EJBR2018v8i1art34-41 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2018; 8 (1): 34-41 Comparative stem anatomy of four taxa of Calycanthaceae Lindl. Niroj Paudel, Kweon Heo* Division of Biological Resource Sciences, Kangwon National University, Chuncheon, 24341, South Korea *Corresponding author: Kweon Heo; Phone: +82-33-250-6412; E-mail: laurus@kangwon.ac.kr ABSTRACT The anatomical character is potential value in Calycanthaceae for their taxonomic study. Four species of Calycanthaceae were collected for this experiment. The experiment was done using the resin methods for preparation of the permanent slide for anatomical studies. The anatomical character like two traces of the unilocular vascular bundle, in the primary vascular cylinder, contains four cortical vascular bundles in the stem, the unilocular structure of primary cylinder, the presence of numerous intercellular space in phloem, the presence of oil cell in the form of scatter in Calycanthus whereas small size in Chimonathus. Calycanthus possess boarder pit with circular aperture while Chimonanthus possess elliptical. The tracheid is a characteristic feature of the spiral band wider in Chimonanthus than that of Calycanthus and Sinocalycanthus. The noted sclerenchymatous cells are grouped of the colony which is a characteristic feature of Sinocalycanthus and Calycanthus but in case of Chimonanthus is the long chain with the layer of the cell. Collenchymatous cell was circular with an intercellular in Calycanthus; ovoid shape with the intercellular in Chimonanthus but in Sinocaly- canthus is elongation with the minor regular shape. The different character of pith cells found in hexagonal and circular shape which is also distinguished feature in Calycanthaceae. The valu- able stem anatomical characters are the importance of their function, ontogeny, and phylogeny. Keywords: Anatomical character; Calycanthaceae; Collenchyma; Sclerenchymatous; Vascular bundle. 1. INTRODUCTION Calycanthus, Chimonanthus, and Sinocaly- canthus are the genus of Calycanthaceae. Sino- calycanthus is native to China. Sinocalycanthus is the synonym of Calycanthus. The literature reveals that long horticulture forms and varieties due to the long cultivation of history. Calycanthaceae is the small family of the plant with four genera and ten species which is the sister group of Laurales [1-7]. Within Calycanthaceae, the deepest split is between the tropical monotypic tree Idiospermum austra- liense and the temperate shrubs of the rest of the family Calycanthoideae (Calycanthaceae) [8, 9], unique in Laurales are features of the gynoecium: ovule number and placentation differ from all other Laurales, and the seeds in Idiospermum have the largest embryos known in angiosperms [5, 10]. Although very old Chinese drawings and Japanese wood figures of Chimonanthus are apparently in existence [11], the first drawing to appear in the taxonomic literature was probably that [12, 13]. Calycanthus floridus Linneaus [14] recognized only the genus Calycanthus with the two species Received: 15 January 2018; Revised submission: 09 March 2018; Accepted: 14 March 2018 Copyright: © The Author(s) 2018. European Journal of Biological Research © T.M.Karpiński 2018. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1199578 35 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 Calycanthus floridus and Calycanthus praecox [13], as well as Lindley [11], considered Calycanthus praecox to represent a new genus Chimonanthus. Some authors [13, 15, 16] maintained this concept of two genera. Others [18, 19] followed Linneaus in recognizing only one genus. Prantl [18], the other hand, recognized two sections, viz., Eucalycanthus and Chimonanthus. There has been some confusion in the past concerning the correct names for these genera. However, the designation of Calycanthus L. and Chimonanthus Lindley as nomina conser- vanda Lanjouwa [19] has solved this problem. The pertinent nomenclatural information and synonymy have been summarized by Kearney [20] and Rehder [21] for Calycanthus and by Rehder and Wilson [13] for Chimonanthus. Chimonanthus was monotypic until the description of Chimonanthus nitens by Oliver [22] based on material from August Henry's collections from central China. With more complete collections, two additional species have been proposed, Chimonanthus yunnanensis Smith [23] and Chimonanthus salcifolius Hu [24]. The situation is somewhat different in Calycanthus. Although C. occidentalis of California has been recognized as comprising a distinct and relatively uniform species, the plants of the southeastern United States have been treated as representing from one to as many as six species. Rafinesque [25] represented the latter extreme by stating to the sp. of Calycanthus L. only one. In most manuals at least two species C. floridus and C. fertilis have been recognized based primarily differences in pubescence and leaf shape. The aim of this study is the histological comparison of the stem of Calycanthaceae for the purpose of discussion and implication of observed anatomical trait for support the classification of the plant. 2. MATERIAL AND METHODS Altogether four species (Table 1) is collected. The stems were fixed with the FAA (formalin: glacial acetic acid: 50% ethanol, 5:5:90, by volume) from each family mature stem were selected then passed alcohol series after that; alcohol: technovit 7100 resin. Serial section of 5-6 µ m thickness was cut using disposable blade knives stuck into glass slides and dried on electrical slide hot plate for Twenty four hour; slides were stained with 0.1% toluidine blue for 60-90 second. After that rinsed with running water, and again dried on the electric hotplate for more than six hours to remove water. The stained slides were then the mounted with Entellen. Four permanent slides were observed under an Olympus BX-50 light microscope (Olym- pus Co. Japan), Photographs were taken with the digital camera system attached to the microscope and multiple image alignment was done using Photoshop. Table 1. Collection information of genus and species used in the present study. Taxa Collection information Calycanthus occidentalis Hook. & Arn. Korea, Cultivated at Kangwon University, K. Heo & N. Paudel s.n. 2016 (KWNU) Chimonanthus praecox Lindl. Korea, Cultivated at Kangwon University, K. Heo & N. Paudel s.n. 2016 (KWNU) Chimonanthus salcifolius S.Y. Hu Korea, Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 Sinocalycanthus chinensis W.C. Cheng & S.Y. Chang Korea, Cultivated at Kangwon University, K. Heo & N. Paudel s.n. 2016 (KWNU) 3. RESULTS Epidermis the single-layered outermost com- posed of tabular parenchyma cells (Table 2) which are compactly arranged without having inter-cellular spaces in Chimonanthus praecox (fig. 2C) and Chimonanthus salcifolius (fig. 2C). In Sinocaly- canthus chinensis and Calycanthus occidentalis were intercellular space in the epidermal cell (figs. 1C, 2I). Outer walls were cuticularised. Collen- 36 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 chymatous cells are a circular shape which is inter- connected with each other layer in Chimonanthus praecox and Chimonanthus salcifolius (figs. 1I, 2I). Sclerenchymatous cells are higher cell grouped in Sinocalycanthus chinensis and Calycanthus occi- dentalis whereas in Chimonanthus praecox, and Chimonanthus salcifolius formation of long 2 layer chain (figs. 1I, 1D) with two traces of unilocular vascular system was noted in all species Sino- calycanthus chinensis, Calycanthus occidentalis, Chimonanthus praecox, and Chimonanthus salci- folius. In primary vascular cylinder, four cortical vascular bundles were noted in all species in Calycanthaceae (figs. 1A, 1G, 2A, 2G). A cortical bundle which is later developed in the central bundle in the stem. Especially unilocular system is in the primary vascular cylinder in all species (figs. 1D, 2H, 2C, 2I). Calycanthus occidentalis has circular border pits (fig. 1E). In the center, pith is a loosely bound hexagonal structure (fig. 1F) with inter- cellular. Parenchymatous cell are in circular and ovoid shaped whereas sclerenchymatous cells are in group colony contains thirteen number of cells (fig.1D) Protoxylem vessels with wider cavities with annular thickening towards the epidermal cell (figs. 1G, 1H). Pentagonal intercellular space gap was seen in Calycanthus occidentalis (fig 1I). The tracheid possesses spiral band (figs.1E, 1K). Chimonanthus praecox possess straight chain border pits which are undergoing towards the epidermis (fig.1K) with elliptical aperture. The pith cell circular with intercellular space was noted in Chimonanthus praecox (fig. 1L). The parenchyma- tous cells were rectangular in shape possess the large intercellular space (figs. 1I, 1J). Chimonanthus salcifolius also possess straight chain bordered pits (fig. 2E). Parenchymatous cells are circular or ovoid shaped with intercellular space (figs. 2C, 2D) the vascular bundle is collateral (figs. 2B) the pith cell are also noted large circular cell with intercellular space (figs. 1L, 2F). Sinocalycanthus chinensis parenchymatous cells are ovoid with some rectangular shape (figs. 2I, 2J). The vascular bundle is noted four in each quadrangular side (fig. 2G). The cortical bundle is also the lateral side of the stem (fig. 2H). The pitch cells are large with intercellular space with circular as well as hexagonal shape (fig. 2L). Table 2. Comparative stem anatomical characters of Calycanthaceae. Taxa Epidermis Collenchyma Parenchyma Sclerenchyma Endodermis Number of vascular bundle Xylem Pith Calycanthus occidentalis Hook.& Arn. Single layered Circular shape, loosely bind Ovoid or circular shape 13-14 cells in the group, scatter Single layered, parenchymatous cell 4 Protoxylem vessels with wider cavities Hexagonal shape with intercellular space, loosely bind each other Chimonanthus praecox Lindl. Single layered Ovoid shape, intercellular space circular shape 2-6 cells in the group, scatter Single layered, parenchymatous cell 4 Protoxylem vessels with smaller cavities Circular shape, loosely bind Chimonanthus salcifolius S.Y. Hu Single layered Ovoid shape, intercellular space Circular shape 2-7 cells in the group, scatter Single layered, parenchymatous cell 4 Protoxylem vessel with smaller cavities Circular shape Sinocalycanthus chinensis W.C. Cheng & S.Y.Chang Single layered Elongation shape, intercellular space Ovoid or elongation shape 14-17 cells in a group, scatter Single layered, parenchymatous cell 4 Protoxylem vessel with wider cavities Hexagonal shape interact with each other 37 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 Figure 1. Calycanthus occidentalis (A-F); A. Cross section of young stem, B. A detail portion of cross section (arrow head shows the vascular bundle), C. Epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), D. Sclerenchyma with the colony, E. Tracheid and vessel, F. Pith. Chimonanthus praecox (G-H); G. Cross section of young stem, H. A detail portion of cross section (arrow head shows the vascular bundle), I. Epidermis and collenchyma (arrow head represents epidermis, and cl shows collenchyma), J. Sclerenchyma with the colony, K. Tracheid and vessel, L. Pith. 4. DISCUSSION The stem of the Calycanthaceae is characte- rized by its quadrangular appearance (figs. 1A, 1H, 2A, 2G). This is usually quite in young stems but less in older ones. The tissues of the mature stem have received a close examination by several researchers. Much of the research has focus on the presence of inverted cortical bundles in the stem. 38 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 Figure 2. Chimonanthus salcifolius (A-F); A. Cross section of young stem, B. A detail portion of cross section (arrow head shows the vascular bundle), C. Epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), D. Sclerenchyma with colony, E. Tracheid and vessel, F. Pith; Sinocalycanthus chinensis (G-L); G. Cross section of young stem, H. A detail portion of cross section (arrow head shows the vascular bundle), I. Epidermis and collenchyma (arrow head represent epidermis, and cl shows collenchyma), J. Sclerenchyma with colony, K. Tracheid and vessel, L. Pith. The literature has been summarized by Metcalf and Chalk [26], Solereder [27] and in part by Bennett [28] and Quinlan [29]. Quinlan [29] found only slight differences between members of the family. The more important of these works are summarized in the following treatment. The work concerning the vascular system of the family is that of Fahn and Bailey [30] who studied the nodal anatomy of both Calycanthus and Chimonanthus. They found the family to possess a two-trace, unilacunar vascular 39 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 system. Such a nodal structure is now recognized to be primitive among angiosperms and also occurs in the Austrobaileyaceae, Monimiaceae, Annonaceae and Winteraceae among others [31]. They have noted some differences between species of Calycan- thaceae in the level of branching and fusion of the vascular bundles of the eustele. This could be utilized as a taxonomic feature which is also supported by our results. In addition to the primary vascular cylinder, four cortical vascular bundles occur in the stem. These were first described [29] and have long drawn the attention of plant anatomists. There has been some disagreement as to their origin and phylogenetic significance and the various descriptions and viewpoints have been summarized by [28] and [29] in a detailed study of the seedling, stated: "Cortical bundles were found to originate from the primary vascular poles of the root after they had diverged from the central cylinder to become the trace to the cotyledon. This is the only point at which the cortical system connects with the central style of the stem. Fahn and Bailey [30] in general agreed with the observations of Bennett. In their study of the eustele as well as the cortical system of the mature stem and seedlings, they found no evidence that the cortical system is a modification of lateral traces of a trilacunar or multilacunar nodal system, as is the apparent case in some dicot families [31], but believed that it is an additional independent system which has been superimposed upon the double trace, unilacunar structure of the primary cylinder. They also indicated that transverse connections between the cortical strands which are present in the nodal region of Calycanthus are less well developed in Chimonanthus. A complete and detailed description of the phloem of the Calycanthaceae has been given by Cheadle and Esau [32]. Our results also show Calycanthus occidentalis, Chimonanthus praecox, Chimonathus salcifolius and Sinocalycanthus chi- nensis in which they found the phloem to be very similar and to possess the following characteristics in common: absence of fibers, sclareids, the presence of numerous intercellular spaces, presence of oil cells in varying degrees of abundance, uniseriate to multiseriate rays, sieve elements with thick nacreous walls, and simple sieve plates located laterally or on the end walls in an oblique or transverse manner. Oil cells were found to be in Calycanthus occidentalis, and infrequent and usu- ally smaller in Chimonanthus praecox, whereas Chimonanthus salcifolius and Sinocalycanthus chi- nensis possess big size. In Sinocalycanthus chinensis we also demonstrated a more irregular cell pattern with sieve tubes in more markedly isolated strands than in either Calycanthus occidentalis or Chimo- nanthus praecox and Chimonanthus salcifolius. Characteristics of the secondary wood of the family have been summarized by Metcalf and Chalk [26]. Lemesle [33] in his rather extensive study, reported differences primarily between the two genera in tracheid characteristics. Those of Calycanthus and Sinocalycanthus possess bordered pits with circular apertures while those of Chimonanthus possess more elliptical apertures. Lemesle [33] considered the wood of Calycanthus to be more primitive, at least in this characteristic. Tracheids are quadrangular in cross section. The internal surface of the tracheids possesses spiral bands which are stated to be slightly wider in Chimonanthus than in Calycanthus. We also supported that the majority of the tracheids possess simple oval or oblong perforations although some tracheids may be devoid of the connecting perforations. The vessels are small, polygonal in cross section and possess simple perforations. The fibers are libriform and almost completely devoid of bordered pits in Calycanthaceae. There was no detail character for the stem in Calycanthaceae. Sclerenchyma possesses chain and colony structure which is a new character for young stem anatomy in Calycanthaceae. Key to the genera of Calycanthaceae based on the stem anatomy: 1. Sclerenchyma is (14-17) cells group formation of the colony, protoxylem vessel is wider cavity ………………........................... Sinocalycanthus 2. Sclerenchyma is (13-14) cells group formation of colony, protoxylem vessel is wider cavity ………........................................…. Calycanthus 3. Sclerenchyma is arranged in the long chain with two layers, protoxylem vessel is smaller cavity ………..........................………..... Chimonanthus 40 | Paudel & Heo Comparative stem anatomy of four taxa of Calycanthaceae Lindl. European Journal of Biological Research 2018; 8 (1): 34-41 AUTHOR’S CONTRIBUTION Both authors have equal contribution. 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