Occurrence of gilled fungi in Puducherry, India

fungi and carrying out part of microscopic analysis. TSM—sequenced the ITS region of three gilled fungi and carried out phylogenetic analysis. SG—assisted in identifying and describing some of the species of agarics mentioned in the present study. Acknowledgements: VK thanks Dr. M. Sangararadje, Director, Dr. B.K. Nayak, Head of the Department of Botany, KMGIPSR, Puducherry for facilities and encouragement, and UGC (MRP-2192/06; MRP-6365/16) for financial assistance. Abstract: Thirty-three species of gilled fungi belonging to 23 genera and 14 families were recorded from Puducherry, southern India. Agaricaceae were represented by eight species, followed by Psathyrellaceae (5), Lyophyllaceae & Marasmiaceae (3 each), Hymenogastraceae , Pleurotaceae , Pluteaceae, & Polyporaceae (2 each), and Biannulariaceae , Bolbitiaceae , Omphalotaceae , Schizophyllaceae , Strophariaceae , & Tricholomataceae (1 each). Fourteen species of agarics are new reports from Puducherry. Chlorophyllum rhacodes , Lactocollybia epia , Leucoagaricus meleagris , and Schizophyllum commune were widely distributed. Phylogenetic relationships of the abundant species C. rhacodes , L. epia , and L. meleagris were inferred by maximum likelihood method.


INTRODUCTION
Gilled fungi belonging to Agaricales Underw. constitute ~10% of fungal species described so far (Kirk et al. 2008). It has been assumed that India hosts onethird of the global fungal taxa (Manoharachary et al. 2005) and hence there is an urgent need to document fungi in the unexplored parts of this country covering all possible habitats and seasonal variations. This will help in maintaining the germplasm of these important fungi, as well as to screen these macrofungi for their unique and versatile metabolic potential.
Gilled fungi in Puducherry have not been extensively studied. Studies on the diversity of macrofungi in adjacent areas are by Mani & Kumaresan (2009a,b). Thirty species of white-spored agarics have been reported from Puducherry (Kumaresan et al. 2011), although their identity was not confirmed by phylogenetic inferences. With the rapid deterioration of natural habitats due to human activity, it has become imperative to record these fungi before they become extinct. The study becomes even more interesting considering the fact that these basidiomata are ephemeral, especially the gilled fungi. Moreover, scientists have taken recourse to molecular techniques for identification of these poorlystudied organisms. Many Indian species are called after their North American or European lookalikes (Cannon & Kirk 2007). Sequencing the internal transcribed spacer region for as many fungi as possible from different regions will help immensely in creating or adding to the existing sequence database, to resolve the identities of species complexes and uncover new taxa.

MATERIALS AND METHODS
Basidiomata were sampled during the rainy season of 2007-2009 and 2016-2019 from different places in Puducherry, located 160 km south of Chennai on the southeastern coast of India. The area has a tropical climate and receives a mean annual rainfall of around 126 cm during the north-east monsoon in the months of October-December. During collection, photographs of fresh specimens were taken and morphological characters of fresh basidiomata such as colour (Kornerup & Wanscher 1978), size, and gill attachment were recorded in the field (Senthilarasu & Kumaresan 2018). Dried basidiomata were sealed in zip lock polythene covers after labeling for further microscopic studies. Samples are maintained in the mushroom herbarium collection in the Department of Botany, Kanchi Mamunivar Government Institute for Postgraduate Studies and Research, Puducherry, India.

Microscopic examination
Thin hand-made sections of the pileus and gills were taken and revived in 5-10 % KOH and stained with phloxine (1 %). Microscopic features were recorded following Largent (1977). Approximately, 30 basidiospores sections were measured, excluding the apiculus. The spore quotient (Q) was obtained by dividing the mean length by the mean width in profile view.

DNA extraction and PCR amplification
Few nuclear ribosomal internal transcribed spacer sequences are available for a majority of the species observed here, thus we isolated whole genomic DNA and amplified the ITS sequence to compare it with available sequences in the NCBI database.
The PCR amplification was performed as follows: 95 ºC for 10 min, 30 cycles of 95 ºC for 30 s, 55 ºC for 30 s and 72 ºC for 60 s; and 72 ºC for 10 min. The PCR products were purified and sequenced using ABI 3130 genetic analyzer using primers ITS1F and ITS4B.

Phylogenetic analyses
Sequences were compared using NCBI Blast. Sequences with significant matches were selected and aligned using ClustalW (Thompson et al. 1994), checked visually and edited as required, and evolutionary trees were inferred using the maximum likelihood approach (Kimura 1980) using MEGA v6.06 (Tamura et al. 2013). Bootstrap analysis (1,000 replicates) was performed to calculate the branch support (Felsenstein 1985).

Phylogenetic analysis
The sequences obtained from Chlorophyllum rhacodes, Leucoagaricus meleagris and Lactocollybia epia have been deposted in GenBank with the accession numbers MT229200, MT229202, KU320581, respectively. We constructed maximum likelihood trees to compare our sequences to understand their phylogenetic relationship with related sequences from the database (Figures 1-3). The phylogenetic tree generated using ITS dataset for C. rhacodes and related species included 28 nucleotide sequences. The tree with the highest likelihood (-2549.8398) is depicted (Figure 1). For constructing the tree, all positions with less than 95 % site coverage were eliminated and the final dataset included 537 positions. The ITS sequence of C. rhacodes from this study (MT229200) was placed in the same subclade containing sequence belonging to ITS sequence of C. rhacodes isolated from Gorakhpur, India (MH820354) with 100 % support. The maximum likelihood tree generated for ITS sequence of L. meleagris and its other related species included 17 nucleotide sequences. The tree with the highest likelihood (-1609.0537) is depicted (Figure 2). The final dataset included 604 positions after removing all positions with less than 95 % site coverage. Our isolate (MT229202) clustered in the same subclade with other L. meleagris isolate (GQ249888) from Rajasthan, India with 100 % bootstrap support. For L. epia and its related isolates, the maximum likelihood tree generated included 20 nucleotide sequences and the tree with the highest likelihood (-3410.7721) is shown (Figure 3). The final dataset included 412 positions after removing all positions with less than 95 % site coverage. Our isolate (KU320581) clustered together with L. epia (MN523272), an isolate obtained from China, and showed 100 % bootstrap support.

DISCUSSION
Puducherry does not have any major forest, but there are patches of tropical dry evergreen forest and small areas of sacred groves and mangroves (Ponnuchamy et al. 2013). Therefore, not much litter deposition occurs to create conditions favourable for litter fungi. Studies on the occurrence of agarics in Puducherry resulted in recording more gilled fungi from soil as substrate including A. endoxanthus, A. trisulphuratus, C. molybdites, C. rhacodes, L. serenus, P. cyanescens, three species of Termitomyces, V. hypopithys, C. lagopus, P. plicatilis, three species of Psathyrella, A. manihotis, L. hyalodes, and M. lobayensis. Most of the dark-spored species recorded in the present study were reported by Natarajan & Raman (1983) in tropical dry evergreen forest areas. This shows that forest type plays an important role in determining agaric species composition (Küffer& Senn-Irlet 2005). The 10 dark-spored species along with four white-spored ones recorded in the present study are reported for the first time from Puducherry ( Table  1). Among the three species of Psathyrella sampled in the present study, P. candolleana is known to be widely distributed (Manjula 1983;Natarajan et al. 2005;Farook et al. 2013;Amandeep et al. 2015a). Interestingly, a total of 53 species of Psathyrella have been recorded from India (Amandeep et al. 2015a); however, P. glaucescens and P. obtusata recorded in the present study have so far not been reported from southern India. Similarly, the genus Termitomyces, one of the mushrooms of tribal importance (Varghese et al. 2010), was represented by three species, of which T. microcarpus has been reported widely (Karun & Sridhar 2013).
Vellinga (2002) based on similarities in morphology and molecular studies transferred a few species previously placed in Macrolepiota Singer or Lepiota (Pers.) Gray, into Chlorophyllum. Most of the Chlorophyllum species occur in arid habitats in subtropical to tropical regions (Ge et al. 2018). In India, C. rhacodes is known to be widely distributed and recorded as Macrolepiota rhacodes earlier (Manjula 1983;Amandeep et al. 2015b). We found C. rhacodes to occur in a number of places in Puducherry and the identity of the species was confirmed through ITS sequence analysis by constructing maximum likelihood based phylogenetic tree (Figure 1). Interestingly, phylogenetic analysis of ITS sequences from two species which occurred widely in Puducherry showed that L. meleagris (Syn: Leucocoprinus meleagris) ( Figure 2) clustered with L. meleagris reported from Rajasthan, India while L. epia (Figure 3)     www.threatenedtaxa.org The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication.

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