First record of fungus Cryptomarasmius T . S . Jenkinson & Desjardin ( Physalacriaceae : Agaricales : Basidiomycota ) from India

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The genus Cryptomarasmius (Agaricales, Basidiomycota) was proposed by Thomas S. Jenkinson & Dennis E. Desjardin (2014) by treating Marasmius hygrometricus (V.Brig.)Sacc.as the type species.Historically, all species belonging to this genus were classified as Marasmius under section Hygrometrici (Kühner), having a combination of marasmioid stature, a smaller pileus that is well-pigmented and broom cells mostly of the Rotalis-type or often in a combination with smooth cells, free to adnate attachment of lamellae, absence of collarium, a central and insititious stipe, usually presence of pleuro-and cheilocystidia, neither amyloid or dextrinoid nature of the pileus, lamellae or stipe trama, and medium-sized basidiospores (Singer 1976).
Phylogenetic approach based on molecular sequence data (nrDNA ITS) including the member of the sect.Hygrometrici was previously attempted by Tan et al. (2009) where four included sequences of Cryptomarasmius micraster (Petch)  Reports on the taxa belonging to the gen.Considerably little works had been carried out on this group from south Asian regions.Pegler (1986) reported two species (M.micraster and M. thwaitesii) from Sri Lanka.Other than that, the presence of the fungi belonging to the group is scarce in literatures.The present paper deals with the new record of Cryptomarasmius exustoides from India.This paper represents the first distributional record of the genus in the Indian context.
The specimen under consideration was collected during July 2014 from Kolkata, West Bengal, India.The material was photographed in the field using a digital camera and extensive notes on the basidiomata were done before drying.Colour terms follow Kornerup & Wanscher (1978).Microscopic features were obtained from free-hand sections of the dried basidiocarp tissues in 5% KOH, Melzer's reagent and Congo Red.Basidiospores size are provided as a mean value (underlined); values in parentheses indicate minimum or maximum measured values.Q value denotes length/ width ratio of the basidiospores.The voucher specimen has been deposited in the Calcutta University Herbarium (CUH) with the accession number CUH AM127.
The extraction of genomic DNA as well as PCR protocol for the amplification of nrDNA regions (nrITS and nrLSU) follows Dutta et al. (2015).PCR products were purified using QIAquick® Gel Extraction Kit (QIAGEN, Germany) and used for automated DNA sequencing on ABI3730xl DNA Analyzer (Applied Biosystems, USA) using primers identical with amplification for nrDNA regions (Vilgalys & Hester 1990;Gardes & Bruns 1993).The generated sequences were edited manually using BioEdit sequence alignment editor version 7.0.9.0 (Tom Hall, Ibis Biosciences, Carlsbad, USA).
The newly generated ITS and LSU sequences were 732 bp and 588 bp respectively.Both the sequences were then used for BLAST searches in the GenBank nucleotide database.The newly generated sequences and those retrieved from GenBank based on a BLAST search (Table 1) were chosen for conducting phylogenetic analysis.

Cryptomarasmius exustoides
Habit and habitat: Solitary or in clusters on rotting bark or wood sticks. Remarks: The characteristic features of Cryptomarasmius exustoides includes a small, plicate pileus coloured light brown to brown at disc with yellowish-white to pale orange or light orange margin; distant (6-7), non-collariate lamellae; an insititious stipe coloured brown to dark brown; absence of rhizomorphs; ellipsoid basidiospores measuring 9-11 × 4-6 µm; presence of dimorphic cheilocystidia i.e. fusoid with capitate to sub-capitate apex and combination with Rotalis-type cells; clavate pileosclerocystdia measuring 31-37 × 5-7.5 µm; and absence of pleurocystidia and caulocystidia.The features of our Indian collection match nicely with that of the type specimen description, except having a bit smaller length of the stipes (5-14 mm vs 10-25 mm; Desjardin & Horak 1997).Previously there were reports on the occurrences of the species from New Zealand (Desjardin & Horak 1997) and Hawaii (USA).The present work constitutes the first report of this taxon from India.Among phenotypically similar taxa: Cryptomarasmius crescentiae (Murrill) T.S. Jenkinson & Desjardin, originally described from Cuba and later subsequently reported from South America and Papua New Guinea, have ochraceous to ochraceous brown pileus with deep ochraceous brown center, presence of more number of lamellae (7-10), and much smaller (6.5 × 2.7 µm), oblong basidiospores (Singer 1976).Marasmius exustus Berk.& M.A. Curtis, originally described from the Bonin Islands, primarily differs in having much smaller basidiospores (6.5-8.0 × 4-5 µm), and absence of pileosclerocystidia on the pileus surface (Berkeley & Curtis 1868).Cryptomarasmius minutus (Peck) T.S.
Phylogenetic analyses were performed on combined dataset (ITS plus nrLSU) of 23 sequences representing six genera viz.Cryptomarasmius (nine sequences), Rhizomarasmius (three sequences), Flammulina (two sequences), Physalacria (four sequences), Omphalotus (three sequences) and Marasmius (two sequences) of which taxa belonging to the gen.Marasmius was used as an outgroup for rooting purposes.The ML analysis resulted the phylogenetic tree (-lnL = 9264.444470)that did not differ significantly in topology from the tree obtained from Bayesian analyses.Bayesian analyses reached a standard deviation of split frequencies of 0.005 after 1,000,00 generations, and the initial 25% trees recovered were excluded as the burn-in.Maximum likelihood bootstrap values (BS) and Bayesian posterior probabilities (PP) support the deeper nodes as well as the terminal nodes with moderate to strong support (Fig. 2).
The phylogenetic analyses clustered all of the members belonging to the family Physalacriaceae within a strongly supported clade (100% BS, 1.00 PP) where nine sequences representing six species of the genus Cryptomarasmius was found to monophyletic with strong support values (97% BS, 1.00 PP).In the resulted phylogenetic analysis, the acquired sequence of Cryptomarasmius aukubae was found to cluster with the deposited two Russian sequences of Cryptomarasmius crescentiae with full support values (100% BS, 1.00  Cryptomarasmius exustoides cluster with the sequence of the same taxon earlier reported from USA with significant support values (92% BS, 1.00 PP).
T.S. Jenkinson & Desjardin (then as Marasmius micraster Petch) clustered together within the clade containing species belonging to the gen.Marasmius sects.Marasmius and Sicciformes with no statistical support.Later, in the phylogenetic analysis performed by Jenkinson et al. (2014), four previously known member of the family Hygrometrici (viz.Marasmius corbariensis (Roum.)Sacc., M. exustoides Desjardin & E. Horak, M. micraster and M. thwaitesii Berk.& Broome) formed a clade together with the well-known representatives of the family Physalacriaceae Corner with low statistical support (BPP 0.81, BS 61%).In the said phylogenetic analysis (Jenkinson et al. 2014), the four taxa were found to form a sister lineage within a clade containing other genera like Cylindrobasidium Jülich, Physalacria Peck, and Rhodotus Maire with moderate to weak statistical support (BPP 0.76, BS <50%).From the overall study (Jenkinson et al. 2014), based on the nuclear ribosomal large subunit sequence data (LSU), insisted Jenkinson and his group to transfer the previously accepted members of the Marasmius sect.Hygrometrici (family Marasmiaceae) to a new genus Cryptomarasmius within the family Physalacriaceae.

Figure 2 .
Figure 2. Maximum likelihood (ML) tree generated using combined datasets of nrITS and nrLSU sequences by employing GTR+I+G model of nucleotide evolution (-lnL = 9264.444470).Numbers to the left of / are ML bootstrap support, and those to the right indicate the Bayesian posterior probabilities (PP).ML >50% and PP > 0.50 values are indicated below or above the branches and the scale bar represents the expected changes per site.The Indian collection of Cryptomarasmius exustoides is placed in bold font to highlight its phylogenetic position in the tree.

Image 1 .
Cryptomarasmius exustoides.a -Natural habitat of the collection place; b -Fresh basidiomes growing in cluster on rotting bark; c -Basidiomes growing on dead wood sticks; d -Basidiomes showing cap surface and lamellae side (scale = 5mm).

Table 1 . Fungal species and GenBank accession number for the sequences used in the phylogenetic analysis. Bold font represents the newly sequenced specimen.
of Threatened Taxa | www.threatenedtaxa.org| 26 March 2018 | 10(3): 11464-11469