Anacamptis and Neotinea cited respectively, by Kretzschmar et al. (2007), and the genus Androrchis contained all the species of the genus Orchis, except the group with an anthropomorphic labellum, which is retained in Orchis (including Aceras). Later, Tyteca and Klein (2008) adopted the enlarged genera Anacamptis and Neotinea sensu Bateman et al. (1997Bateman et al. ( , 2003, but reaffi rmed the segregated genus Androrchis (Gamarra et al. 2012). Delforge (2009) published a new classifi cation of Orchis s.l. and accepts the taxonomical position of Orchis and Neotinea sensu Bateman et al. (1997Bateman et al. ( , 2003; however, he did not support the expanded genus Anacamptis, considering this genus as monotypic (A. pyramidalis), and segregating the rest of the species into the genera Herorchis, Vermeulenia, Anteriorchis E.Klein & Strack and the new genus Paludorchis P.Delforge (Gamarra et al. 2012). In this study, we have chosen the species delimitation of Bateman et al (1997), because it requires the fewest change in nomenclature.
According to Kretzschmar et al. (2007), the genus Anacamptis has three part lip, but undivided middle lob, at base, in front of the spur entrance are two raised disks or longitudinal ridges; bracts from at least half as long to (mainly) longer than the ovary. Th e genera Orchis and Neotinea have three part lip with +/-divided middle lob, without raised disks or ridges at the base; bracts either clearly shorter or at most as long as the ovary. Th e genus Orchis diff ers from Neotinea with uniform, round or trapezoid stigmatic cavity, longish column and without genuine winter rosette. Th e distribution area of the genus Anacamptis reaches to the Atlantic in the west and to the Hebrides and southern Scandinavia in the north. It includes the North African mountains in its southwest border, whereas other parts of North Africa and the Canaries remain blank, although it penetrates along the Levant considerably further to the south. Th e genus in the east reaches to Lake Balchaš in central Asia and its representatives are also found on all the larger islands of the Mediterranean. Th e ecological demands of the diff erent species are various, but all commonly prefer to settle within biotopes that have seasonal changes, really humid winters, which temporarily become very dry in summer (Delforge 2006, Kretzschmar et al. 2007, Govaerts et al. 2016. Th e genus Anacamptis have 11 accepted species and 20 subspecies (Kretzschmar et al. 2007, Govaerts et al. 2016.
Th e genus Orchis (Orchidaceae, Orchidinae) is limited in its distribution exclusively to the northern hemisphere. Its mainly distribution area is Mediterranean Basin where the maximum density of species is reached; however, other part of Europe are also settled to great extent. In addition the genus with some species, divert out of its main range and reaches northwards to Scandinavia, whilst in an easterly direction to Mongolia and reaches last Lake Baikal. On the north coast of Africa the eastern part is blank to great extent due to the absence of suitable biotopes; however, areas of Asia Minor and further on to Iraq and Iran are included. Th e ecological demands of the diff erent species are various (Delforge 2006, Kretzschmar et al. 2007, Govaerts et al. 2016. Th e genus Orchis have 21 accepted species and 16 subspecies (Kretzschmar et al. 2007, Govaerts et al. 2016.
Th e genus Neotinea is limited to Europe, Asia Minor, the Caucasus and the northwest coastal regions of North Africa. Th e ecological demands of the diff erent species are various (Delforge 2006, Kretzschmar et al. 2007, Govaerts et al. 2016. Th e genus Neotinea comprises four accepted species and two subspecies (Kretzschmar et al. 2007, Govaerts et al. 2016.
Seed morphology is one of the important taxonomic characters of orchids. Beer (1863) published the fi rst study about the seed morphology in Orchidaceae, while, the taxonomic importance of the seed characteristics was fi rst pointed out by Cliff ord and Smith (1969). Arditti et al. (1979) established the methodology for quantitative analyses, related to the sizes and volumes of seeds and embryos. Orchid seeds are characterized by minute and consist of an elliptical embryo enclosed within a generally transparent and often fusiform testa. Testae and embryos of diff erent genera and species may vary in size, shape, color or the ratios between their volumes. Th e walls of testa cells can be smooth or reticulate and when reticulation is present, its patterns may be distinctive (Arditti 1967, Arditti et al. 1979, Chase and Pippen 1988. Th e rather small sizes of seeds make them diffi cult to study their details and to compare some features with only light microscopy. Th erefore, making comparisons and determining details that could be used as taxonomical characters without SEM techniques appear to be a challenging task (Arditti et al. 1979). However, if some characters are investigated only by SEM, then this may lead to obtaining of some wrong data. Th erefore, relying on the use of both techniques, light microscopy and SEM, complementary to each other will be a better option for a researcher to get a clear picture of the studied question.
Most of the studies performed on orchid seeds were based on tropical orchids whereas the non-tropical species were generally neglected (Arditti 1967, Arditti et al. 1979, Chase and Pippen 1987, 1988, Rasmussen and Whigham 1993, Kurzweil 1993, Molvray and Kores 1995, Swamy et al. 2004, 2007, Gamarra et al. 2007, 2010, 2012, Chaudhary et al 2014, Galán Cela et al. 2014. Several authors published diff erent papers about seed morphology in the genera of Orchis, Anacamptis and Neotinea. Wildhaber (1972) initiated the morphological study of the seeds in the genera Orchis and Neotinea using light microscopy to obtain a key for the species based principally on the morphology and length of the seeds. Barthlott (1976) confi rmed the taxonomic value of the periclinal walls in the genera Orchis and Neotinea. Ziegler (1981) recognized the characteristic seeds of the genus Orchis as Orchis-type. Tohda (1983) analyzed the diff erences in the sculpturing of the testa seeds in some Orchis species using SEM images and recognized three groups, two with slanting stripes and one with smooth periclinal walls. Mrkvicka (1994) analyzed quantitative and qualitative data of European Orchis using light microscopy, revealing a high diversity in the seed coat micromorphology. Molvray and Kores (1995) provided data on the number of testa cells in Orchis spectabilis (L.) Raf. Arditti and Ghani (2000) reviewed the purely numerical and physical characteristics of orchid seeds and their biological implications; among of them Anacamptis collina  Gamarra et al. (2007) analyzed the morphology of the seed and of the anticlinal and periclinal walls using SEM in the genus Neotinea. Gamarra et al. (2012) analyzed seeds of 24 taxa belonging to the genera Anacamptis and Orchis.
Few studies exist on seed morphology of Turkish orchids. One of them was performed by Olgun and Aybeke (1996) on Edirne Ophrys L. species using SEM. Th ere are also light microscopy studies on Ophrys species (see Aybeke 1997) and Orchis species (see Güler 1997) in Edirne Province. Th e present study aimed to reveal the relationship between Orchis and allied genera Anacamptis and Neotinea species growing naturally in Edirne region and to contribute to species classifi cation based on seed measurement and morphological data.

Materials and methods
We analyzed seeds of eight taxa belonging to the genera Orchis, Anacamptis and Neotinea. Th e study material consisting of specimens of eight orchid taxa were collected from the region within Edirne provincial borders in 1995 and 1996 and are kept in EDTU Herbarium. A list of voucher specimens and localities is given in the Table 1. Fresh seeds were dried and stored in small paper envelopes. Th e identifi cation of the specimens was performed according to local fl ora and monographs (Tutin et al. 1980, Sezik 1984, Renz and Taubenheim 1984, Buttler 1986, Kreutz 1998, Delforge 2006. Th e seeds obtained from mature and opened fruits were used for seed morphology investigations. For this purpose, permanent slides of seeds were prepared with glycerin jelly solution on a heating plate (Ozban and Ozmutlu 1994) and the slides were investigated under a light microscope for morphological evaluations. Th e seeds were measured and then photographed. Th e color of the seeds were observed and described in annotated subjective terms with the help of optical microscope (Gamarra et al 2012, Chaudhary et al 2014, Galán Cela 2014. Th e specimens used for SEM were dried and examined for fi ne structure details. Th e terminology and methods were adopted from those of Arditti (1967), Arditti et al. (1979Arditti et al. ( , 1980, Healey et al. (1980), Pippen (1987, 1988), Kurzweil (1993), Molvray and Kores (1995) and Arditti and Ghani (2000). Measurements of seed embryos for morphometric data were taken using an Olympus BH2 light microscope equipped with a micrometric ocular. Statistical analyses were performed by NCSS 2013 (Version 9.0.5) for Windows. Seed and testa volumes were calculated using the formulations in Arditti et al. (1979). Since all seeds studied were fusiform, closely approximating two cones joined at their bases, their volumes were calculated using the formula: V t = 2[( W / 2 ) 2 (½ L)(1.047)] where w is the seed width, L is the seed length, and 1.047 is equal to p/ 3 . Th e volumes of the embryos elliptical in their cross section were calculated by using the formula: V e = 4 / 3 pab 2 where a is ½ of embryo length, b is ½ of embryo width, and 4 / 3 p is equal to 4.188. Percentage air space was calculated by using the formula: [(V t -V e )/V t ].100.

Results and discussion
All investigated orchid seeds were fusiform in shape and had transparent and elliptical embryos (Figures 1−4). Th eir testae colors were diff erent shades of brown. Th e measurements of the seeds as revealed by light microscopy investigations are given in Table 2.
When testae and embryos were investigated for their colors, the following patterns were obtained: Orchis mascula subsp. mascula and Anacamptis laxifl ora subsp. laxifl ora were light brown, A. coriophora, A. morio subsp. morio and A. papilionacea were brown, O. purpurea subsp. purpurea and Neotinea tridentata subsp. tridentata were dark brown and O. simia subsp. simia was darker brown than the rest.
When the reticulations were analyzed, it appeared that they showed minute anastomosis. Some orchids, especially the tropical ones, have conspicuous reticulations such as Calypso bulbosa (L.) Oakes (Arditti and Ghani 2000), but this was not the case in the Turkish species we included in the present study. Reticulation directions showed diff erences among species. It was more or less transverse in Neotinea tridentata subsp. tridentate (Fig. 4f ), diagonal in Anacamptis coriophora (Fig. 1C) and longitudinally diagonal in A. papilionacea (Fig. 2F). Reticulations in these species were conspicuous particularly in their periclinal walls. On the other hand, reticulations in A. morio subsp. morio were inconspicuous since they were thin and transversely diagonal (Fig.  2C). Testa cells of A. laxifl ora subsp. laxifl ora appeared to be diff erent from those of the other species. Anticlinal walls of their testa cells were fairly thick and showed unbranched thickenings (Fig. 1F). Th e periclinal wall investigations showed that the walls were smooth in some species while in some others they had fi ne reticulations. Additionally, in some seeds, one could barely see fi ne and inconspicuous reticulations, and then only in basal cells. Testa cell walls of the species with no reticulations generally showed thickenings in their joining regions (Orchis mascula subsp. mascula (Fig. 3C), O. purpurea subsp. purpurea (Fig. 3F) and O. simia subsp. simia (Fig. 4C)). Among these, folds in periclinal walls could sometimes be observed. Table 2. Measurement data of orchid seeds and embryos.    Seed lengths and widths ranged between 0.263-0.640 mm and 0.118-0.208 mm, respectively. Th e length and width measurements for embryos were 0.118-0.225 mm and 0.079-0.140 mm, respectively. All species are listed in Table 2.
When the mean values of orchid seed morphometry obtained in the present study were compared to those reported in Arditti and Ghani (2000), it appeared that both data were similar. Th e measurement data given for orchids in Arditti and Ghani (2000) is as follows; testa length 0.49 (± 0.17) mm, width 0.17 (± 0.06) mm and volume 3.93 ± 3.24 mm 3 , embryo length 0.18 (± 0.05) mm, width 0.12 ± 0.04 mm and volume 1.22 (± 0.77) x 10 -3 mm 3 and percentage air space 43.01 (± 35.16) mm 3 . When these measurement data are compared to the present fi ndings (Table 2), one can see that they are quite similar and support each other. Similarly, the current morphometric data on Anacamptis coriophora, A. morio subsp. morio, Orchis purpurea subsp. purpurea and O. simia subsp. simia was found to be almost identical, with only a few diff erences, to the ones reported in Arditti and Ghani (2000). L / W ratios provide data on the relative degree of truncation (Arditti 1979). Th e lowest L / W of 1.674 in Orchis mascula subsp. mascula showed that seeds of this species were the most truncate seeds. Th is species is followed by O. simia subsp. simia, Anacamptis coriophora and O. purpurea subsp. purpurea with their low L / W ratios implying a high truncate nature. On the other hand, higher L / W values were obtained for A. papilionacea, A. laxifl ora subsp. laxifl ora and Neotinea tridentata subsp. tridentata indicating that they have more elongate seeds. Th e highest L / W ratio of A. morio subsp. morio seeds (4.197) shows that the seeds of this species are elongate.
Th e mean lengths and widths of the embryos of the investigated eight taxa were 0.151 mm and 0.106 mm, respectively. Th e embryos were found to be elliptical with an average L / W value of 1.43. Th e lowest L / W value of O. mascula subsp. mascula led us to conclude that the embryos of this species were sphere-like. Th is species is followed by Anacamptis papilionacea. Th e high L / W values of the other species is an indication that their embryos are elliptical rather than spherical.
Percentage air space aff ects the length of time the orchid seeds are in air. Specimens with high percentage air space values are known to spread over longer distances via wind (Arditti 1967, Chase and Pippen 1988, Kurzweil 1993. Th e highest percentage air space determined for the seeds investigated ranged from 56% to 80%. Anacamptis morio subsp. morio seeds, a taxon sampled in most of the visited localities, had both the highest and the lowest percentage air space values. Th e mean air space value for orchid taxa in Edirne province is 70% and A. morio subsp. morio, A. coriophora and A. laxifl ora subsp. laxifl ora were determined to have the lowest value of approximately 65%. Orchis mascula subsp. mascula, on the other hand, whose seeds were short and wide, had the highest mean value of 78%.
As shown in previous studies on orchids, there are a number of diagnostic and phylogenetically informative characters present in orchid seeds. In this study, seed morphologies of eight orchids taxa growing in Edirne province were investigated and criteria that could be used to diff erentiate the seeds are presented. Also, a key is constructed below, based on seed morphology.  Identification key of the eight orchid taxa growing in Edirne province