Comparative anatomical studies in relation to taxonomy of Sedum s.l. (Crassulaceae) in Iran

This study aimed to characterize the stem, peduncle, and leaf anatomy of 22 species of Sedum senso lato distributed in Iran. The results showed that the presence of tanniniferous storage cells in stems and leaves, distinct xylem vessels in stems, and the shape of peduncle cross sections, were taxonomically informative evidence, and isolated Phedimus from the other studied taxa of Sedum s.l., whereas hairy leaves and peduncles containing starch storage cells were identified as Prometheum and Hylotelephium respectively. In addition, the current anatomical evidence confirmed the alliance of 2 sections of Sedum and Epeteium within Sedum sensu stricto. The result of numerical analysis (including 31 qualitative and quantitative anatomical characters) supported complete separation of the fourallied genera and revealed significant influence of anatomical traitsin taxonomy of Sedum s.l.

were carried out for at least 5 cross-sections, selected from 2 to 3 populations per species. A list of voucher specimens is given in Table 1. Leaf samples were selected from the 2nd to 6th nodes of the stems. Stem samples were cut from about 1cm in the middle of the 2nd to 4th nodes. Fresh materials were fixed in 50% FAA (formaldehyde/acetic acid/ethanol) for 48 h, then washed with distilled water, dehydrated in an ethanol series (30, 50, 70, 95 and 100%), and transferred to 70% ethanol for long-term preservation. Herbarium specimens were rehydrated in water before fixing in FAA. Stem peduncle and leaf cross-sections were prepared by hand cutting and then stained with methyleneblue and Congo red. Observations were carried out using an Olympus BX-51 light microscope under 100× and 400× magnifications. For cluster analysis (CA), NTSYS software (version 2.02) (Rohlf, 1997) was used to construct the dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) (Sneath and Sokal 1973). Cophenetic correlation coefficient was calculated to find out to what extent cophenetic matrix fits the original similarity matrix. In this analysis, a total of 46 samples (Table 1) and 31 anatomical characteristics comprising 20 qualitative (Tables 2 and 3) and 11 quantitative characteristics (i.e. diameter of tanniniferous storage cells in stem; thickness of stem cuticle, endodermis and parenchyma layer; ratio of xylem thickness to phloem in stem; number of collenchyma layers in stem and peduncle; thickness of epidermal layer and cortex in peduncle, ratio of xylem thickness to phloem and ratio of pith thickness to cross section in peduncle) were involved. Figure 1, Figure 2) Cross-sections were undulating circular to circular in shape with 2 lateral wings. There was a single layer of epidermal cells surrounded by a layer of cuticle on the surface. Stomata were present on the epidermis of Ph. obtusifolious (Figure 1e), S. tenellum, S. sabulatum and S. annum but absent in the other studied taxa. Trichomes were observed in the epidermis of the representatives of the genus Prometheum (Figures 1f-1h) and 2 taxa of Sedum s.s.: S.tenellum ( Figure 1l) and S. elbursense ( Figure  2o). Underneath the epidermis was a collenchymatous region consisting of 1-3 cell layers. This region was absent in S. caespitosum (Figure 2j) A parenchymatous cortex comprising circular compact cells was located between the collenchyma and the endodermis. Cortical bundles were observed in the parenchymatous tissue of S. tetramerum ( Figure 2a) and S. pallidum (Figure 2c). One layer of endodermis separating the cortex from vascular cylinder was recognized in the stem of all examined taxa, except in Prometheum taxa Vascular cylinder consisted of a poorly developed phloem (which were not easily recognized) and a well-developed xylem. Xylem vessels were in a continuous ring in all studied species except in Ph. stoloniferus (Figure 1a), Ph. spurius ( Figure 1c) and Ph. obtusifolius (Figure 1d), which exhibitedcluster vessels of unequal size. The pith region was observed in the central part of the stem of all examined taxa, except S. callichroum (Figure 2l). In some species, parenchymatous tissue of cortex and pithcontainedtanniniferous and starch storage cells that varied in size and density among the studied taxa (Figures 1a-1d, 2e, 2g-2i).

Cross-section of peduncle (Table3, Figure3, Figure4)
Cross-sections had different shapes including circular, undulating circular, circular with 2 wings, semicircular with 3 wings and stellate. The outer surface was covered by 1 layer of cutinized epidermal cells and in some species with uni-and multicellular glandular trichomes 4i). The cortex consisted of 1-3 layers of collenchyma and strongly developed parenchyma layers (especially in the representatives of the genus Prometheum). Some storage cells were observed in the parenchymatous region of H. caucasicum (Figure 3g).The vascular cylinder consisted of xylem, which was found in a closed ring or in clusters, a poorly developed phloem, and a pith zone. (Table 3, (Figure 6d), adaxial and abaxial sides were indistinguishable. In all taxa, there was a single layer of ovate-circular epidermal cells on both leaf surfaces. Trichomewas observed on the epidermis of P. pilosum and P. sempervivoides. Prometheum pilosum had multicellular glandular hairs on the lower epidermis (Figures 5e-5f), while P. sempervivoides showed a higher density of trichomes on both lower and upper epidermises (Figures 5g-5h). In some species (e.g. S. lenkoranicum and S. gracile), lower epidermal cells were larger than upper cells (Figures 5m-5o). Hypoderm was observed in the studied taxa of Phedimus, Prometheum, Hylotelephium and 3species of Sedum s.s. (S. lenkoranicum, S. rubens and S. annum). In all examined taxa, mesophyll was isobilateral and not differentiated into palisade and spongy layers. There were some large mucilaginous cells associated with parenchymatus cells in some species (e.g. S. hispanicum, S. pentapetalum, S. album etc.), but these cells were not constant in the different populations of the same species. In the representatives of Phedimus, storage cells were found in the mesophyll (especially around the vascular bundles) (Figures 5a-5d). The midrib region was not clearly recognized in some species of Sedum s.s., while it was obvious and prominent in the examined taxa  Figure 7) showed a high cophenetic correlation (98%), indicating a great proportion between the dendrogram and original matrix, and a complete separation of the genera into2 majorclusters; the 1st cluster contained representatives of Phedimus, Promethium and Sedum s.s., and the 2nd comprised Hylotelephium species. Two sections of Sedum and Epeteium could not be delimitated in this dendrogram.

Discussion
Environmental influences can simulate the appearance of various adaptations in plants and thus strongly affect their morphoanatomical differentiation (Anacker, 2014). These differences usually represent an adaptive response to certain habitat conditions, but in cases of highly distinct differences may have important taxonomic significances (kruckeberg, 1951;Anacker et al., 2011). Herein we used different accessions for each taxon and selected features that were constant in them.

Important anatomical characteristicss used for the intergeneric delimitation and taxonomic relationships
The characteristics that contributed most to the separation of genera were the presence or absence of hairs and tanniniferous secretory cells in the stem and leaf; the number of collenchyma layers; the presence or absence of endodermis and types of xylemvessels (distinct or continuous) in the stem; the shape of the cross-section; and the presence or absence of starch storage cells in peduncles. Based on Metcalf and Chalk (1950), tanniniferous secretory cells are commonly present in stemnonlignified tissues, especially the cortex, pith, and phloemof the family Crassulaceae. In majority of these plants, distinct vascular bundles are rare, and the xylem forms a continuous cylinder. Our finding revealed the importance of stem and peduncle anatomical charactersin the separation of Phedimus from Prometheum, Hylotelephium and Sedum  (Jansson and Rechinger, 1970;Akhiani, 2000) and the flora of Turkey (Chamberlain, 1972) have treated these species under the genus Sedum.
The presence of starch storage cells in the cortex of the peduncle (Figure 3g) and 2-3 collenchyma layers in the Table 3. Selected qualitative characters of peduncle and leaf for anatomical comparison of the examined species. + represents presence of character, -represents absence of character, numbers indicate cell layers. CS = cross-section, T = trichome, SSC = starch storage cells, XyV = xylem vessel, Cl = collenchyma, Hy = hypodermis, TSC = tanniniferous storage cell, Mid = midrib region.   The studied anatomical characters present a strong variation within Sedum s.s.; however, these traits do not show obvious synapomorphies in the genus.

Importantanatomical characters used for infragenericdelimitation and taxonomic relationships
Within the genus Phedimus, Ph. stoloniferus was distinguished by its starch storage cells (Figure 1b), and Ph. obtusifolious was differentiated from the other 2 examined taxa in having stomata in its stem cross section (Figure 1e).
Following Berger's classification (1930), some local flora, such as Flora Europea (Webb, 1964), Flora Iranica  (Jansson and Rechinger, 1970) and flora of Turkey (Chamberlain, 1972) arranged Sedums.s. into 2 sections, Sedum and Epeteium. However, the 2 sections were merged by recent studies (Clausen, 1975;Ohba, 1978;̓ t Hart, 1982;Akhiani, 2000). Our anatomical evidences support the later investigation concerning the alliance of 2 sections. The 2 sections have several common anatomical traits, e.g. the shape of the stem cross-section (circular, undulating circular, and circular with 2 wings); number of (0-1) collenchyma layer in the stem and peduncle; the absence of storage cells in the stem, peduncle, and leaf; the presence or absence of trichome in the stem and peduncle; the presence of endodermis in the stem; the shape ofxylem vessels in the stem (a ring) and leaf (circular/crescent), and the presence of pith in the stem and peduncle. According to Berger's classification (1930), the Sedum rubens group (including S. hispanicum, S. pentapetalum, and S. rubens) has a controversial systematic position and their delimitation is not clearly understood (̓ t Hart, 1985). Some previous authors (Froderstrum, 1932;Jansson and Rechinger, 1970) considered S. pallidum as a synonym of S. hispanicum, while others (Borrisova, 1939;Webb, 1964;Chamberlain, 1972;Akhiani, 2000) treated it as a distinct species. Furthermore, Zaffran (1976) considered S. pallidum to be conspecific with S. rubens (̓ t Hart 1985). Sedum pallidum and S. hispanicum have identical morphological characters,such asterete leaves; 10 stamens; triangular sepals with equal size; and white petals with a reddish median line. However, based on palynological (Giuliani, 2017) and phylogenetic evidence (Nikulin, 2016), S. rubens, S. hispanicum, and S. pallidum are distinct species occurring in the Leucosedum clade. According to our anatomical data, these 3 closely related taxa can be readily isolated by the cross-section shape of their stem and peduncle (circular with 2 wings in S. hispanicum (Figures 2d and 3o), but undulating circular in S. pallidum (Figure 2b, Figure 3n) and S. rubens (Figures  2h and 4c). In addition, S. pallidum is characterized by the presence of cortical bundles in the stem (Figure 2c). Metcalf and Chalk (1950) and Abdel-Raouf (2012) have reported this character in some other taxa of Crassulaceae. The absence of starch storage cells in the stem of S. pallidum and a convex cross-section of the leaf with a defined midrib region in S. rubens ( Figure 6E) are other important differentiating characters in this group. S. hispanicum and S. pentapetalum have an ambiguous taxonomic position. They were treated as 2 distinct species in both Flora Iranica (Jansson and Rechinger, 1970) and flora of Iran (Akhiani, 2000),whereas Sarvar (2004) combined the 2 later species and considered S. pentapetalum as a synonym of S. hispanicum. These species have several palynological and morphological similarities (Sarvar, 2004). However, the result of present study did not show significant anatomical differences between the 2 later species.

Conclusion
Our study shows anatomical traits can provide helpful data in solving the current problem of the taxonomy and nomenclature of Sedums.l. in Iran. The representatives of the genera Phedimus, Prometheum, and Hylotelephium can be easily delimitated from the species of Sedum s.s. using anatomical features. The results also support the alliance of 2 sections Sedum and Epeteium due to several identical anatomical characters described within the sections. An identification key was provided based on the most important diagnostic characters: