International Journal of Plant Biology 2018; volume 9:7525 Pericarp, seed coat anatomy ily and Chimonanthus is sister to the and seed morphology of Calycanthus-Sinocalycanthus.1,25 In Correspondence: Kweon Heo, Department of angiosperms, floral phyllotaxis is often Applied Plant Sciences, Kangwon National Calycanthaceae variability of various taxonomic feature.27 University, Chuncheon 24341, South Korea. Gynoecium is unique to Idiospermum pres- Tel.: +82.33.2506412. Niroj Paudel, Kweon Heo ence of only one carpel, rarely up to five.1. E-mail: [email protected] Floral phyllotaxis and architecture were Department of Applied Plant Sciences, Key words: Calycanthaceae; pericarp; seed described which is further supported, devel- Kangwon National University, coat; SEM; trichome. opment of gynocium.28,29 Chuncheon, South Korea Structure and evolution of embryology Funding: none. in Laurales is described.30 Seed coat anato- my and trichome morphology of Contributions: the authors contributed equally. Abstract Calycanthacae have not studied. We focus Pericarp, trichome, and seed coat anato- on the seed morphology, seed anatomy, and Conflict of interest: the authors declare no my display great features of taxonomic trichome morphology on Calycanthaceae, potential conflict of interest. value in the Calycanthaceae. The present which support for the phylogenetic implica- tion. The main objective of this study is to Received for publication: 7 December 2017. study about trichome and seed coat anato- Accepted for publication: 27 June 2018. my has based on external and internal deduce their phylogenetic relationship. observation. Detail anatomical study of This work is licensed under a Creative seeds provides valuable information for fur- Commons Attribution-NonCommercial 4.0 ther study about their function, ontogeny, Materials and Methods International License (CC BY-NC 4.0). and phylogeny. Therefore, the goal of the The collected seeds were fixed with the study is to investigate trichome morphology ©Copyright N. Paudel and K. Heo, 2018 FAA (formalin: glacial acetic acid; 50% Licensee PAGEPress srl, Italy and seed coat anatomy in Calycanthaceae to ethanol, 5:5:90, by volume) from each International Journal of Plant Biology 2018; 9:7525 provide more detail characterization. Seeds species (Table 1). Mature seeds passed onlydoi:10.4081/pb.2018.7525 were collected after that preserved with through the alcohol series. After complete FAA. Furthermore, alcohol series applied dehydration, the seeds passed through com- for SEM and light microscopy. The unicel- bination alcohol/Technovit and then embed- lular trichome morphology is common in all ded in Technovit 7100 resin. use species in Calycanthaceae. Density of tri- Serial sections of 5-6 µm thickness 11.45×5.2-6.65). The variable seed feature chome is highest in Calycanthus occiden- were cut using the disposable blade knives of the species in order to measurement of talis. Different variation of seed coat and stuck into glass slides, and dried on electri- seed size (length-width, length/width ratio) pericarp layers are characteristics of poten- cal hot plate for 24 hours. Dried slides were, these are the significant dissimilarities of tial phylogenetic significance in the family. stained with 0.1% Toluidine blue for 60-90 the species we concluded That the number second, ringed with running water, and of species in genus Chimonanthus is quite again dried on the electric hotplate for more large than that of Calycanthus, but in case than 6 hour to remove water. The stained of Sinocalycanthus is medium size The Introduction slides then mounted with Entellan (Merck morphological character is little value for Calycanthaceae is small family compro- Co., Germany). Four permanent slides generic delamination in Calycanthaceae. mised four genera and ten species.1-6 observed under the Olympus BX-50 light There is difference on seed coat layer, peri- Calycanthaceae is temperate shrubs or trop- microscope (Olympus Co., Japan). carp layer cell shape structure. The seed sur- ical trees have relatively large flowers, The pre-treatment applied for SEM. The face, the seed coat and pericarp character which are pollinated by beetles.7-10 The fos- preserved seeds were passed through the certainly have significant in phylogeny. sil record of reproductive structures of alcohol series then immersed in 100% Detail individual description of the nature Calycanthaceae is relatively abundantNon-commercial in ethanol after that used critical point dryer of the seed surface and seed coat is given earlier.11-15 Some of these fossils are (CPD) then sample were attached from cop- (Tables 2-3). In this studies, seed shape was 15 16 per tape. SEM image was carried out from extremely well preserved. Van Heel quite variable large, the range of the helium KBSI, Chuncheon at (EHT= 3.00 KV). The studied carpel development. Within is concave, long or medium, the range 14.4- multiple image alignments did by using Calycanthaceae, gynoecium character is 16.4×6.4-7.5 mm the shape of the testal Photoshop CS6. unique to Idiospermum.10 Early studies rec- cells either, polygonal or sub polygonal, ognized the affinity of Calycanthaceae to irregular (Figures 1-3). The wall ornamen- Monimiaceae and Lauraceae.16 This affinity tation is roughly, most of the cell have con- was acknowledging in the major classifica- struction on the center in (Figure 2A, 2B, tion systems of the past 50 years.17-24 Results 2C) the color of seed was brown in Molecular phylogenetic analyses settled the Out of one Idiospermum genera we Calycanthaceae (Figure 1), the seed were family as sister to the rest of Laurales.2- include three genera for this study; enclosed in the fleshy hypanthium. 4,25,26 These analyses also divided the Sinocalycanthus Calycanthus and Pericarp separated in three histological remaining Laurales into two clades of three Chimonanthus. Of the 3 genera and eight zones: exocarp, mesocarp, and endocarp families each: the Siparunaceae- species included, the seed of Chimonanthus developing from the outer epidermis, meso- Atherospermataceae-Gomortegaceae clade3 fragrans (19.2-20×6.1-7.25 mm) is largest phyll and inner epidermis. The exocarp is and the Monimiaceae- Hernandiaceae- and we found that the size and shape were represented by the single layer and tangen- Lauraceae clade.3 Within Calycanthaceae, highly variable with in the study of genus tially attached with mesocarp (Figures 4C, Idiospermum is sister to the rest of the fam- small in Calycanthus occidentalis (10.1- 4L), thick (Figure 4F). The parenchyma of [International Journal of Plant Biology 2018; 9:7525] [page 19] Article mesocarp regular shape in Ch. nitens, Ch. Sinocalycanthus. Endocarp was highly lig- layer in Sinocalycanthus chinensis (Figure fragrans, Ch. salicifolius, Ch. yunnanensis, nified and well developed in 4B). The cell shape was circular or sub-cir- and Ch. luteus but irregular shape in Ch. Calycanthaceae. cular (Figure 5E). The cell shape was irreg- praecox, Sinocalycanthus chinensis and Ca. Seed coat divided into three layers as ular, more than two layers of exotesta in Ch. occidentalis. The intercellular spaces devel- exotesta, mesotesta, and endotesta. fragrans (Figure 5B). Cell shape is circular oped in the region of mesocarp (Figures 4C, Mesotesta was irregular and thick, endotes- which is attached with mesotesta in Ch. 4F, 4I, 4L, 5C, 5F, 5I, 5L, 6L). The vascular ta and exotesta were polygonal or sub luteus. In Ch. salicifolius, the cell was bundle developed in mesocarp (Figures 6A- polygonal in Ch. salicifolius (Figure 5E). arranged with each other formation of sin- 6H, 6K). Mesocarp layers were thickest in Exotestal layer was single, circular or sub- gle layered with elongation cell shape Chimonanthus than that of Calycanthus and circular, which is connected the mesotestal (Figure 5E). Single layer was defined in Ch. Table 1. Collection information of genus and species used in present study. Taxa Collection information Calycanthus occidentalis HooK. & Arn. Korea. Cultivated at Kangwon University, K. Heo & N. Paudel s.n. 2016 (KWNU) Chimonanthus fragrans (Loisel.) Lind. Korea. Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 Chimonanthus luteus (G.Don) Biel. Korea. Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 (KWNU) Chimonanthus nitens (Oliv.) Rehder Korea. Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 Chimonanthus praecox (L.) Link Korea. Cultivated at Kangwon University, K. Heo & N. Paudel s.n. 2016 (KWNU) Chimonanthus salicifolius S.Y.Hu Korea. Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 Chimonanthus yunnanensis (W.W.Sm.) 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. Heoonly & N. Paudel s.n. 2016 (KWNU) Table 2. Seed morphology and measurement. Taxa Seed shape and size Seed character Cell shape Number Seed coat Tegmen use of layer width in µm Pericarp Seed coat Calycanthus occidentalis Elliptical: Relatively large, Relatively large, 4-5 3-4 124-128 2 layered Hook. & Arn. 10.1-11.45×5.2-6.65 concave, Hairy concave Chimonanthus fragrans Elliptical: Relatively large, Relatively large, 4-5 4-5 111-114 2 layered (Loisel.)Lind. 19.2-20×6.1-7.25 concave concave, Hairy Chimonanthus luteus Elliptical: Relatively large, Narrow, subpolygonal 4-5 4-5 120-124 2 layered (G.Don) Biel. 14.4-16.4×6.4-7.5 concave, Hairy to irregular Chimonanthus nitens Elliptical: Relatively large, Relatively large, 5-6 3-4 110-115 2 layered (Olive.) Rehder 10-11.35×5-6.1 concave, Hairy concave Chimonanthus praecox Elliptical: Relatively large, Relatively large, 2-3 3 124-129 2 layered (L.) Link 11.1-14×5-6.2-7.1 concave, Hairy concave Chimonanthus Elliptical: Relatively large, Narrow, sub polygonal 6-7 3 124-128 2 layered salicifolius S.Y.Hu 11.55-13.75×6.15-7.6 concave, Hairy to irregular Chimonanthus Elliptical: Relatively large, Narrow, polygonal 3-4 3 125-128 2 layered yunnanensis (W.W.Sm.)Hu 12.5-16.35×5.2-7.35 concave, Hairy to circular Sinocalycanthus Elliptical: Non-commercial Relatively large, Narrow, elongated 4-5 3-4 118-122 2 layered chinensis W.C.Cheng 12.3-14.35×7- 7.65 concave, Hairy to irregular & S.Y.