- Email: [email protected]
Additional characters for taxonomic treatment on Chimonanthus praecox (L.) Link (Calycanthaceae)
Flora 249 (2018) 150–155
Contents lists available at ScienceDirect
Flora journal homepage: www.elsevier.com/locate/flora
Additional characters for taxonomic treatment on Chimonanthus praecox (L.) Link (Calycanthaceae) Niroj Paudel, Kweon Heo
T
⁎
Department of Applied Plant Science, Kangwon National University, Chuncheon, 24341, South Korea
A R T I C LE I N FO
A B S T R A C T
Edited by Alessio Papini
The detailed study of Chimonanthus praecox investigated to contribute in Calycanthaceae. Especially for the detail, characters are for further phylogeny as proper placement of the plant. The study reveals that presence of trichome, crystals in mesophyll, seed size, pericarp, seed coat, and ovule structure and pollen character are important for present study. Experiment conducted out from the resin method. Leaf, petiole, and flowers are used for the preparation of permanent slides. The valuable additional characters of Chimonanthus praecox are vascular bundles in the petiole, crystals presence in mesophyll, furrow like pollen grain, and U-shape vascular bundle. These anatomical characters are important for taxonomic treatments and phylogeny of Calycanthaceae.
Keywords: Anatomical character Mesophyll cell Ovule Petiole Resin method
1. Introduction Chimonanthus praecox belongs to the family Calycanthaceae which is sister of order Laurales (Cheng and Chang, 1964; Renner, 1999; Nicely, 1965; Staedler et al., 2007, 2009). It is also known as winter-sweet or Japanese allspice. Chimonanthus praecox is a deciduous shrub and native in China. Initially, the taxonomic figure was described as two species in Calycanthus floridus and Chimonanthus praecox in Calycanthaceae (Linnaeus, 1759). Complete description of phloem has given in Calycanthaceae (Cheadle and Esau, 1958). Calycanthus and Chimonanthus are differentiate between colony and chain structure on the base of sclerenchyma (Paudel and Heo, 2018). Unique characters in Laurales are for the gynoecium: ovule number and placentation differ from all other Laurales (Endress, 1983). Lindley (1819) considered that the C. praecox represents the new genus in Chimonanthus. Chimonanthus is commonly cultivated in Asia, North America, and Europe (Bygrav, 1996; Jelitto, 1971; Lasseigne et al., 2001; Ranney, 2004; Zhang and Liu, 1998). The genus is characterized the opposite leaves, numerous tepals, stamen and achene that closed concave receptacle (Cronquist, 1981; Dahlgren, 1983; Hailler, 1905). Comparative study of flower vessel is noted (Wilson, 1976). Aglycones quercetin and kaempferol were isolated and identified in Calycanthus occidentalis, Calycanthus floridus, and Chimonanthus sp. (Sterner and Young, 1981). The phylogeny of the Calycanthaceae supported from the molecular data (Chase et al., 1993; Renner, 2005; Soltis et al., 2000; Zhou et al., 2006). For cladistics study, Li and Li (2000a, b) is supported for relation of Chimonanthus and its chloroplast DNA restriction (Wen and Zimmer,
⁎
1996). The designation of Calycanthus and Chimonanthus is for nomenclature for conservation (Lanjouwa, 1961). The Chinese endemic Chimonanthus is the third genus of Calycanthaceae (Cheng and Chang, 1964). In detail, study of monograph was accepted only three species, C. nitens, C. praecox, and C. salicifolius (Nicely, 1965). In addition, Chinese workers are described six additional species, C. campanulatus from southeastern Yunnan, C. grammatus from Jiangxi and C. zhejiangensis from Zhejiang and Fujian (Liu, 1984). The classification of Chimonanthus is a subject of debate for a long time (Dai et al., 2012). C. anhuiensis (Chen et al., 1987) and some of these names are probably synonyms. For example, C. baokanensis has considered as C. praecox but other entities appear to be good species. Six species of Chimonanthus were recognized for that study. In this study, we aim to present the leaf morphology and anatomy, embryology, pollen and seed of C. praecox by observing the different characters of petiole, leaf, ovule, anther, pollen, and seeds. These were previously not reported. This information will help the comparative study of closely related species within the Chimonanthus and the taxonomic relationships among them. 2. Materials and methods Chimonanthus praecox was collected during the Kangwon National University (KNU); in 2016 and 2017. The collected leaf, petiole, flower, and seeds were fixed with the FAA (formalin: glacial acetic acid; 50% ethanol, 5:5:90, by volume). Collected samples passed through the
Corresponding author. E-mail address: [email protected] (K. Heo).
https://doi.org/10.1016/j.flora.2018.11.004 Received 16 May 2018; Received in revised form 12 October 2018; Accepted 2 November 2018 Available online 03 November 2018 0367-2530/ © 2018 Elsevier GmbH. All rights reserved.
Flora 249 (2018) 150–155
N. Paudel, K. Heo
alcohol series. After complete dehydration, the samples passed through combination of alcohol/Technovit and then embedded in Technovit 7100 resin. Serial sections were cut 5–6 μm thickness using disposable knives and stuck into the glass slides, and dried on electrical hot plate for 24 h. Dried slides were stained with 0.1% Toluidine blue O for 60–90 second, ringed with running water, and again dried on the electrical hot plate for more than 6 h to remove water. The stained slides were mounted with Entellan (Merck Co., Germany). Mounted permanent slides were observed under the Olympus BX-50 light microscope (Olympus Co.,
Table 1 Morphological characteristic features of Chimonanthus praecox (L.) Link. Characters
Description
Habitat Root Stem Leaf Flower Fruit Seed
Originally from China, perennial shrub, deciduous Main and lateral roots Erect, quadrangular in young stem, circular in old stem, brownish Light green, leaf blade elliptic to broadly elliptic, petiolate Purplish red pigment at the base, yellow tepals Pseudo fruit Number of seed 10-15, brown, elliptical shape
Fig. 1. Transverse section (TS) of petiole: A, TS of petiole shows in detail; B, TS of petiole shows the U-shape vascular bundle; C, Sclerenchyma; D, Detail structure of ventral vascular bundle; E, Collenchyma; F, Magnified xylem. 151
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Fig. 2. Transverse section (TS) of leaf: A, TS of leaf mid rib ; B, TS of leaf veinlet; C, TS of leaf; D, Xylem; E, Collenchyma and upper epidermis; F, Collenchyma and lower epidermis; G, Sclerenchyma; H, Stomata and epidermal layer; I, Unicellular trichome. (Abbreviation; UE: Upper epidermis, LE: Lower epidermis: MC: Mesophyll cell, CR: Crystals).
Transverse sections (TS) of leaf in main vein and lateral vein are distinguished features (Fig. 2A, B). TS of the leaf shows the unique character, crystals in mesophyll cell. The mesophyll cells have 2–3 layers of palisade cells loosely bind with intercellular space (Fig. 2C). Upper epidermis has a single layer, which is slightly thicker than that of the lower epidermis (Fig. 2C). Sclerenchyma tissues were connected with other downward edges, which is the long chain with 6–7 layers of cell (Fig. 2G). In upper edge, there is large cells with intercellular space for the collenchyma cells (Fig. 2E). Lower epidermis represents the small circular collenchyma tissue with loosely presented in intercellular space (Fig. 2F). Many proto-xylem and meta-xylem vessels are characters in Chimonanthus praecox (Figs. 1F, 2 D). Stomata are of the paracytic type (Fig. 2H). Unicellular trichomes are distributed in the vein region (Fig. 2I).
Japan) For the study of stomata, leaves were peeled out the epidermal layer then stained with 0.1% Toluidine blue O. The multiple image alignment was done by using Photoshop CS6. 3. Results 3.1. Leaf morphology Chimonanthus praecox is variable in leaf shape and size. The leaves are typically petiolate and elliptical to broadly elliptic. The length of the petiole is 0.5–2.0 cm (Table 1). They are persistent during the time of flowering. 3.2. Anatomy of petiole and leaf
3.3. Anther and ovule Transverse section of petiole shows the three bundles, of which one is dorsal whereas the other two are ventral bundles (Fig. 1A). The vascular bundle is well-managed xylem and phloem tissue (Fig. 1B). Sclerenchyma cells are scattered in the outside of vascular bundles (Fig. 1C). The vascular bundles are surrounded the collenchyma tissues which are loosely bind, ovoid shape, somewhat in circular shape (Fig. 1D). Upper epidermis is a single layer of cells in the petiole. There is circular and ovoid shape with intercellular space of collenchyma tissues (Fig. 1E).
Flowers are opposite and tepals with yellow color. Stamens attached with the base and base gynoecium i.e. tetra-sporangia, basified, and scattered anther with anther sac as numerous pollen grains (Fig. 3A). Transverse section of anther wall is represented the epidermis, endothecium, and degenerated middle layers (Fig. 3B). Pollen grains are the monosulcate and large grain, which is measuring the 50–55 μm (Fig. 3C). The ovule is bitegmic and anatropous (Fig. 3D–G). The inner carpels 152
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Fig. 3. Anther, pollen, ovule, young seed, seed coat, and pericarp: A, Tetra-sporangia; B, Anther wall; C, Pollen grain; D–E, Longitudinal section of ovule; F–G, TS of ovule; H, TS of young seed; I, TS of pericarp and seed coat in young stage; J, TS of seed coat and embryo in mature stage; K, TS of mature pericarp. (Abbreviation; COT: Cotyledon; EP: Epidermis; ET: Endothecium; EXT: Exotesta; MST: Mesotesta; ENT: Endotesta; EXC: Exocarp; ENC: Endocarp; TEG: Tegmen).
the mature pseudo-fruit. Seed size was 10.1–11.45 × 7.8-7.72 mm in C. praecox. The pericarp differentiated into the exocarp, mesocarp, and endocarp, which develops from the outer epidermis, mesophyll and inner epidermis of the ovary (Fig. 3I, K). The exocarp is one layer in mature fruit (Fig. 3I, K). Exocarp from the intercellular space due to the loss of protoplast but in young fruit, it is single layer. In the young fruit stage, the exocarp is single layer (Fig. 3H, I). The entire mature seed coat was represented testa and tegmen (Table 2). Ovule was bitegmic,
are the long, which is the part of ovary length. The carpels is usually contain 3–4 ovules (Fig. 3D, F). The outer integument is originated from the base of the lower ovule (Fig. 3D). The carpel is located in the central of the gynoecium. 3.4. Pericarp and seed coat Pseudo-carp, hairy achene, seed development were 10–12 fruits in 153
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Table 2 Anatomical characteristic features of Chimonanthus praecox (L.) Link. Characters
Description
Petiole Leaf Pericarp Seed coat
Number of vascular bundle is three, collenchyma is circular, vessels are hexagonal structure Presence of crystals in mesophyll cell, sclerenchyma cell is circular compact adaxial side Endocarp developed into palisade sclerenchyma cells Bitegmic and one layer of exotesta, several layers of mesotesta, one layer of endotesta, and two layers of tegmen
bitegmic/unitegmic, crassinucellate/tenuinucellate ovules, nuclear/ cellular endosperm development, large/small seed size, woody/herbaceous habit and tropical/temperate. The pseudocarp in Chimonanthus is garnish bright with vary diameter (Nicely, 1965). Initially, pseudocarp was considered as possible taxonomic character, but the observation in Calycanthus floridus varies due to the abortion of achenes. The presence of two sterile carpels, bisporic Allium type embryo sac, single-seeded fruit, palisade endocarp, and compressed exotesta were considered as possible taxonomic characters to compare with species (Ghimire et al., 2018). The ovule is bitegmic and anatropous (Staedler et al., 2009). In most angiosperms, the carpels become close before the ovules are visible from the outside of the still incompletely closed carpels (Endress, 2015). The micropyle is an architectural necessity for the closure of the originally circular opening (Endress and Igersheim, 1997, 2000). We present here additional characters for the C. praecox. They are the unicellular trichome hair originated from the vein; the collenchyma tissue that surrounds the vascular bundle, which is loosely bind ovoid shape somewhat in circular. Therefore, we concluded that the presence of vascular bundles in petiole, crystals present in mesophyll of leaf, Ushaped vascular bundle, and furrow-like pollen grains are the additional taxonomic characters for C. praecox. These anatomical characters give us good understanding for their taxonomy and phylogeny.
and testal cells divided into exotesta, mesotesta, and endotesta (Fig. 3J). Testal cells are not distinguished in young stage (Fig. 3I). Exotestal cells were characterized as ovate shape (Fig. 3J). Tegmen remains into thin two layers (Fig. 3J).
4. Discussion The leaf trace pattern and nodal anatomy of Calycanthaceae and Idiospermaceae (one-trace and unilacunar) are very similar (Wilson, 1976). In our result, the leaf anatomy is completely developed xylem and phloem compact form. Many researchers focused on their description of the plant for molecular phylogeny in Laurales with molecular data (Renner, 1999; Zhou et al., 2006). However, Liu et al. (2014) concluded the transcriptomic analysis present in the development of the pathway for the Chimonanthus praecox. The floral phyllotaxis and architecture are represented in Calycanthaceae (Staedler et al., 2007, 2009). First report is cultivated varieties of C. praecox from the genetic improvement, identification and germplasm (Zhou et al., 2006; Li and Li, 1999). Calycanthaceae is originated from Qinling Mountain to the East Hengduan Mountain (Smith, 1928). A transection of the internodal region of Calycanthus and Idiospermum shows that the vascular bundle is organized in what has been termed a pseudo-siphono stele (Bailey and Nast, 1948). It appears as a continuous ring of primary xylem and phloem. In our results, however, the primary xylem and phloem for C. praecox arranged compactly. The air cavity is within the mesophyll cells. Structure of xylem and phloem, crystals in mesophyll tissue, and presence of cuticle layer are relevant characters for the phylogeny. Chimonanthus is more primitive than other genera of Calycanthaceae (Liu, 2000b). The Calycanthaceae are somewhat unique in woody Ranales, since the seed contains a large embryo and no endosperm at maturity (Eames, 1961). The petiole possess bundle which is the three to five enter the blade (Nicely, 1965). We observed three bundles in petiole with one dorsal and two ventral bundles. They occur throughout the angiosperms (Cheadle and Esau, 1958). The vascular bundles between Chimonanthus and Calycanthus are different in shape and size (Metacalfe and Chalk, 1950). Smith (1928) has described the floral anatomy of this genus. He noted that the species of Chimonanthus are more or less similar due to observation of vascular bundle trace in perianth. Perianth of Chimonanthus is three main trace entering the basal portion. Stamens are arranged in cup like receptacles. The vascular anatomy of the carpels and ovule is described. According to his results, two anatropous ovules are present in the carpels (Schaeppi, 1953). We noted more than three anatropous ovules in C. praecox. Gynoecium structure is composed of four types according to the mode of carpel closure: secretion, without postgenital fusion; with a continuous secretory canal but partial postgenital fusion; with an incomplete secretory canal and complete postgenital fusion; and by complete postgenital fusion in periphery (Endress and Igersheim, 2000). Grant (1950) observed that the pollination in Calycanthus occidentalis is carried out by the beetle (Colopterus truncates). Nicely (1965) also observed the Carpophilus lugubris of Nitidulidae. The possible evolutionary status of the endothelium, hypostase, pachychalaza and the recalcitrant viability behavior of seed is related to
Acknowledgement This study was supported by 2017 research grant from Kangwon National University (No. 520170190). References Bailey, I.W., Nast, C.G., 1948. Morphology and relationships of Illicium, Schisandra and Kadsura. I. Stem and leaf. J. Arnold Arbor. 29, 77–89. Bygrav, P., 1996. Allspices and winter-sweets: an account of Calycanthaceae in the wild and cultivation. New Plantsman. 3, 40–49. Cheadle, V.I., Esau, K., 1958. Secondary phloem of Calycanthaceae. Univ. Calif. Publ. Bot. 29 (397-), 510. Chen, Z.X., Ding, B.Z., Chao, T.B., Song, L.G., Li, Z.Q., Liu, C.Y., Yu, S.Z., Ren, S.L., Zhou, F.M., Jiao, S.D., 1987. Study on plant resource of Chimonanthus Lindl. Henan. Acta Agric. Univ. Henan. 21, 413–426. Chase, M.W., Soltis, D.E., Olmsted, R.G., Morgan, D., Les, D.H., Mishler, B.D., Duvall, M.R., 1993. Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Mo. Bot. Gard. 80, 528–580. Cheng, W.C., Chang, S.Y., 1964. Genus novum Calycanthacearum chinaeorientalis. Acta Phytotax Sin. 9, 137–138. Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York. Dahlgren, R., 1983. General aspects of angiosperm evolution and macro systematics. Nord. J. Bot. 3, 119–149. Dai, P.F., Yang, J., Zhou, T.H., Huang, Z.H., Feng, L., Su, H.L., Liu, Z.L., Zhao, G.F., 2012. Genetic diversity and differentiation in Chimonanthus praecox and Ch. salicifolius (Calycanthaceae) as revealed by inter-simple sequence repeat (ISSR) markers. Biochem. Syst. Ecol. 44, 149–156. Eames, A.J. (Ed.), 1961. Morphology of the Angiosperms. McGraw-Hill. Endress, P.K., 1983. Dispersal and distribution in some small archaic relic angiosperm families (Austrobaileyaceae, Eupomatiaceae, Himantandraceae, Idiospermoideae, Calycanthaceae). Sundered. Naturists ver. Hamburg 7, 201–217. Endress, P.K., 2015. Patterns of angiospermy development before carpel sealing across living angiosperms: diversity, and morphological and systematic aspects. Bot. J. Linn. Soc. 178, 556–591. Endress, P.K., Igersheim, A., 1997. Gynoecium diversity and systematics of the Laurales. Bot. J. Linn. Soc. 125, 93–168. Endress, P.K., Igersheim, A., 2000. Gynoecium structure and evolution in basal angiosperms. Int. J. Plant Sci. 161, S211–S223.
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Ranney, T.G., 2004. Reinventing sweet shrubs. Proc. Int. Plant Propagators’ Soc. 53. Renner, S.S., 1999. Circumscription and phylogeny of the Laurales: evidence from molecular and morphological data. Am. J. Bot. 86, 1301–1315. Renner, S.S., 2005. Variation in diversity among Laurales, early cretaceous to present. Biologiske Skrifter/ Kongelike Danske Videnskabernes Selskab. 55, 441–458. Schaeppi, H., 1953. Morphologische Untersuchungen an den Karpellen der Calycanthaceae. Phytomorphology 3, 112–118. Smith, G.H., 1928. Vascular anatomy of Ranalian flowers II. Ranunculaceae (Cont.), Menispermaceae, Calycanthaceae. Annonaceae. Bot. Gaz. 85, 152–177. Staedler, Y.M., Weston, P.H., Endress, P.K., 2007. Floral phyllotaxis and floral architecture in Calycanthaceae (Laurales). Int. J. Plant Sci. 168, 285–306. Staedler, Y.M., Weston, P.H., Endress, P.K., 2009. Comparative gynoecium structure and development in Calycanthaceae (Laurales). Int. J. Plant Sci. 170, 21–41. Sterner, R.W., Young, D.A., 1981. Flavonoid chemistry and the phylogenetic relationships of the Idiospermaceae. Syst. Bot. 5, 432–437. Soltis, D.E., Soltis, P.S., Chase, M.W., Mort, M.W., Albach, D.C., Zanis, Mj., Savolainen, V., 2000. Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB sequences. Bot. J. Linn. Soc. 133, 381–461. Wen, J., Zimmer, E.A., 1996. Phylogeny an, d biogeography of Panax L. (the ginseng genus Araliaceae): inference from ITS sequences of nuclear ribosomal DNA. Mol. Phylogen. Evol. 6, 167–177. Wilson, C.L., 1976. Floral anatomy of Idiospermum australiense (Idiospermaceae). Am. J. Bot. 63, 987–996. Zhang, R.H., Liu, H. (Eds.), 1998. Wax Shrubs in the World (Calycanthaceae). China Science & Technology Press, Beijing. Zhou, S., Renner, S.S., Wen, J., 2006. Molecular phylogeny and intra- and intercontinental biogeography of Calycanthaceae. Pers. - Int. Mycol. J. 1, 1–15.
Ghimire, B., Suh, G.U., Lee, C.H., Heo, K., Jeong, M.J., 2018. Embryological studies on Abelia tyaihyoni Nakai (Caprifoliaceae). Flora 242, 79–88. Grant, V., 1950. The pollination of Calycanthus occidentalis. Am. J. Bot. 37 294–29. Hailler, H., 1905. Ein zweiter entwurf des natürlichen (phylogenetischen systems der Blütenp Xanzen. Bot. Acta 23, 85–91. Jelitto, C.R., 1971. Calycanthus floridus, der Gewurzstrauch. Palmengarten 35, 91–92. Lasseigne, F.T., Fantz, P.R., Raulston, J.C., Straley, G.B., 2001. X Sinocaly-calycanthus raulstonii (Calycanthaceae): a new intergeneric hybrid between Sinocalycanthus chinensis and Calycanthus floridus. J. Am. Soc. Hortic. Sci. 36, 765–767. Lanjouwa, J., 1961. International Code of Botanical Nomenclature. Utrecht, Netherlands. . Lindley, J., 1819. Edwards’ Bot. Reg 5, 404. Linnaeus, C., 1759. Syst. Nat. ed. 10, 1066. Li, Y., Li, P.T., 1999. Epidermal features of the leaves of Calycanthaceae. J. Trop. Subtrop. Bot. 7, 202–206. Li, Y., Li, P.T., 2000a. Cladistics analysis of Calycanthaceae. J. Trop. Sub-trop. Bot. 8, 275–278. Li, Y., Li, P.T., 2000b. Origin, evolution and distribution of the Calycanthaceae. Guihaia 20, 295–300. Liu, D., Sui, S., Ma, J., Li, Z., Guo, Y., Luo, D., Li, M., 2014. Transcriptomic analysis of flower development in wintersweet (Chimonanthus praecox). PLoS One 9, e86976. Liu, M.C., 1984. Study of the genus Chimonanthus. J. Nanjing Inst. For. (China) 2, 78–82. Metacalfe, C.R., Chalk, L., 1950. Anatomy of the Dicotyledons 1. Clarendon Press, Oxford, pp. 13–16. Nicely, K.A., 1965. A monographic study of the Calycanthaceae. Castanea 30, 38–81. Paudel, N., Heo, K., 2018. Comparative stem anatomy of four taxa of Calycanthaceae Lindl. Eur. J. Biol. Res. 15, 34–41.
155
Contents lists available at ScienceDirect
Flora journal homepage: www.elsevier.com/locate/flora
Additional characters for taxonomic treatment on Chimonanthus praecox (L.) Link (Calycanthaceae) Niroj Paudel, Kweon Heo
T
⁎
Department of Applied Plant Science, Kangwon National University, Chuncheon, 24341, South Korea
A R T I C LE I N FO
A B S T R A C T
Edited by Alessio Papini
The detailed study of Chimonanthus praecox investigated to contribute in Calycanthaceae. Especially for the detail, characters are for further phylogeny as proper placement of the plant. The study reveals that presence of trichome, crystals in mesophyll, seed size, pericarp, seed coat, and ovule structure and pollen character are important for present study. Experiment conducted out from the resin method. Leaf, petiole, and flowers are used for the preparation of permanent slides. The valuable additional characters of Chimonanthus praecox are vascular bundles in the petiole, crystals presence in mesophyll, furrow like pollen grain, and U-shape vascular bundle. These anatomical characters are important for taxonomic treatments and phylogeny of Calycanthaceae.
Keywords: Anatomical character Mesophyll cell Ovule Petiole Resin method
1. Introduction Chimonanthus praecox belongs to the family Calycanthaceae which is sister of order Laurales (Cheng and Chang, 1964; Renner, 1999; Nicely, 1965; Staedler et al., 2007, 2009). It is also known as winter-sweet or Japanese allspice. Chimonanthus praecox is a deciduous shrub and native in China. Initially, the taxonomic figure was described as two species in Calycanthus floridus and Chimonanthus praecox in Calycanthaceae (Linnaeus, 1759). Complete description of phloem has given in Calycanthaceae (Cheadle and Esau, 1958). Calycanthus and Chimonanthus are differentiate between colony and chain structure on the base of sclerenchyma (Paudel and Heo, 2018). Unique characters in Laurales are for the gynoecium: ovule number and placentation differ from all other Laurales (Endress, 1983). Lindley (1819) considered that the C. praecox represents the new genus in Chimonanthus. Chimonanthus is commonly cultivated in Asia, North America, and Europe (Bygrav, 1996; Jelitto, 1971; Lasseigne et al., 2001; Ranney, 2004; Zhang and Liu, 1998). The genus is characterized the opposite leaves, numerous tepals, stamen and achene that closed concave receptacle (Cronquist, 1981; Dahlgren, 1983; Hailler, 1905). Comparative study of flower vessel is noted (Wilson, 1976). Aglycones quercetin and kaempferol were isolated and identified in Calycanthus occidentalis, Calycanthus floridus, and Chimonanthus sp. (Sterner and Young, 1981). The phylogeny of the Calycanthaceae supported from the molecular data (Chase et al., 1993; Renner, 2005; Soltis et al., 2000; Zhou et al., 2006). For cladistics study, Li and Li (2000a, b) is supported for relation of Chimonanthus and its chloroplast DNA restriction (Wen and Zimmer,
⁎
1996). The designation of Calycanthus and Chimonanthus is for nomenclature for conservation (Lanjouwa, 1961). The Chinese endemic Chimonanthus is the third genus of Calycanthaceae (Cheng and Chang, 1964). In detail, study of monograph was accepted only three species, C. nitens, C. praecox, and C. salicifolius (Nicely, 1965). In addition, Chinese workers are described six additional species, C. campanulatus from southeastern Yunnan, C. grammatus from Jiangxi and C. zhejiangensis from Zhejiang and Fujian (Liu, 1984). The classification of Chimonanthus is a subject of debate for a long time (Dai et al., 2012). C. anhuiensis (Chen et al., 1987) and some of these names are probably synonyms. For example, C. baokanensis has considered as C. praecox but other entities appear to be good species. Six species of Chimonanthus were recognized for that study. In this study, we aim to present the leaf morphology and anatomy, embryology, pollen and seed of C. praecox by observing the different characters of petiole, leaf, ovule, anther, pollen, and seeds. These were previously not reported. This information will help the comparative study of closely related species within the Chimonanthus and the taxonomic relationships among them. 2. Materials and methods Chimonanthus praecox was collected during the Kangwon National University (KNU); in 2016 and 2017. The collected leaf, petiole, flower, and seeds were fixed with the FAA (formalin: glacial acetic acid; 50% ethanol, 5:5:90, by volume). Collected samples passed through the
Corresponding author. E-mail address: [email protected] (K. Heo).
https://doi.org/10.1016/j.flora.2018.11.004 Received 16 May 2018; Received in revised form 12 October 2018; Accepted 2 November 2018 Available online 03 November 2018 0367-2530/ © 2018 Elsevier GmbH. All rights reserved.
Flora 249 (2018) 150–155
N. Paudel, K. Heo
alcohol series. After complete dehydration, the samples passed through combination of alcohol/Technovit and then embedded in Technovit 7100 resin. Serial sections were cut 5–6 μm thickness using disposable knives and stuck into the glass slides, and dried on electrical hot plate for 24 h. Dried slides were stained with 0.1% Toluidine blue O for 60–90 second, ringed with running water, and again dried on the electrical hot plate for more than 6 h to remove water. The stained slides were mounted with Entellan (Merck Co., Germany). Mounted permanent slides were observed under the Olympus BX-50 light microscope (Olympus Co.,
Table 1 Morphological characteristic features of Chimonanthus praecox (L.) Link. Characters
Description
Habitat Root Stem Leaf Flower Fruit Seed
Originally from China, perennial shrub, deciduous Main and lateral roots Erect, quadrangular in young stem, circular in old stem, brownish Light green, leaf blade elliptic to broadly elliptic, petiolate Purplish red pigment at the base, yellow tepals Pseudo fruit Number of seed 10-15, brown, elliptical shape
Fig. 1. Transverse section (TS) of petiole: A, TS of petiole shows in detail; B, TS of petiole shows the U-shape vascular bundle; C, Sclerenchyma; D, Detail structure of ventral vascular bundle; E, Collenchyma; F, Magnified xylem. 151
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Fig. 2. Transverse section (TS) of leaf: A, TS of leaf mid rib ; B, TS of leaf veinlet; C, TS of leaf; D, Xylem; E, Collenchyma and upper epidermis; F, Collenchyma and lower epidermis; G, Sclerenchyma; H, Stomata and epidermal layer; I, Unicellular trichome. (Abbreviation; UE: Upper epidermis, LE: Lower epidermis: MC: Mesophyll cell, CR: Crystals).
Transverse sections (TS) of leaf in main vein and lateral vein are distinguished features (Fig. 2A, B). TS of the leaf shows the unique character, crystals in mesophyll cell. The mesophyll cells have 2–3 layers of palisade cells loosely bind with intercellular space (Fig. 2C). Upper epidermis has a single layer, which is slightly thicker than that of the lower epidermis (Fig. 2C). Sclerenchyma tissues were connected with other downward edges, which is the long chain with 6–7 layers of cell (Fig. 2G). In upper edge, there is large cells with intercellular space for the collenchyma cells (Fig. 2E). Lower epidermis represents the small circular collenchyma tissue with loosely presented in intercellular space (Fig. 2F). Many proto-xylem and meta-xylem vessels are characters in Chimonanthus praecox (Figs. 1F, 2 D). Stomata are of the paracytic type (Fig. 2H). Unicellular trichomes are distributed in the vein region (Fig. 2I).
Japan) For the study of stomata, leaves were peeled out the epidermal layer then stained with 0.1% Toluidine blue O. The multiple image alignment was done by using Photoshop CS6. 3. Results 3.1. Leaf morphology Chimonanthus praecox is variable in leaf shape and size. The leaves are typically petiolate and elliptical to broadly elliptic. The length of the petiole is 0.5–2.0 cm (Table 1). They are persistent during the time of flowering. 3.2. Anatomy of petiole and leaf
3.3. Anther and ovule Transverse section of petiole shows the three bundles, of which one is dorsal whereas the other two are ventral bundles (Fig. 1A). The vascular bundle is well-managed xylem and phloem tissue (Fig. 1B). Sclerenchyma cells are scattered in the outside of vascular bundles (Fig. 1C). The vascular bundles are surrounded the collenchyma tissues which are loosely bind, ovoid shape, somewhat in circular shape (Fig. 1D). Upper epidermis is a single layer of cells in the petiole. There is circular and ovoid shape with intercellular space of collenchyma tissues (Fig. 1E).
Flowers are opposite and tepals with yellow color. Stamens attached with the base and base gynoecium i.e. tetra-sporangia, basified, and scattered anther with anther sac as numerous pollen grains (Fig. 3A). Transverse section of anther wall is represented the epidermis, endothecium, and degenerated middle layers (Fig. 3B). Pollen grains are the monosulcate and large grain, which is measuring the 50–55 μm (Fig. 3C). The ovule is bitegmic and anatropous (Fig. 3D–G). The inner carpels 152
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Fig. 3. Anther, pollen, ovule, young seed, seed coat, and pericarp: A, Tetra-sporangia; B, Anther wall; C, Pollen grain; D–E, Longitudinal section of ovule; F–G, TS of ovule; H, TS of young seed; I, TS of pericarp and seed coat in young stage; J, TS of seed coat and embryo in mature stage; K, TS of mature pericarp. (Abbreviation; COT: Cotyledon; EP: Epidermis; ET: Endothecium; EXT: Exotesta; MST: Mesotesta; ENT: Endotesta; EXC: Exocarp; ENC: Endocarp; TEG: Tegmen).
the mature pseudo-fruit. Seed size was 10.1–11.45 × 7.8-7.72 mm in C. praecox. The pericarp differentiated into the exocarp, mesocarp, and endocarp, which develops from the outer epidermis, mesophyll and inner epidermis of the ovary (Fig. 3I, K). The exocarp is one layer in mature fruit (Fig. 3I, K). Exocarp from the intercellular space due to the loss of protoplast but in young fruit, it is single layer. In the young fruit stage, the exocarp is single layer (Fig. 3H, I). The entire mature seed coat was represented testa and tegmen (Table 2). Ovule was bitegmic,
are the long, which is the part of ovary length. The carpels is usually contain 3–4 ovules (Fig. 3D, F). The outer integument is originated from the base of the lower ovule (Fig. 3D). The carpel is located in the central of the gynoecium. 3.4. Pericarp and seed coat Pseudo-carp, hairy achene, seed development were 10–12 fruits in 153
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Table 2 Anatomical characteristic features of Chimonanthus praecox (L.) Link. Characters
Description
Petiole Leaf Pericarp Seed coat
Number of vascular bundle is three, collenchyma is circular, vessels are hexagonal structure Presence of crystals in mesophyll cell, sclerenchyma cell is circular compact adaxial side Endocarp developed into palisade sclerenchyma cells Bitegmic and one layer of exotesta, several layers of mesotesta, one layer of endotesta, and two layers of tegmen
bitegmic/unitegmic, crassinucellate/tenuinucellate ovules, nuclear/ cellular endosperm development, large/small seed size, woody/herbaceous habit and tropical/temperate. The pseudocarp in Chimonanthus is garnish bright with vary diameter (Nicely, 1965). Initially, pseudocarp was considered as possible taxonomic character, but the observation in Calycanthus floridus varies due to the abortion of achenes. The presence of two sterile carpels, bisporic Allium type embryo sac, single-seeded fruit, palisade endocarp, and compressed exotesta were considered as possible taxonomic characters to compare with species (Ghimire et al., 2018). The ovule is bitegmic and anatropous (Staedler et al., 2009). In most angiosperms, the carpels become close before the ovules are visible from the outside of the still incompletely closed carpels (Endress, 2015). The micropyle is an architectural necessity for the closure of the originally circular opening (Endress and Igersheim, 1997, 2000). We present here additional characters for the C. praecox. They are the unicellular trichome hair originated from the vein; the collenchyma tissue that surrounds the vascular bundle, which is loosely bind ovoid shape somewhat in circular. Therefore, we concluded that the presence of vascular bundles in petiole, crystals present in mesophyll of leaf, Ushaped vascular bundle, and furrow-like pollen grains are the additional taxonomic characters for C. praecox. These anatomical characters give us good understanding for their taxonomy and phylogeny.
and testal cells divided into exotesta, mesotesta, and endotesta (Fig. 3J). Testal cells are not distinguished in young stage (Fig. 3I). Exotestal cells were characterized as ovate shape (Fig. 3J). Tegmen remains into thin two layers (Fig. 3J).
4. Discussion The leaf trace pattern and nodal anatomy of Calycanthaceae and Idiospermaceae (one-trace and unilacunar) are very similar (Wilson, 1976). In our result, the leaf anatomy is completely developed xylem and phloem compact form. Many researchers focused on their description of the plant for molecular phylogeny in Laurales with molecular data (Renner, 1999; Zhou et al., 2006). However, Liu et al. (2014) concluded the transcriptomic analysis present in the development of the pathway for the Chimonanthus praecox. The floral phyllotaxis and architecture are represented in Calycanthaceae (Staedler et al., 2007, 2009). First report is cultivated varieties of C. praecox from the genetic improvement, identification and germplasm (Zhou et al., 2006; Li and Li, 1999). Calycanthaceae is originated from Qinling Mountain to the East Hengduan Mountain (Smith, 1928). A transection of the internodal region of Calycanthus and Idiospermum shows that the vascular bundle is organized in what has been termed a pseudo-siphono stele (Bailey and Nast, 1948). It appears as a continuous ring of primary xylem and phloem. In our results, however, the primary xylem and phloem for C. praecox arranged compactly. The air cavity is within the mesophyll cells. Structure of xylem and phloem, crystals in mesophyll tissue, and presence of cuticle layer are relevant characters for the phylogeny. Chimonanthus is more primitive than other genera of Calycanthaceae (Liu, 2000b). The Calycanthaceae are somewhat unique in woody Ranales, since the seed contains a large embryo and no endosperm at maturity (Eames, 1961). The petiole possess bundle which is the three to five enter the blade (Nicely, 1965). We observed three bundles in petiole with one dorsal and two ventral bundles. They occur throughout the angiosperms (Cheadle and Esau, 1958). The vascular bundles between Chimonanthus and Calycanthus are different in shape and size (Metacalfe and Chalk, 1950). Smith (1928) has described the floral anatomy of this genus. He noted that the species of Chimonanthus are more or less similar due to observation of vascular bundle trace in perianth. Perianth of Chimonanthus is three main trace entering the basal portion. Stamens are arranged in cup like receptacles. The vascular anatomy of the carpels and ovule is described. According to his results, two anatropous ovules are present in the carpels (Schaeppi, 1953). We noted more than three anatropous ovules in C. praecox. Gynoecium structure is composed of four types according to the mode of carpel closure: secretion, without postgenital fusion; with a continuous secretory canal but partial postgenital fusion; with an incomplete secretory canal and complete postgenital fusion; and by complete postgenital fusion in periphery (Endress and Igersheim, 2000). Grant (1950) observed that the pollination in Calycanthus occidentalis is carried out by the beetle (Colopterus truncates). Nicely (1965) also observed the Carpophilus lugubris of Nitidulidae. The possible evolutionary status of the endothelium, hypostase, pachychalaza and the recalcitrant viability behavior of seed is related to
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