A detailed anatomical approach to taxonomically problematic endemic Seseli L. (Apiaceae) taxa in Türkiye

In this study, the vegetative anatomical features of the taxa, which were previously named as Seseli gummiferum subsp. ilgazense A.Duran, Ö.Çetin & M.Öztürk and S. paphlagonicum Pimenov & Kljuykov, were compared with a closely related species, S. resinosum Freyn & Sint., which is also endemic in Türkiye. The root, the stem, the leaf segment, the petiole, and the rachis parts were examined anatomically and explained in detail for the first time to discuss the results of recent morphological and molecular studies with the anatomy of the taxa. The results show that S. gummiferum subsp. ilgazense and S. paphlagonicum have anatomically similar characters. However, in terms of the same characters, these taxa quite differ from a closely related species, S. resinosum. The most prominent different characters can be listed as the shape of stems, petioles and rachis, the presence of sclereids in the roots, the collenchyma arrangement of stems and petioles, the surface view of rachis and the sclerenchyma above floem in the stems and rachis. This revisional and detailed descriptive study highlights the importance of anatomy in the taxonomy of Apiaceae, and it is obvious that it will assist future taxonomic research in the genus.

According to Lyskov et al. (2018), within Seseli sect.Seseli s. str., there are two distinct groups of species, namely the "S.tortuosum" complex and the "S.gummiferum" complex.However, the taxonomic treatment of species within both of these groups has been a subject of some controversy.The "S. gummiferum" complex consists of the species S. gummiferum, S. paphlagonicum, S. resinosum, and some other related species (Lyskov et al., 2018).Among the members of this species complex, Seseli paphlagonicum was first described from Kastamonu province (Mountain Ilgaz) as an endemic species to Türkiye by Pimenov andKljuykov in 2010. However, Do gan Güner andDuman (2013) has synonymized the species under Seseli gummiferum by using the characters of fruit anatomy.Later S. gummiferum subsp.ilgazense was described from the same locality of S. paphlagonicum by Çetin et al. (2015).Lyskov et al. (2018) conducted a recent study using molecular data that confirms Pimenov and Kljuykov (2010) that S. gummiferum is not distributed in Türkiye and endemic to Crimea, and furthermore, that S. paphlagonicum is a distinct species, rather than a subspecies or synonym of S. gummiferum as opposed to Do gan Güner and Duman (2013).Also, S. gummiferum subsp.
In this study, it is aimed to evaluate the results of the previous studies, based on molecular and morphological data, with an anatomical approach to discuss whether the anatomy of the taxa will support them.While some anatomical characteristics of the species discussed in this article have been partially studied in previous works (Çetin et al., 2015;Do gan Güner, 2006;Pimenov & Kljuykov, 2010), the detailed anatomical properties of the roots, stems, leaves, and rachis of S. paphlagonicum, S. gummiferum subsp.ilgazense, and S. resinosum are presented for the first time in this study.This revisional and descriptive study underscores the importance of anatomy in the taxonomy of Apiaceae.It is evident that this research contributes to the present taxonomic relationships of these species and will also serve as a valuable resource for future taxonomic and evolutionary studies within the genus.Also, the anatomical descriptions presented in detail may also constitute a subordination for other taxa of the genus and closely related genera.

| MATERIALS AND METHODS
The taxa used in this study in Table 1, were freshly obtained and kept in 70% ethyl alcohol (EtOH) to be fixed.For sectioning, Paraffin sectioning method was modified, and 5-15 μm thickness of slices were gathered from the samples (Johansen, 1940).Sections, taken from the same level in the tissues of the species, were obtained by using the Thermo Shandon Finesse325 microtome.After staining with safranin and fast green, the figures were captured using a Zeiss Axiocam 208 color camera attached to a Zeiss Axioscope 5 microscope.Measurements were obtained by using ZEN imaging software.A polygon counter application of the software was used for area measurements.
At least 30 measurements from at least 10 sections belonging to the same species were taken.

| RESULTS
It is clearly seen from all the studied taxa that, raphide crystals of calcium oxalate are found in all tissues of the species.

| Root anatomy
In general, under the multilayered periderm, there are disordered parenchymatic cells in the cortex, including secretory canals.Under the cortex, secondary phloem rays extend into the inner sides of the sections, which are followed by secondary xylem.And lastly, primary xylem cells are located in the middle of the roots.The periderm of Seseli resinosum has a very thick phellem.However, the roots of the topotype of Seseli gummiferum subsp.ilgazense and Seseli paphlagonicum have only 2-3 layers of phellem cells.Also, phellogen and phelloderm can be easily observed from the root sections of S. resinosum (Figure 1).Under the periderm, there are large secretory canals between the parenchyma cells of the cortex, surrounding the secondary phloem elements.In the S. gummiferum subsp.ilgazense and the S. paphlagonicum roots, a zone of radially seriate xylem and phloem elements can easily be distinguished.Prominently, there are big T A B L E 1 The locations and the collector of the taxa.groups of sclereids located with the xylem elements in S. resinosum roots (Figure 1a).Although the overall cell order is the same, there are no sclereids in the roots of S. gummiferum subsp.ilgazense (Figure 1c) and S. paphlagonicum (Figure 1d).Moreover, in the central part of the S. resinosum roots, there are prominent and large secretory canals embedded in irregular and fragmented parenchymatic cells (Figure 1b), while in the middle of the roots of the other taxa, primary xylem elements are located and the canals are indistinct.

| Stem anatomy
The stem cross sections of the taxa are covered with cuticula and short, simple hairs.Under the epidermis, having raphide crystals, there are 2-3 cell layers of collenchymatic tissue that continuously cover the parenchymatic cortex.Collenchyma cell layers increase to cover the area between the epidermis and the secretory canals that are closest to the epidermis.The vascular bundles and sclerenchymatic cells between them make a ring in the sections.The vascular ring covers the pith, which is composed of parenchymatic cells and large secretory canals.The number of secretory canals decreases toward the pith.
In the stem sections of S. resinosum (Figure 2a), under the collenchyma, 2-3 rows of chlorenchyma cells are lined up to the large parenchyma cells surrounding the secretory canals in the cortex.The hairs of S. resinosum stems seem to be shorter than the stem hairs of the other taxa.However, S. resinosum has the widest epidermis cells, covered by the thickest cuticle (Table 2).Also, distinctively, the sclerenchyma makes a cap above the floem of the vascular bundles of S. resinosum, but in S. gummiferum subsp.ilgazense and S. paphlagonicum, the sclerenchyma above the phloem is absent or present with 3-4 cells.In the general appearance of the sections, S. gummiferum subsp.ilgazense (Figure 2b) and S. paphlagonicum 2c) have recessed and wavy-shaped stems.The vascular bundle rings also look wavy in the stem sections of these species.However, S. resinosum has a smooth-shaped stem.In general, the schizogenic secretory canals located between the parenchymatic cells in the vascular ring of the stems are smaller than the secretory canals located in the cortex (Table 2).The inner canals nearly have the same size within all taxa.However, in the cortex, S. resinosum has the largest secretory canals (Table 2).The pith is completely filled with parenchymatic cells.

| Leaf segment, petiole and leaf rachis anatomy
For the leaf segment anatomy, sections were taken from the middle part of the basal leaves (Figure 3).The leaf segments seem to be mono For the petiole anatomy, cross sections were taken from the part between the basal leaves and their leaf sheaths (Figure 4).Under the epidermis, collenchyma cells making 1-3 cell layers surround the entire petiole in S. gummiferum subsp.ilgazense (Figure 4b) and S. paphlagonicum (Figure 4c).This collenchyma layer thickens between the secretory canals above the 3-4 vascular bundles in the middle of the peripheral side and the epidermis.But in S. resinosum (Figure 4a), collenchyma only occurs between the epidermis and secretory canals closest to the epidermis.In the parts that are not connected to the secretory canal, the collenchyma has been replaced by chlorenchyma in S. resinosum petiole.The vascular bundles, which are getting smaller to the edges and are arranged only in the peripheral region, have sclerenchyma both above the floem and below the xylem.Secretory canals are arranged around the vascular bundles.Considering the petioles, the most prominent difference between the species is their shapes.The petiole shape of S. resinosum is ovoidal and adaxially flattened.However, the petiole shape of the other taxa is adaxially slight furrow and has long margins filled with collenchyma on either side (Figure 4b).
The sections of rachis also have different shapes within the species (Figure 5).The rachis of all the taxa are grooved.Under the epidermis, the collenchyma covers the entire section in 2-3 layers and it makes ribs in the sections where it is thicker.These ribs give a wavy surface appearance in S. resinosum (Figure 5a,b), but sharply in S. gummiferum subsp.ilgazense and S. paphlagonicum (Figure 5c,d).The floem and the sclerenchyma of the vascular bundles of the latter two taxa surround almost the entire xylem in the rachis.In this way, a U-shape is formed especially in the largest bundle in the middle.
Moreover, a hadrocentric vascular bundle is formed in the edges of some rachis sections of S. gummiferum subsp.ilgazense and S. paphlagonicum.

| DISCUSSION
The most significant differences and some measurements of the species are summarized in Table 2. Ulusoy et al. (2017) emphasized that the presence of sclereids in the roots of a genus from the Apiaceae family is a taxonomically important character and they resolved the infra-generic discrimination of the genus with the presence or absence of sclereids of the roots.From this point of view, it can be said that the most distinctive root character among the studied taxa is sclereids, which are located among xylem elements in S. resinosum.
Also, S. resinosum has a thick phellem with more than 30 cell layers.
However, the other taxa do not have sclereids in their root sections and they have thin phellem with 2-10 cell layers.T A B L E 2 Characters of the taxa.
fascial.The irregularly shaped epidermal cells are covered by a thin layer of cuticle.Palisade parenchyma tissue surrounds the vascular bundles and secretory canals.The secretory canals are located around the vascular bundles.There is a collenchymatic tissue between the epidermis and the secretory canal that is embedded near the midrib xylem elements.Also, this central vein, which is the biggest one of the leaf segments, has sclerenchyma above its floem.The sections of leaf segments of all the taxa seem to have the same structural properties.F I G U R E 1 Root sections (a) S. resinosum (5X) (b) sclereids of S. resinosum (20X) (c) S. gummiferum subsp.ilgazense (5X) (d) S. paphlagonicum (10X).pd, periderm; ph, phloem; phe, phellem; r, raphids; sc, secretory canal; scl, sclereid; xl, xylem.

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I G U R E 3 Sections of basal leaf segments (a) S. resinosum (20X), (b) S. gummiferum subsp.ilgazense (10X) (c) S. paphlagonicum (10X).c, cuticle; col, collenchyma; e, epidermis; ph, phloem; pp, palisade parenchyma; s, stoma; sc, secretory canal.According to Saltan et al. (2021), there are 5-8 layers of chlorenchyma located below the epidermis and also collenchyma is located between the epidermis and secretory canals of the stems of Glaucosciadium cordifolium (Boiss.)B.L. Burtt & P.H. Davis (Apiaceae).The results support this previous study that, the stem sections of the taxa have chlorenchyma below epidermis and they have collenchyma between the secretory canals and epidermis.Moreover, epidermal cells adjacent to collenchyma cells seem to be smaller than the other epidermal cells.Do gan Güner (2006) made a revisional study of the genus Seseli distributed in Türkiye.In Do gan Güner (2006), stem, leaf, and petiole anatomy are mentioned in S. gummiferum subsp.gummiferum collected from Ankara province and S. resinosum collected from Amasya province in Türkiye.According to this previous study, the stem shape of S. resinosum has flate circle shape and S. gummiferum subsp.gummiferum has recessed and wavy circle shape, which is also seen of its vascular bundle array (Do gan Güner, 2006).The results of the current study support Do gan Güner's study in 2006 that, despite being collected from different locations, both samples of S. resinosum have the same stem structure.Moreover, the stem shape and the vascular bundle arrangement of the stems of S. gummiferum subsp.gummiferum (Do gan Güner, 2006), S. gummiferum subsp.ilgazense, and S. paphlagonicum seem to be similar.Furthermore, the previous study indicated that the sclerenchyma above floem is a general stem anatomical character for the genus.When the figures were examined, it was seen that there were 2-3 sclerenchyma cells above floem of the vascular bundles in the S. gummiferum subsp.gummiferum (Do gan Güner, 2006) stem and a cap was formed in the S. resinosum stem, which is consistent with the results of the current research study.Accordingly, S. gummiferum subsp.gummiferum(Do gan Güner, 2006), S. gummiferum subsp.ilgazense, and S. paphlagonicum seem to be similar in this character.Pimenov and Sdobnina (1975) indicated that the petiole anatomy is an important character for the taxonomy of the genus Seseli.Moreover,Pimenov et al. (1982) published a comprehensive study of the petiole anatomy and its taxonomic importance among taxa in the Caucasian Apiacea family.In this previous study, characters, such as the shape of the petioles, the features of the adaxial side, the rib structure, the presence and structure of the collenchyma under the epidermis, the hairiness, and the number or arrangement of the vascular bundles were mentioned.Also, Do gan Güner (2006) indicated that S. gummiferum subsp.gummiferum can be distinguished from its close species by petiole anatomy.The current study supports all these previous studies that, the petiole anatomy is completely different in S. resinosum considering these mentioned petiole characters.However, S. gummiferum subsp.ilgazenseand S. paphlagonicum and even S. gummiferum subsp.gummiferum (Do gan Güner, 2006), have more or less similar petiole shapes.

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I G U R E 4 Sections of petiols (a) S. resinosum (b) S. gummiferum subsp.ilgazense (c) S. paphlagonicum (10X).chl, chlorenchyma; col, collenchyma; ph, phloem; phs, sclerenchyma above phloem; r, raphids; sc, secretory canal; x, xylem.In a descriptive study, Lobiuc et al. (2012) examined some anatomical characters of three species of Romanian Angelica genus from the family Apiaceae and mentioned the similarities and differences of the arrangement of ribs of leaf rachis.Considering the studied taxa, the collenchymatic ribs of S. resinosum rachis make a wavy surface appearance.However, in S. gummiferum subsp.ilgazense and S. paphlagonicum, the ribs give a sharp appearance to the rachis.