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Research Article Available Online at http://www.recentscientific.com International Journal of CODEN: IJRSFP (USA) Recent Scientific International Journal of Recent Scientific Research Research Vol. 10, Issue, 11(C), pp. 35918-35924, November, 2019 ISSN:0976-3031 DOI: 10.24327/IJRSR Research Article SYSTEMATIC SIGNIFICANCE OF POLLEN MORPHOLOGY IN SOUTH INDIAN SPECIES OF CINNAMOMUM SCHAEFFER (LAURACEAE) Remya Krishnan R.V1*, Santhoshkumar E.S2 and Radhamany P.M1 1Department of Botany, University of Kerala, Kariavattom P.O., Kariavattom, Kerala-695581, India 2Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Karmankode P.O., Palode Thiruvananthapuram district, Kerala-695562, India DOI: http://dx.doi.org/10.24327/ijrsr.2019.1011.4196 ARTICLE INFO ABSTRACT Article History: Cinnamomum Schaeffer belongs to the family Lauraceae. Pollen morphological characters were studied in 25 taxa of Cinnamomum Schaeffer, which include 22 wild and 3 cultivated ones. All the Received 4th August, 2019 th taxa showed variation inpollen size, exine ornamentation, exine thickness and inter spinular Received in revised form 25 distance. The first Principal Component (PC) accounted for 97.6% of phenotypic variance followed September, 2019 by second for another 0.935%. Major pollen traits that accounted for more variability in PC1, PC2 Accepted 18th October, 2019 th and PC3 include polar length, equatorial diameter, exine thickness, nature of granules and Published online 28 November, 2019 arrangement of spinules. The unweighted pair- group method with mathematical averaging (UPGMA) clustering method revealed two principal clusters which separated all the accessions Key Words: between euclidean distances of 0.109 – 0.741. Both cluster analysis and principal co-ordinate Cinnamomum, pollen morphology, PCA, analysis revealed that Cinnamomum moozhiyarense (CMz) collected from Pathanamthitta, UPGMA, PCoA. Cinnamomum travancoricum (CT), collected from Idukki and Cinnamomum walaiwarense (CWa) from Thrissur have morphologically distinct pollen grains. The phenogram and PCoA scatter plot showed the grouping of accessions with species delimitation, which emphasize the importance of pollen morphology as a key character in identifying Cinnamomum at species level. Copyright © Remya Krishnan R.V, Santhoshkumar E.S and Radhamany P.M,2019, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. INTRODUCTION of taxonomic uncertainty. The genus is usually recognised by trinerved and fragrant leaves, paniculate inflorescence, flower The genus Cinnamomum Schaeffer belongs to the family with nine stamens and fruits seated on a cupule. But there are a Lauraceae. The term ‘Cinnamomum’ is derived from the Greek lot of taxonomic disputes regarding the identification of plants word Kinnamon or kinnamomum meaning sweet wood. It at species level (Abeysinghe et al., 2009). Morphology of consists of about 350 species distributed in East Asia, South pollen is an efficient marker in identifying plants at all levels of East Asia, Australia, Samoa, Fiji and Tropical America taxa. But, the pollen morphology of Cinnamomum were (Mabberley, 2008).To date, The Plant List includes 893 scarcely described and discussed. Therefore, in the present scientific plant names of species rank for the genus study, pollen morphology has been studied to delimit and Cinnamomum (http://www.theplantlist.org, last accessed 11 unravel the species complexities to solve the taxonomic April 2019) and 48 of these are attributed to species occurring disputes of Cinnamomum. in India, which are mainly distributed in the Western Ghats, Eastern Ghats and the Himalayas (Baruah & Nath, 2007; MATERIALS AND METHODS Geethakumary et al., 2015; Gangopadhyay, 2008). South India In the present study, twenty five taxa of Cinnamomum were houses about 26 species and they constitute 20 endemics, 4 collected from different localities of Kerala. Specimens were cultivated species (Cinnamomum verum, C. cassia, C. identified using authentic literature and comparing with the camphora & C. tamala) and two distributional records, like C. relevant type specimens deposited at various herbaria. Flower dubium and C. litseaefolium (Geethakumari et al., 2007; buds just before the anthesis were collected and fixed in glacial Geethakumari et al., 2012). acetic acid. The pollen grains were acetolysed according to the following procedure proposed by Erdtman (1960). Cinnamomum is a highly complicated genus due to overlapping phenotypic characters in many species and become a complex Acetolysis *Corresponding author:Remya Krishnan R.V Department of Botany, University of Kerala, Kariavattom P.O., Kariavattom, Kerala-695581, India International Journal of Recent Scientific Research Vol. 10, Issue, 11(C), pp. 35918-35924, November, 2019 The floral buds fixed in glacial acetic acid were crushed with a RESULTS AND DISCUSSION glass rod in centrifuge tube. Three milliliter of freshly prepared Palynological features have, however, attracted little attention acetolysis mixture (9 parts acetic anhydride to 1 part for defining the closely related species of Cinnamomum. The concentrated Sulphuric acid) was added to the content in the pollen grains of Cinnamomum were found to be spheroidal, tubes. The content was heated in a water bath from 70˚C to prolate-spheroidal or sub-prolate; apolar and inaperturate, with boiling point and stirred occasionally. The centrifuge tubes and highly reduced exine consisting of a thin coherent layer content were left in boiling water for 3 minutes and then ornamented with spinules. Spinules are usually broad at base centrifuged at 4000 r.p.m. for 5 minutes while still hot. The and with pointed, acuminate, acute or blunt tips. They are supernatant was decanted into acetolysis waste bottle before the characterised by the presence of a conspicuous, circular, addition of some water to the sediments in the tubes, and was cushion- like base which invariably protrudes proximally. Its shaken vigorously using a whirl mixer. Few drops of surface is studded with granular to globular elements (Figs. 1, methylated spirit were added to remove the foam formed and 2, 3 & 4). Out of the 25 taxa investigated, the largest pollen centrifuged again. The supernatant was decanted, washed with grains are those of C. moozhiyarense, followed by C. water, and centrifuged repeatedly four times. Fifty percent travancoricum and smallest are those of C. walaiwarense. The glycerin was added and left standing for two hours. The tubes pollen grains of the species differ in the exine ornamentation, were shaken vigorously using a whirl mixer and centrifuged at exine thickness, inter- spinular distance and size of pollen 4000 rpm. for 10 minutes. The supernatant was finally (Tables 1 & 2). Hence these characters can be used to support decanted off, and the tube was inverted over filter paper and the taxonomy of Cinnamomum at species level. left overnight. 100% glycerol was added to the tubes, shaken and poured into labelled storage vials. The pollen grains were mounted in unstained glycerin jelly. The slides were examined with a Fisher scientific illumination microscope under (E 40; 0.65), oil immersion (E 100; 1.25) using 10× eye piece. The measurements were based on 20 readings from each specimen. Photomicrographs were taken using Leica CME with Digital Microscope. Light microscopic preparations were made by mounting the acetolysed pollen grains in glycerine jelly. The quantitative measurements were done at x100 magnification for 20 pollen grains of each taxon. Light microscopic studies were carried out by using Olympus CX21i. Scanning Electron Microscopic (SEM) Analysis Pollen grains for the SEM analysis were mounted directly on graphite tape and sputter coated for 45 seconds with gold in Fine Coater (JFC-1200, Joel, Japan), analysed and photomicrographed by SEM (JSM-5600 LV, Joel, Japan) at a voltage ranging 10-12 kV. The terminology for pollen description were adapted from Nair (1970) and Walker and Doyle (1975). Considering the longest diameter, the size classes of pollen have been determined following the classification proposed by Erdtman (1970). A total of 15 characters (9 qualitative and 6 quantitative) were selected for the study. Fig 1SEM Photographs of pollen of Cinnamomum. A & D- C. agasthyamalayanum, B & E- C. cassia, C & F- C. chemungianum, G & J- C. The pollens were observed, measured and recorded using 20 dubium, H & K- C. filipedicellatum, I & L- C. goaense. samples for each taxon. Observations on 15 morphological Analysis of variance traits were scored including both quantitative and qualitative characters. All data were processed using one way ANOVA Analysis of variance and Kruskal Wallis Test performed on and Duncan Multiple Range Test and the significance of quantitative and qualitative data respectively showed qualitative characters were tested using Kruskal Wallis Test significant (P <0.05) variation among the 25 taxa of using SPSS 16.0. A probability value of 0.05 was used as a Cinnamomum studied (Table 1). Polar length and equatorial bench mark for significant difference between parameters. diameter of pollen were significantly high (P <0.05) in C. Multivariate analysis was performed by numeric taxonomic moozhiyarense, while, C. walaiwarense has the lowest mean techniques using the procedure of Principal Component value for polar length and equatorial diameter of pollen. Analysis
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