Indian J. Applied & Pure Bio. Vol. 36(1), 167-179 (2021). ISSN: 0970-2091 A web of Science Journal Exomorphological Variation in Gaultheria fragrantissima Wall. (Ericaceae: Vaccinioideae) in India: A Micromorphological Solution from Leaf Stomata and Pollen Morphology 1,3S. Panda, 2,3T. Majumder and 3D. S. Mahanty 1Angiosperm Taxonomy & Biodiversity Conservation Laboratory, Botany Department, Maulana Azad College, University of Calcutta, Kolkata-700013 (India) (present address) 2Nimgram Beluri H.S. School, PO. Beluri, Murshidabad-742226, West Bengal (India) (present address) 3Post-Graduate Department of Botany, Barasat Govt. College, Kolkata-700124 (India) Corresponding Author: [email protected] Abstract Gaultheria fragrantissima Wall., a variable species in the family Ericaceae, is restricted to some south-eastern Asian countries viz., India, Nepal, Bhutan, China, Myanmar, Sri Lanka, Malaysia and Vietnam. This work includes field and herbarium-based exomorphology and Light as well as Scanning Electron Microscopic studies of leaf stomata and pollen morphology of six distinct populations of G. fragrantissima Wall. collected from three phytogeographical regions in India including tables of comparative studies of different populations to stabilize the species delimitation based on leaf stomata and pollen morphological data. Gaultheria fragrantissima Wall., a Gaultheria L. were studied by workers like variable species in the family Ericaceae Juss., Clarke4, Airy Shaw1, Middleton15, Kron et was first described and named by Nathalien al.,12, Ruizheng & Stevens23, Apte et al.,2, Wallich28 based on the plant collected from Panda19,20, S. Panda et al.,21 and Panda & Nepal Himalaya. The etymology of the Sanjappa22. In India, the genus is studied in specific epithet, “fragrantissima” was due to detailed by S. Panda & Sanjappa22 who revised its wintergreen fragrance. The species is the genus and described 23 species including restricted to some south-eastern Asian G. fragrantissima Wall. countries viz., India, Nepal, Bhutan, China, Myanmar, Sri Lanka, Malaysia and Vietnam. Apte et al.,2 studied genetic diversity In India, the species is distributed in Eastern analysis in Gaultheria fragrantissima Wall. Himalaya, North eastern India and hill tops of based on Western Ghats and Meghalayan South western Ghats. populations using ISSR markers. They observed high gene flow within Western Ghat Detailed investigations of the genus population, which may be enough to prevent (168) genetic drift, compare to very low gene flow stomata (LM) and pollen morphology (LM & within Meghalayan populations, while Panda19 SEM) of six distinct populations of Gaultheria revised the genus Gaultheria L. including fragrantissima Wall. in India. Leaf-stomatal G. fragrantisima Wall. based on herbarium- and polliniferous materials used in this based exomorphology. investigation were taken from total 26 dried duplicate herbarium specimens deposited in The present work embodies field and Barasat Govt. College Herbarium (BGC) as herbarium-based exomorphology and Light well as from live materials collected from Microscopic studies of leaf-stomata (LM) as Arunachal Pradesh, Sikkim and Meghalaya well as Light and Scanning Electron (deposited in BGC). This work was carried Microscopic studies of pollen morphology (LM out partly in the Taxonomy and Biosystematics & SEM) of six distinct populations of G. Laboratory, Post Graduate Department of fragrantissima (in case of Pollen morphology, Botany, Barasat Government College (first five populations were studied due to author supervised second author under PG unavailability of flower buds in one) collected Dissertation Project) and partly in Botany from three phytogeographical regions (Eastern Department, Maulana Azad College, Kolkata. Himalaya, North eastern India and Western All measurements are given in metric system. Ghats) in India to stabilize delimitation of G. The dimensions “D”, “(d)” and “2f” corres- fragrantissima Wall. based on leaf-stomata ponding to the tetrad diameter, diameter of and pollen morphological data to show that individual pollen grains and colpi lengths exomorphological variations among these six respectively were measured according to populations are not soundly observed in Oldfield18. These pollen measurements are micromorphological data, which may justify based on at least 10 grains from each that all variable populations are within the specimen. circumscription of the same species, G. fragrantissima Wall. This work also includes Methodology for stomatal study: tables of comparative studies of six different populations under three phytogeographical Mature leaves were obtained from regions in India. Nair and Kothari16 described duplicate herbarium specimens deposited in pollen morphology of this species based on CAL and BGC Herbarium as well as from herbarium material collected only from South live collections from Arunachal Pradesh. Small India (DD, no 26147). Workers like Niedenzu17, cubical pieces (c. 1 sq.cm) were excised from Cox5, Hagerup11, Watson29,30, Lems13 and the base, middle and apical regions of the blade. 27 Stevens contributed a little or no works on Several existing methods viz., 10% HNO3- leaf-stomata. boiling for 10 minutes, 5% KOH overnight (12– 24 hours) treatment without boiling and with Present work is the result of detailed boiling were done. Pieces were ringed in Light (Olympus, Tokyo) as well as Scanning sterilized water until clear. After clearing, Electronic Microscopic (Hitachi-S530, Japan pieces were dehydrated in an ethanol series at Burdwan University Centre) studies of leaf followed by staining with 10% safranin and (169) mounted onto microscope slide in DPX (pieces Asiat. Res. 13: 397. 1820 & Numer. List: no. of basal, middle and apical regions in one slide). 765. 1829; G. Don, Gen. Syst. 3: 840. 1834; C. The slide was examined under Olympus B. Clarke4 in Hook. f., Fl. Brit. India 3: 457. (Tokyo, Japan) light microscope using 40X and 1882; Hara in Hara et al., Enum. Fl. Pl. Nepal 100X objectives and Camera Lucida drawings 3: 55. 1982; Rae in A. J. C. Grierson & D. G. were made with the help of drawing prism. Long, Fl. Bhutan 2 (1): 388. 1991; Ruizheng 23 The descriptive terminology follows Metcalfe & P. F. Stevens in Wu et al., Fl. China 14: and Chalk14, Dilcher6, Stace25,26, Fahn10 and 472. 2005; S. Panda, W. Kameng Ericaceae: Carpenter3. 155. 2013; Panda & Sanjappa in Sanjappa & Sastry, Fasc. Fl. India 25: 196. 2014. Panda et 21 Preparation of Pollen slides: al., Div. Gen. Gaultheria India: 105 – 113. 2014. G. fragrans D. Don, Prodr. fl. nepal.: The method used in this study was by 151. 1825. Type: Nepal, 1818, Wallich s.n. (K, Erdtman7-9. Dry polliniferous materials Cibachrome image!). Arbutus laurifolia (mature flower buds) were taken from Buch.Ham. ex D. Don, Prodr. fl. nepal.: 151. duplicate herbarium specimens deposited in 1825. Gaultheria ovalifolia Wall., Numer. CAL and BGC Herbarium as well as from List: no. 1523. 1829. Gaultheria leschenaultii DC., Prodr. 7 (2): 593. 1839. Leucothoe? fresh flower buds collected from Arunachal katagherensis DC., Prodr. 7 (2): 606. 1839. Pradesh. The descriptive terminology follows Andromeda katagherensis Hook., Icon. pl. Erdtman9 and Sarwar et al.,24. 3: t. 246. 1839. Gaultheria forrestii Diels, Notes Roy. Bot. Gard. Edinburgh 5 (25): 210. Slide preparation for SEM : 1912. (Figs. 1-7). Vernacular names: Chanchhewaa, Dhasingare, Goenhli Leaf samples for stomata and acetolysed (Nepalese of Sikkim); Shep-Sheng (Monpas pollen grains (following Erdtman’s method8) of Bomdila); Jirhapkynthai, Jirhap, Soh- were prepared for Scanning Electron Microscope lyngthrait (Khasis of Meghalaya); Kolakkaai, observation. Pollen grains at least from 10 Moolai (Tamil of Tamil Nadu). flowers of each species were acetolysed and studied. Observations were made with Hitachi Stout, erect shrub, 0.3–2 (–3.5) m S530 (SEM, Tokyo, Japan at Burdwan high, or drooping down from rock crevices. University Instrument Centre) in the high Stems always glabrous, greyish-brown to light vacuum mode at an applied voltage of 15 KV. brown, profusely branched; branchlets blood For SEM, above samples were mounted on red to deep pink, glabrous, glaucous, winged the metallic stub using double stick tape. or triangular. Leaves coriaceous, lamina ovate- elliptic, oblong-elliptic, ovate-lanceolate to Taxonomy and Exomorphological rarely ovate or obovate, 3–12×2–4 (–6) cm, observation : serrate to serrulate at margin, broadly cuneate at base, always mucronate at apex, deep green, Gaultheria fragrantissima Wall., glabrous above, light green, punctate beneath; (170) (171) petioles stout, 5–11 mm long, glabrous or Hill-tops of SW Ghats: Tamil Nadu, Kerala punctate beneath. Racemes axillary; rachis 2– and Karnataka); Nepal; Bhutan; China; 6 (–10) cm long, 10–26-flowered, puberulous. Myanmar and Sri Lanka. Flowers always 5-merous, 8–14 mm long; pedicels greyish-white, 2–8 mm long, puberulous; Flowering: March–May; December–January. bract 1, basal, light green, broadly ovate, 2– Fruiting: May – October. 4×1.5–2.5 mm long, ciliate at margin, acute or subacute at apex, glabrous; bracteoles 2, Habitat : grown in moist rocky soil, opposite, mostly apical, rarely subapical to often hanging down from rock crevices, rarely median on the pedicel, pink with greenish in loose humus-covered boulders or in landslide areas at altitudes ranging from 1600–2300 m. stripes, broadly ovate, 1.5–2.5×1–2 mm, ciliolate at margin, acute at apex, puberulous. Results (Micromorphological observation)