International Journal of Biology 2018; volume 9:7525

Pericarp, seed coat anatomy ily and is sister to the and seed morphology of -Sinocalycanthus.1,25 In Correspondence: Kweon Heo, Department of angiosperms, floral phyllotaxis is often Applied Plant Sciences, Kangwon National 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 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 (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 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 or trop- microscope (Olympus Co., Japan). carp layer cell shape structure. The seed sur- ical trees have relatively large , 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 . 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) (Oliv.) Rehder Korea. Cultivated in Chollipo Arboritum, K. Heo s.n. 2009 (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. Chang

Table 3. Trichome morphology and seed surface in Calycanthaceae. Taxa Trichome Surface and direction Seed surface Density Calycanthus occidentalis Hook. & Arn. Dense Smooth, Erect Rough, irregular Chimonanthus fragrans (Loisel.) Lind. Dense Smooth, Upward bending (30-45) degree to surface Slightly pentagonal Chimonanthus luteus (G.Don) Biel. Dense Smooth, Upward Rough, Irregular Chimonanthus nitens (Oliv.) Rehder Dense Smooth, Upward bending (45-60) degree to surface Rough, Irregular Chimonanthus praecox (L.) Link Dense Smooth, Erect Pentagonal Chimonanthus salcifolius S.Y.Hu Dense Smooth, Upward Smooth, pentagonal Shape Chimonanthus yunnanensis (W.W.Sm.) Hu Dense Smooth Rough, Irregular Sinocalycanthus chinensis W.C.Cheng & S.Y.Chang Moderate Smooth, Upward and parallel to surface Rough, Irregular

[page 20] [International Journal of Plant Biology 2018; 9:7525] Article nitens (Figure 4K). Seed surface was found pentagonal shape in systematic relationship among the species. Endotesta layer was 1, 2 or more con- Ch. praecox, Ch. nitens, and Ch. salicifolius Our result is also similar to the previous nected with each other that formed the inter- (Figures 2H, 2I, 3A, 3F). In case of finding.31,32 The primary vascular cylinder space gap (Figure 4B, 4E, 4H, 5K). Crystal Sinocalycanthus and Calycanthus occiden- of Calycanthaceae is notable for peculiar was observed all Species on Calycanthacae. talis, seed surface was rough and irregular. system of four-inverted cortical bundle. The Tegmen was two layers that formed stomata are rubiaceous. The trichome is bitegment. unicellular. We noted that the unicellular Trichome and seed surface of hair on the surface of which is the dis- Calycanthaceae has summarized (Table 3). Discussion tinguish character of Calycanthaceae. Non-glandular and unicellular trichomes Unicellular trichomes were variable in were representative characters in all species This study represents the most compres- length.6 Our results were also support den- of Calycanthaceae. The density of trichome sive investigation of trichome micromor- sity and size of the trichomes, which was was dense in Ca. occidentalis and phology, seed surface, and seed anatomy in important for for Chimonanthus species but moderate in Calycanthaceae. Our data confirm the struc- Calycanthaceae. The surface of seed and Sinocalycanthus chinensis. The trichome ture of seed coat and seed surface of differ- angle of direction of trichomes were also was erect and bending to the seed surface. ent genus. In addition, it is possible to the supported for the phylogeny (Table 3). Non-

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Figure 1. Seed morphology OF Calycanthaceae from stereo microscope. A, Sinocalycanthus chinensis; B, Calycanthus occidentalis; C, Chimonanthus praecox; D, Chimonanthus nitens ; E, Chimonanthus fragrans; F, Chimonanthus salicifolius; G, Chimonanthus yunna- nensis; H, Chimonanthus luteus (Without scale; A-H).

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Figure 2. Seed morphology of Calycanthaceae from SEM. A-C, Sinocalycanthus chinensis; D-F, Calycanthus occidentalis; G-I, Chimonanthus praecox; J-L, Chimonanthus nitens. (Trichome, E, H, K; Seed surface C, F, I, L).

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Figure 3. Seed morphology of Calycanthaceae from SEM. A-C, Chimonanthus fragrans; D-F, Chimonanthus saliicifolius; G-I, Chimonanthus yunnanensis; J-L Chimonanthus luteus. (Trichome, E, H, K; Seed surface C, F, I, L).

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Figure 4. Seed anatomy of Calycanthaceae. A-C, Sinocalycanthus chinensis; D-F, Calycanthus occidentalis; G-I, Chimonanthus praecox; J-L, Chimonanthus nitens. Abbreviation: end, endotesta; ext, exotesta; mst, mesotesta; exc; exocarp; enc, endocarp; cr, crystal.

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Figure 5. Seed anatomy of Calycanthaceae. A-C, Chimonanthus fragrans; D-F, Chimonanthus salicifolius; G-I, Chimonanthus yunna- nensis; J-L, Chimonanthus luteus. Abbreviation: ent, endotesta; ext, exotesta; mst, mesotesta; exc, exocarp; enc, endocarp; cr, crystal .

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Figure 6. Seed anatomy of Calycanthaceae. A, Sinocalycanthus chinensis; B and K, Calycanthus occidentalis; C, Chimonanthus praecox; D and L, Chimonanthus nitens; E, Chimonanthus fragrans; F and G, Chimonanthus salicifolius; H and I, Chimonanthus luteus; J, Chimonanthus yunnanensis. Abbreviation: Vb, vascular bundle; cr, crystal.

[page 26] [International Journal of Plant Biology 2018; 9:7525] Article glandular trichome is a little diagnostic fea- and thin than that of other. 11. Mai HM. Neue Arten nach Fru chten ture of the Calycanthaceae.33 Unicellular Our data show that trichome micromor- und Samen aus dem Tertiar von trichome is common in all species. The phology and seed surface are useful for the Nordwestsachsen und der Lausitz. density and position of the trichome play separation of species within characterizing Feddes Report 1987;98:105-26. importance role on systematic in of genus and species. There are only on reli- 12. Friis EM, Eklund H, Pedersen KR, Calycanthaceae. We found smooth and able of molecular phylogenetic known as Crane PR.Virginianthus calycanthoides erect trichome in all species. They are vary- the sister group of Laurales.6 One important genet sp. nov. - a calycanthaceous ing in their arrangement to the seed surface. result of the above-mentioned surface was from the Potomac Group (Early Variation of seed surface structure is anoth- rough in both Sinocalycanthus and Cretaceous) of eastern North America. er strong character for the Calycanthaceae. Calycanthus. Non-glandular trichome was Int J Plant Sci 1994;155:772-85. Pentagonal shape recorded in dense in Calycanthus occidentalis. In this 13. Herendeen PS, Magallon-Puebla S, Chimonanthus praecox, Chimonanthus fra- study, seed morphology and seed coat Lupia R, et al . A preliminary conspec- grans, and Chimonanthus salicifolius anatomy provide some useful information tus of the Allon flora from the Late (Figures 2L, 2C, 2F). Sinocalycanthus chi- in the genetic delimitation of Cretaceous (Late Santonian) of central nensis and Calycanthus occidentalis repre- Calycanthaceae. Number of pericarp layer, Georgia, USA. Ann Mo Bot Gard sented as rough and irregular shape (Figures number of seed coat layer, and shape of cell 1999;86:407-71. 2C, 2F). Chimonanthus nitens, were distinguished features. Nature of the 14. Mohr BA, Eklund H. Araripia florifera, Chimonanthus yunnanensis, and tegmen, seed shape and seed size are for a magnoliid angiosperm from the Chimonanthus luteus are irregular and species delineation. Presence of crystal in Lower Cretaceous Crato Formation rough (Figures 2L, 3I, 3L). all species has no value for the species (Brazil). Rev Palaeobot Palynol The seed of Chimonanthus fragrans is delimited. Trichome density and seed sur- 2003;126:279-92. largest. Seeds feature of the species in order face were great value for the genus level. 15. Crepet WL, Nixon KC, Gandolfo MA. to measurement of the seed size was the sig- An extinct calycanthoid taxon, nificant dissimilarities of the species. We Jerseyanthus calycanthoides, from the concluded genus Chimonanthus is quite onlyLate Cretaceous of New Jersey. Am J larger than that of Calycanthus, but References Botany 2005;92:1475-85. Sinocalycanthus is medium size. The mor- 1. Zhou S, Renner SS, Wen J. Molecular 16. Van Heel, WA .Variation in the develop- phological characters are for generic delam- phylogeny and intra-and intercontinen- ment of ascidiform carpels. Blumea ination in Calycanthaceae. There is differ- tal biogeography of Calycanthaceae. 1984;29:443-52. ence seed characters have significant for use Mol Phylogenet Evol 2006;39:1-15. 17. Cronquist, A. Outline of a new system phylogeny. The great consultancy of seed 2. Renner SS. Circumscription and phy- of families and orders of the dicotyle- coat feature has observed in logeny of the Laurales: evidence from dons. Bull Jard Bot Brux 1957; 27:13- Calycanthaceae. The all species of molecular and morphological data. Am 40. Calycanthus have specialized mesocarp J Botany 1999;86:1301-15. 18. Cronquist A. An Integrated System of feature. In the seed coat anatomy, all species 3. Renner SS. Variation in diversity Classification of Flowering . have thicker mesotestal layer. The vascular among Laurales, Early Cretaceous to Columbia University Press, New York bundle is in cortex, which is independent to present. Biologiske Skrifter/Kongelike 1981. the stellar system through the 19. Cronquist A. The Evolution and 34 Danske Videnskabernes Selskab Calycanthaceae. nd 2005;55:441-58. Classification of Flowering Plants 2 The exotesta was arranged ovate shape 4. Qiu YL, Dombrovska O, Lee J, et al. Ed. New York Botanical Garden, New in Chimonanthus praecox, Ch. salicifolius, Renner SS. Phylogenetic analyses of York 1988. and Ch. luteus. In case of Chimonanthus basal angiosperm based on nine plastid, 20. Takhtajan AL. Outline of the classifica- fragrans, Ch. nitens, and Ch. yunnanensis, mitrochondrial, and nuclear genes. Int J tion of flowering plants the seed shapes were elliptical shape. Plant Sci 2005;166:815-42. (Magnoliophyta). The Bot Rev 1980; Calycanthus occidentalis and 5. Cheng WC, Chang SY. Genus novum 46:225-359. Sinocalycanthus show the circular exotesta. Calycanthacearum chinae orientalis. 21. Thorne RF. A phylogenetic classifica- Exotesta is highly developedNon-commercial in the Acta Phytotax 1964;9:137-8. tion of the Annoniflorae. Aliso: A Chimonanthus than Sinocalycanthus and 6. Nicely KA. A monographic study of the Journal of Systematic and Evolutionary Calycanthus. Calycanthaceae. Castanea 1965; 38-81. Botany 1974; 8:147-209. 7. Grant B. The pollination of Calycanthus 22. Thorne RF. Classification and geogra- occidentalis. Am J Bot 1950;37:294-7. phy of the flowering plants. The Bot 8. Badrutt S. Struktur und Funktion der Rev 1992;58:225-327. Conclusions Blüten bei Calycanthaceae. Diploma 23. Throne RF. The classification and geog- Seed coat anatomy is different in thesis. University of Zurich; 1992. raphy of the flowering plants: Calycanthaceae. From the results, seed coat 9. Vogel S. Remarkable nectaries: struc- Dicotyledons of the class Angiospermae developed in the Chimonanthus than ture, ecology, organophyletic perspec- (subclasses Magnoliidae, Sinocalycanthus, and Calycanthus endotes- tives. II. Nectarioles. Flora 1998;193:1- Ranunculidae, Caryophyllidae, ta shows broad. 29. Dilleniidae, Rosidae, Asteridae,and The mesotesta is thicker than exotesta 10. Worboys SJ, Jackes BR. Pollination Lamiidae). Bot Rev 2000; 66:441-647. and endotesta in Sinocalycanthus and processes in Idiospermum australiense 24. Dahlgren R, Bremer K. Major clades of Calycanthus .In Calycanthus pericarp con- (Calycanthaceae), an arborescent basal the angiosperms. Cladistics tain hard comprise the stony cells. The peri- angiosperm of Australia’s tropical rain 1985;1:349-368. carp divided as the exocarp, mesocarp, and forests. Plant Syst Evol 2005;25:107- 25. Soltis DE, Solits PS, Chase MW, Mort endocarp. The exocarp is single layer cells 17. ME, Albach DC, Zanis Savolainen V,

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