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Studies on Morphological and Molecular Identification of From 50 KAVAKA54: 50-54 (2020) .doi:10.36460/Kavaka/54/2020/50-54 Studies on morphological and molecular identification ofGeastrum from Gujarat, India Ravi S. Patel,Ajit M.Vasava and Kishore S. Rajput* Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara 390002, India *Corresponding author Email: [email protected] (Submitted on February 13, 2020;Accepted on May 10, 2020) ABSTRACT TheGenusGeastrum Pers.( Geastraceae ),isacosmopolitangastroidmushroompossessingenclosedhymenophore.Itsspeciesarecommonly known as earthstars because of the splitting exoperidium giving the fungus a star like morphology. In the Western part of India, it is represented by three species, viz.Geastrumsaccatum Fr., G.rufescens Pers. and G.triplex Jungh. These are being recorded for the first time from the Gujarat State. Specimens were collected from Vansda National Park and Jambughoda Wildlife Sanctuary during field survey undertaken from 2014- 2019. Identification was carried out based on the morphological features and further confirmation was done by molecular phylogenetic analyses using nuclear rDNA ITS sequencing. Molecular data has been submitted to BOLD system for DNAbarcoding. Key words: Fungal diversity,Geastraceae, Geastrum, ITS, DNAbarcoding. INTRODUCTION 73°39’57.85” E) and Mota Raska, Jambughoda Wildlife Sanctuary (22°21’26.06” N, 73°41’28.12” E) in Gujarat The genusGeastrum was erected by Persoon (1801) with G. State, India and their morphological characters were noted in coronatum Pers., as the type species, which is commonly the field. The collected specimens were preserved for known as earthstar. It is one of the most widely distributed and undertaking macroscopic and microscopic investigations. complex genus of gasteroid fungi with enclosed The additional specimens have been submitted under BARO hymenophore within the exoperidium. Different species of in the Herbarium of Department of Botany, The Maharaja this genus can be easily recognised by their distinct star-like Sayajirao University of Baroda, Vadodara. basidiomata at maturity. According to Zamoraet al . (2014a, b), the genus comprises more than 100-120 species around the Molecular analysis: Genomic DNAwas extracted from fresh world. The taxonomy of the earthstars, as in many other fruiting bodies of collected specimens using a Plant/Fungi macro-fungi, has been traditionally based on morphological DNA isolation kit (Sigma Cat# E5038). ITS region was features of the basidiomata (Sunhede, 1989). This genus is amplified using ITS1 and ITS4 primer pairs (Whiteet al ., reported to harbor some of the medicinally important species 1990). The PCR reactions were performed in a 20μLvolume includingG. saccatum , the extract of which is reported to be containing 1x final concentration of DreamTaq Green PCR anti-inflammatory, antioxidant and cytotoxic in actions (Dore Master mix (Cat# K1081), 50 ng of genomic DNA and 10 et al., 2007) while the extract of G. triplex is reported to be pmol of both primers i.e. ITS1 and ITS4. PCR reactions were quite effective against several plant and human pathogenic carried out by using Veriti® thermal cycler (Applied bacteria (Chittaragiet al ., 2013). BioSystems) under the following conditions: initial 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 In India, species of geaster are reported to be quite common in sec, annealing at 55 °C for 30 sec and extension at 72 °C for moist-deciduous forests, semi-evergreen forests, sacred 1:30 min., with a final extension at 72°C for 10 min. The PCR groves, and coffee plantations of the Western Ghats of product was visualized on 2% agarose gel and amplified PCR Karnataka and Kerala and Shola forests in the Western Ghats. products were purified using PurelinkTM Quick PCR Most of these are usually terrestrial, rarely lignicolous or even Purification kit (Cat# K310001). The purified PCR products coprophilous in habit (Bhagwatet al ., 2005; Mohanan, 2011). were sequenced by Eurrofins Genomics India Pvt. Ltd., However, some of its species are also reported from leaf litter, Bangalore. The obtained sequences were compared with the termite mounds, humus, decomposing twigs or even bark of database sequences available in the NCBI using the Basic Pongamia pinnata, Acacia auriculiformis and decaying twigs Local Alignment Search Tool. The Barcode of Life Data ofSapium insigne (Karun and Sridhar, 2014). In all about 24 System (BOLD) was used to generate DNAbarcodes. different species ofGeastrum are reported from India till date by the previous researchers (Vermaet al ., 2018) and none of Phylogenetic analysis: The phylogenetic tree is generated by the documented species are from Gujarat. using nucleotide sequences of ITS gene. Nucleotide sequences ofGeastrum species are downloaded from In this manuscript summarized account of morphological and GeneBank in FASTA format. Downloaded sequences were molecular studies undertaken during the present study on G. supplemented with the newly generated sequences of G. saccatumFr., G. rufescens Pers., and G. triplex Jungh., has saccatum, G. rufescensand G. triplex , whereas Myriostoma been presented. colifrome was used as outgroup taxa. The nucleotide MATERIALSAND METHODS sequences were aligned with ClustalW (Thompsonet al ., 2002) embedded in MEGA X (Kumaret al ., 2018). Aligned Collection of Fungi: During extensive field visits in different data was analysed with Partition Finder (Lanfearet al ., 2012) forest regions of Gujarat in the years 2014-2019, specimens for optimal partitioning strategy and evolutionary ofG. saccatum., G. rufescens and G. triplex were collected substitution model. Maximum Likelihood (ML) analyses from Vansda National Park, (20°45’31.86” N, 73°28’28.77” were employed to infer phylogenetic relationships in RAxML E), Jambughoda Wildlife Sanctuary (22°22’19.02” N, 51 (Silvestro and Michalak, 2012). An ML analysis was run for sessile endoperidium, fibrillose and delimited peristome. 1000 bootstrap replicates under the GTR + I model to assess According to Trierveiler-Pereiraet al ., (2011), this species is clade support. very similar toG. lageniforme . Both species have saccate basidiomata, sessile endoperidium, fibrillose and delimited TAXONOMIC DESCRIPTIONS peristome.G. lageniforme usually has longer and more slender 1.GeastrumsaccatumFr.,Syst.Mycol. 3 :16,1829 Fig.1(A-D) rays and longitudinal ridges in the external layer. However, G. saccatum may also have such ridges. According to Sunhede Commonly known as the rounded earthstar, bulblight (1989)thesetwospecies canbedistinguished bythepresenceof brown, surmounting a star-shaped base, which raises the clamped hyphae in the external mycelial layer, which only spore sac above the surrounding substrate (Fig.1A ). occur inG. lageniforme . In India G. s accatum is was previously Immature basidiomata epigeous, light brown, sub-globose reported from Kerala (Karun and Sridhar, 2014), Uttar Pradesh to oval, fibrous to squamulose. On maturity, exoperidium (Khare, 1976)andAssam(GogoiandVipin, 2015). splits at the apex into 5-8-pointed non-hygroscopic starfish- like rays, involute and saccate (Fig.1B ). Mature 2. Geastrum rufescens Pers.,Syn. Meth. Fung. ( Go¨ttingen ) sporophores measured from 0.6–2.5 cm in diameter, 1–1.2 1:134, 1801. Fig. 2 (A-D) cm in height (Fig.1C ); peristome slightly darker than Popularly known as the rosy earthstar, bulb up to 2 cm in size, endoperidium, delimited, pale, fimbrillate, conically light greyish, coarsely scaly, sessile and brittle. At maturity, protruding, surrounded by a finely depressed circular halo exoperidium splitting into 5-8 pointed starfish like rays, region (Fig.1D ). Mycelial layer felty but does not encrust which reflect back to reveal the light flesh colour layer (Fig. litter and there is a basal attachment point to the below 2A, B). Fully grown fruiting body 3-5 cm in diameter, ground mycelium; endoperidium pale brown, sessile, thin endoperidium (spore sac) sub spherical, papery, opening by a and papery slight elevated apical pore (Fig.2 C, D ). Gleba (spore mass) at first pallid and firm, becoming brown. World distribution:G.r ufesc e ens is well distributed in different parts of the world including Asia, Europe, Central America and NorthAmerica (Jeppsonet al ., 2013). Fig. 1: Morphology of Geastrum saccatum. A: Immature brown bulb and growing fruiting bodies. B: Mature fruiting bodies with 5-8-pointed non-hygroscopic saccate rays. C: Exposed endoperidium. D: Mature fruit body measuring 0.6 –2.5 cm in diameter.Scale bars :A=5 mm, B = 5 mm, C = 1 mmD = scale indicating cm marking. World distribution: This species is well distributed in Africa, Asia, Australia, Europe, North America, South America andWest Indies (Jeppsonet al ., 2013). Collection Examined: Gujarat: Navasari, Vansda National park. Growing on soil along with mixed leaf litter of Bamboo, Dr. K.S. Rajput, Ravi S. Patel and Ajit M. Vasava. BARO Fig. 2: Morphology of Geastrum rufescens. A&B: Mature 123450030716,August 14, 2016. fruiting bodies with 5-8-pointed starfish like rays. C&D: Remarks:Geastrum saccatum was described based on Endoperidium with Peristome surrounding the slight Brazilian specimens. According to Baseiaet al .,( 2003), G. elevatedapicalpore.Scale Bars :A=4mm,B=4mm,C= saccatum can be distinguished by its saccate exoperidium, 2 mm, D=1 mm 52 Collection Examined: Gujarat, Panchmahal, Jambughoda 3D).At first, gleba pallid and firm, with age it becomes brown wildlife Sanctuary. Growing on soil along with mixed leaf and powdery. litter ofMadhuca
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