Pericarp , seed coat anatomy and seed morphology of Calycanthaceae

Pericarp, trichome, and seed coat anatomy display great features of taxonomic value in the Calycanthaceae. The present study about trichome and seed coat anatomy has based on external and internal observation. Detail anatomical study of seeds provides valuable information for further study about their function, ontogeny, and phylogeny. Therefore, the goal of the study is to investigate trichome morphology and seed coat anatomy in Calycanthaceae to provide more detail characterization. Seeds were collected after that preserved with FAA. Furthermore, alcohol series applied for SEM and light microscopy. The unicellular trichome morphology is common in all species in Calycanthaceae. Density of trichome is highest in Calycanthus occidentalis. Different variation of seed coat and pericarp layers are characteristics of potential phylogenetic significance in the family.


Introduction
2][13][14][15] Some of these fossils are extremely well preserved. 15Van Heel 16 studied carpel development.Within Calycanthaceae, gynoecium character is unique to Idiospermum. 10Early studies recognized the affinity of Calycanthaceae to Monimiaceae and Lauraceae. 16][19][20][21][22][23][24] Molecular phylogenetic analyses settled the family as sister to the rest of Laurales. 2- 4,25,26These analyses also divided the remaining Laurales into two clades of three families each: the Siparunaceae-Atherospermataceae-Gomortegaceae clade 3 and the Monimiaceae-Hernandiaceae-Lauraceae clade. 3Within Calycanthaceae, Idiospermum is sister to the rest of the fam-ily and Chimonanthus is sister to the Calycanthus-Sinocalycanthus. 1,25n angiosperms, floral phyllotaxis is often variability of various taxonomic feature. 27ynoecium is unique to Idiospermum presence of only one carpel, rarely up to five. 1 .Floral phyllotaxis and architecture were described which is further supported, development of gynocium. 28,29tructure and evolution of embryology in Laurales is described. 30Seed coat anatomy and trichome morphology of Calycanthacae have not studied.We focus on the seed morphology, seed anatomy, and trichome morphology on Calycanthaceae, which support for the phylogenetic implication.The main objective of this study is to deduce their phylogenetic relationship.

Materials and Methods
The collected seeds were fixed with the FAA (formalin: glacial acetic acid; 50% ethanol, 5:5:90, by volume) from each species (Table 1).Mature seeds passed through the alcohol series.After complete dehydration, the seeds passed through combination alcohol/Technovit and then embedded in Technovit 7100 resin.
Serial sections of 5-6 µm thickness were cut using the disposable blade knives stuck into glass slides, and dried on electrical hot plate for 24 hours.Dried slides were, stained with 0.1% Toluidine blue for 60-90 second, ringed with running water, and again dried on the electric hotplate for more than 6 hour to remove water.The stained slides then mounted with Entellan (Merck Co., Germany).Four permanent slides observed under the Olympus BX-50 light microscope (Olympus Co., Japan).
The pre-treatment applied for SEM.The preserved seeds were passed through the alcohol series then immersed in 100% ethanol after that used critical point dryer (CPD) then sample were attached from copper tape.SEM image was carried out from KBSI, Chuncheon at (EHT= 3.00 KV).The multiple image alignments did by using Photoshop CS6.

Results
Out of one Idiospermum genera we include three genera for this study; Sinocalycanthus Calycanthus and Chimonanthus.Of the 3 genera and eight species included, the seed of Chimonanthus fragrans (19.2-20×6.1-7.25 mm) is largest and we found that the size and shape were highly variable with in the study of genus small in Calycanthus occidentalis (10.1-11.45×5.2-6.65).The variable seed feature of the species in order to measurement of seed size (length-width, length/width ratio) these are the significant dissimilarities of the species we concluded That the number of species in genus Chimonanthus is quite large than that of Calycanthus, but in case of Sinocalycanthus is medium size The morphological character is little value for generic delamination in Calycanthaceae.There is difference on seed coat layer, pericarp layer cell shape structure.The seed surface, the seed coat and pericarp character certainly have significant in phylogeny.Detail individual description of the nature of the seed surface and seed coat is given (Tables 2-3).In this studies, seed shape was quite variable large, the range of the helium is concave, long or medium, the range 14.4-16.4×6.4-7.5 mm the shape of the testal cells either, polygonal or sub polygonal, irregular (Figures 1-3).The wall ornamentation is roughly, most of the cell have construction on the center in (Figure 2A, 2B, 2C) the color of seed was brown in Calycanthaceae (Figure 1), the seed were enclosed in the fleshy hypanthium.
Pericarp separated in three histological zones: exocarp, mesocarp, and endocarp developing from the outer epidermis, mesophyll and inner epidermis.The exocarp is represented by the single layer and tangentially attached with mesocarp (Figures 4C,  4L), thick (Figure 4F).The parenchyma of Seed coat divided into three layers as exotesta, mesotesta, and endotesta.Mesotesta was irregular and thick, endotesta and exotesta were polygonal or sub polygonal in Ch. salicifolius (Figure 5E).Exotestal layer was single, circular or subcircular, which is connected the mesotestal layer in Sinocalycanthus chinensis (Figure 4B).The cell shape was circular or sub-circular (Figure 5E).The cell shape was irregular, more than two layers of exotesta in Ch. fragrans (Figure 5B).Cell shape is circular which is attached with mesotesta in Ch. luteus.In Ch. salicifolius, the cell was arranged with each other formation of single layered with elongation cell shape (Figure 5E).Single layer was defined in Ch.  nitens (Figure 4K).Endotesta layer was 1, 2 or more connected with each other that formed the interspace gap (Figure 4B, 4E, 4H, 5K).Crystal was observed all Species on Calycanthacae.Tegmen was two layers that formed bitegment.

Article
Trichome and seed surface of Calycanthaceae has summarized (Table 3).Non-glandular and unicellular trichomes were representative characters in all species of Calycanthaceae.The density of trichome was dense in Ca. occidentalis and Chimonanthus species but moderate in Sinocalycanthus chinensis.The trichome was erect and bending to the seed surface.

Discussion
This study represents the most compressive investigation of trichome micromorphology, seed surface, and seed anatomy in Calycanthaceae.Our data confirm the structure of seed coat and seed surface of different genus.In addition, it is possible to the systematic relationship among the species.Our result is also similar to the previous finding. 31,32The primary vascular cylinder of Calycanthaceae is notable for peculiar system of four-inverted cortical bundle.The stomata are rubiaceous.The trichome is unicellular.We noted that the unicellular hair on the surface of leaf which is the distinguish character of Calycanthaceae.
Unicellular trichomes were variable in length. 6Our results were also support density and size of the trichomes, which was important for taxonomy for Calycanthaceae.The surface of seed and angle of direction of trichomes were also supported for the phylogeny (Table 3).Non-    glandular trichome is a little diagnostic feature of the Calycanthaceae. 33Unicellular trichome is common in all species.The density and position of the trichome play importance role on systematic in Calycanthaceae.We found smooth and erect trichome in all species.They are varying in their arrangement to the seed surface.2L, 3I, 3L).
The seed of Chimonanthus fragrans is largest.Seeds feature of the species in order to measurement of the seed size was the significant dissimilarities of the species.We concluded genus Chimonanthus is quite larger than that of Calycanthus, but Sinocalycanthus is medium size.The morphological characters are for generic delamination in Calycanthaceae.There is difference seed characters have significant for phylogeny.The great consultancy of seed coat feature has observed in Calycanthaceae.The all species of Calycanthus have specialized mesocarp feature.In the seed coat anatomy, all species have thicker mesotestal layer.The vascular bundle is in cortex, which is independent to the stellar system through the Calycanthaceae. 34he exotesta was arranged ovate shape in Chimonanthus praecox, Ch. salicifolius, and Ch.luteus.In case of Chimonanthus fragrans, Ch. nitens, and Ch.yunnanensis, the seed shapes were elliptical shape.Calycanthus occidentalis and Sinocalycanthus show the circular exotesta.Exotesta is highly developed in the Chimonanthus than Sinocalycanthus and Calycanthus.

Conclusions
Seed coat anatomy is different in Calycanthaceae.From the results, seed coat developed in the Chimonanthus than Sinocalycanthus, and Calycanthus endotesta shows broad.
The mesotesta is thicker than exotesta and endotesta in Sinocalycanthus and Calycanthus .In Calycanthus pericarp contain hard comprise the stony cells.The pericarp divided as the exocarp, mesocarp, and endocarp.The exocarp is single layer cells and thin than that of other.
Our data show that trichome micromorphology and seed surface are useful for the separation of species within characterizing of genus and species.There are only on reliable of molecular phylogenetic known as the sister group of Laurales. 6One important result of the above-mentioned surface was rough in both Sinocalycanthus and Calycanthus.Non-glandular trichome was dense in Calycanthus occidentalis.In this study, seed morphology and seed coat anatomy provide some useful information in the genetic delimitation of Calycanthaceae.Number of pericarp layer, number of seed coat layer, and shape of cell were distinguished features.Nature of the tegmen, seed shape and seed size are for species delineation.Presence of crystal in all species has no value for the species delimited.Trichome density and seed surface were great value for the genus level.
International Journal of Plant Biology 2018; 9