( Agaricales , Entolomataceae ) : A new distributional record from India

This study reports Entoloma shandongense (first described from China) as a new record for the macrofungal mycota from India. A phylogenetic analysis based on ITS show that E. shandongense can be readily distinguished from other blue coloured morphologically similar species, such as E. atrum. A detailed taxonomic description with illustrations and an artificial key to blue-coloured Entoloma species from India and surrounding areas are provided.

The genus Entoloma P. Kumm. s.l. (Basidiomycota, Agaricales) comprises approximately 1,500 species (Noordeloos and Morozova 2010) and is considered to be worldwide in distribution (Noordeloos 1981).Its cosmopolitan nature is observed through studies in South America (Horak 1982), the Pacific Northwest and Alaska (Largent 1994), the Netherlands (Noordeloos 1980), the Mascarenes and the Seychelles (Noordeloos and Hausknecht 2007), Tasmania (Noordeloos andGates 2007, 2009), Primorsky Territory of the Russian Far East (Noordeloos and Morozova 2010), Brazil (Putzke and Putzke 2000;Alves and Nascimento 2012), China (Li et al. 2009;He et al. 2012;Wang and Bau 2013), and Sri Lanka (Pegler 1986).However, vast areas such as India, Africa, South America and South East Asia are still under-explored (Noordeloos and Morozova 2010).The diagnostic features of angular spores, basidiospore wall evenly cyanophilic, and pinkish spore-print make the genus easy to recognize.Previous molecular works suggest that the genus is monophyletic (Baroni and Matheny 2011;Co-David et al. 2009).As of this date there are reports of ca.76 species of Entoloma from India (Pegler 1986;Manimohan et al. 1995Manimohan et al. , 2002Manimohan et al. , 2006;;Raj and Manimohan 2012;Farook et al. 2013;Pradeep et al. 2012Pradeep et al. , 2013)), most of which were described as new to sci-ence.During a 10-year-long project (Khatua et al. 2015), the authors collected an interesting blue coloured species of Entoloma from the state of West Bengal, eastern India.After thorough consultation of available literature (Pegler 1986;Manimohan et al. 1995Manimohan et al. , 2002Manimohan et al. , 2006;;Raj and Manimohan 2012;Farook et al. 2013;Pradeep et al. 2012Pradeep et al. , 2013;;Wang and Bau 2013), the species was identified as Entoloma shandongense T. Bau & J. R. Wang and further confirmed by molecular (ITS sequence) data.This collection constitutes the second record of the species and expands its geographical distribution to India.We present morphological and molecular studies of the collected taxon, and an artificial key to blue-coloured Entoloma species from India and surrounding areas to aid in future identification by others.
The specimen under consideration was collected during August 2014 from Kolkata, West Bengal, India.The material was photographed in the field using a digital camera and extensive notes on the basidiomata were accomplished before drying.Colour terms (mostly) follow the Royal Botanic Gardens Edinburgh colour chart (Henderson et al. 1969).Microscopic features were obtained from reviving dried material in 95% ETOH and then dH 2 O and then by mounting free-hand sections of basidiocarp tissues in 5% KOH, Melzer's reagent and Congo Red.Basidiospores size are based on 30 measurements from each of the three collected basidiomata of a single collection and given 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).
Genomic DNA was extracted following Dutta et al. (2014).Selective PCR amplification of nrDNA ITS region was performed according to the modified method of Gardes and Bruns (1993) using the primer pairs ITS1F (forward) and ITS4B (reverse).A hot start of 4 min.at 94°C was followed by 30 cycles consisting of 30 sec. at equally, gaps treated as missing data, search level 1 where the initial trees were obtained by the random addition of sequences (10 replicates), tree bisection reconnection (TBR), and collapse of zero length branches.The branch lengths were calculated using the average pathway method (Nei and Kumar 2000) and are in the units of the number of changes over the whole sequence.
Besides MP, phylogenetic analyses were also carried out using the maximum likelihood (ML) method to determine whether different methods (MP vs. ML) alter the resulting phylogenetic tree.ML bootstrapping analysis was done based on the general time reversible model (Nei and Kumar 2000) where the initial tree(s) for the heuristic search was obtained by applying the neighbour-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach.A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4770)).The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 0.0000% sites).In both analysis, all positions containing gaps and missing data were eliminated and a bootstrap test of 1,000 replicates were performed to obtain the percentage of replicate trees for clustering the associated taxa (Felsenstein 1985).Data obtained from both ML and MP analyses (Bootstrap percentages of ≥ 50%) has been indicated in Figure 1 (next to the branches).94°C, 30 sec. at 56°C, 1 min.at 72°C, and a final elongation step of 5 min.at 72°C.PCR products were purified using QIAquick® Gel Extraction Kit (QIAGEN, Germany) and were used for automated DNA sequencing on ABI3730xl DNA Analyzer (Applied Biosystems, USA) using primers identical with amplification for nrDNA ITS region.The generated sequence was edited manually using BioEdit sequence alignment editor version 7.0.9.0 (Tom Hall, Ibis Biosciences, Carlsbad, USA).The edited sequence was then used for BLAST searches in the GenBank database.The newly generated sequence was deposited in GenBank (KP241852).The newly generated ITS sequence (615 bp) and those retrieved from GenBank based on a BLAST search of KP241852 (19 ITS sequences) were chosen for purpose of phylogenetic analysis.The closest hit of KP241852 was Entoloma shandongense T. Bau & J.R. Wang (GenBank KC257440; sequence identity = 602/607 (99%); gaps = 4/607(0%)).Tricholoma vaccinum (Schaeff.)P. Kumm. and Lyophyllum decastes (Fr.)Singer were selected as outgroup taxa for rooting purpose following He et al. (2012).Altogether twenty-three sequences were aligned with ClustalX (Thompson et al. 1997) using default settings, followed by additional manual adjustments.The end of the final alignment was trimmed to create a dataset of 626 bp that included 458 positions.
Maximum Parsimony (MP) analysis was conducted using MEGA6 (Tamura et al. 2013).Searches employed a heuristic search method with all characters weighted Basidiomata small, omphalinoid.Pileus 13-22 mm diameter, convex with a slightly central depression when young, becoming upturned with maturity; surface entirely blue with somewhat purple tint, no colour change with KOH and NH 4 OH, nonhygrophanous, smooth to minutely pubescent all over, non-striate; context 1 mm thick, white, no colour change on exposure to air.Lamellae short decurrent to decurrent when old, close, creamywhite, becoming yellowish in KOH, 1 mm broad, with lamellulae of 2-3 lengths; edge entire, concolorous with the sides.Stipe 2-5 cm long × 3 mm wide, central, equal, fleshy, slightly curved; surface white, smooth; context white, no colour change with KOH, FeSO 4 and NH 4 OH.Odour none.Taste mild.
Distribution: Previously there was single report on the occurrence of E. shandongense from China, Shandong Province, Dezhou City, Botanical Garden of Dezhou, where it was found to grow on soil in Poa pratensis L. grassland.The occurrence of this species in India, West Bengal, Kolkata, Golpark Lake, expands its geographical distribution from China to India.As compared to the mentioned ecosystem of the Chinese collection, the Indian specimen was found to grow in a different ecosystem, covered by mostly deciduous trees like Polyalthia longifolia (Sonn.)Hook.f.& Thomson (Annonaceae), Lagerstroemia speciosa Pers.(Lythraceae) and Ficus religiosa.
The MP analysis resulted in five equally parsimonious trees with a tree length of 368 steps, CI=0.579288,RI=0.730849, and composite index=0.472669(0.423372).Figure 1 shows one of the most parsimonious trees.The ML analysis (-lnL = 2394.8031)resulted same topology and relationships for the species groups as like MP.In both analysis (MP and ML), the newly generated sequence of our collected specimen (KP241852) cluster with the type specimen sequence of E. shandongense with full support values (Figure 1; MLBS = 100%, MPBS = 100%).Concordant with the micro-morphology, the phylogenetic analysis easily differentiate E. shandongense from similar blue coloured E. atrum (Figure 1).Unfortunately, there are misidentification problems in some Genbank sequences (see E. undatum and E. byssisedum in Figure 1).
With regard to the blue colouration of the pileus, morphologically E. shandongense resembles some of the previously reported taxa from India and its surrounding countries (Manimohan et al. 1995(Manimohan et al. , 2006;;Li et al. 2009;Katumoto 2010;He et al. 2012;Pradeep et al. 2012;Wang and Bau 2013).However, there are morphological differences that permit its identification.An artificial key to distinguish E. shandongense from its related species is provided.Authors' contribution statement: KA identified the species, wrote the text, SP performed the microscopic work, identified the species, AKD identified the species, drawn the microscopic plate, made the phylogenetic analysis, wrote the text, and IB collected the specimen, prepared macroscopic data.

Figure 1 .
Figure 1.Maximum parsimony tree inferred from internal transcribed spacer nuclear ribosomal DNA sequences showing the position of Entoloma shandongense.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Values to the left of / are Maximum Likelihood bootstrap (BS) support, and those to the right indicate the MP bootstrap support (in bold) of that clade.BS values ≥ 50% are shown.Entoloma shandongense is placed in bold font to highlight its phylogenetic position.Blue text in the tree represents blue coloured species.