Gymnopilus purpureosquamulosus Høil . ( Agaricales , Basidiomycota ) : a new distributional record from India

Gymnopilus purpureosquamulosus Høil (Stroph ari aceae) is reported for first time from India. A comprehensive description, a photograph, and comparisons with morphologically similar and phylogenetically related species are provided.

The genus Gymnopilus P. Karst. is comprised of approximately 200 species of wood-inhabiting fungi that are worldwide in distribution (Holec 2005;Kirk et al. 2008).Morphologically the genus is well characterized by a combination of features that include a brightly coloured fruitbody with adnexed to decurrent lamellae, the presence of a membranous veil, rust-brown coloured basidiospores that are dextrinoid in nature with verrucose to rugulose ornamentation, well-developed cheilocystidia that are fusiform or lageniform with their apices capitate to subcapitate or obtuse, and the presence of clamp connections in all the tissues (Holec 2005;Guzmán-Dávalos et al. 2008;Kaur et al. 2015).
Presently, the genus is included within the family Strophariaceae as proposed by Kühner (1980).However, Singer (1986) placed the genus in the family Cortinariaceae primarily due to the ferruginous spore-print and basidiospores that possess a compound wall ornamented with warts.
During a field trip in August 2015 for the purpose of macrofungal inventory, a specimen was collected from Katlia, Howrah, West Bengal, India.The morphologi-cal and ecological (habit and habitat) characters of this specimen were noted in the field.Colour codes and terms follow the Kornerup & Wanscher (1978).Dried basidiomata were sectioned by hand, mounted in a mixture of 5% KOH, Congo red, and Melzer's reagent and microscopic features viewed using a Carl Zeiss AX10 Imager A1 phase contrast microscope.Measurements of basidiospores were noted from 30 randomly chosen basidiospores from each of the collected basidiomata (n = 5); values in parentheses indicate minimum or maximum measured values and Q value of the basidiospore denotes length/width ratio of the spores excluding ornamentation (Acharya et al. 2015).A voucher specimen was preserved using the protocol described by Pradhan et al. (2015) and deposited in the Calcutta University Herbarium (CUH).
Genomic DNA was extracted from the dried fruitbody following Dutta et al. (2015).PCR amplification of the nuclear ribosomal internal transcribed spacer sequence (nrITS) region was performed using fungal universal primers pair ITS1 and ITS4 (White et al. 1990) on an Applied Biosystems 2720 automated thermal cycler using the thermal profile as described by Dutta et al. (2015).PCR products were then purified using QIAquick® Gel Extraction Kit (QIAGEN, Germany) and were subjected to automated DNA sequencing on ABI3730xl DNA Analyzer (Applied Biosystems, USA) using the same primer pairs used for the amplification of rDNA ITS region.
The newly generated sequence of G. purpureosquamulosus was then edited using CodonCode Aligner software (CodonCode Corporation, Dedham, Massachusetts) and used for a BLAST search in the NCBI database.Altogether 31 nrDNA ITS sequences of Gymnopilus representing 20 species were chosen for the phylogenetic analyses based on the BLAST search and the previous study of Guzmán-Dávalos et al. (2008).Dermocybe sanguinea (Wulfen) Wünsche [currently Cortinarius sanguineus (Wulfen) Fr.], Galerina clavata (Velen.)Kühner, and Psilocybe cubensis (Earle) Singer were selected as out-group taxa for rooting purpose following Guzmán-Dávalos et al. (2003).
All these sequences were then aligned with ClustalX (Thompson et al. 1997) using default settings.The alignment was then imported into MEGA v. 6.0 (Tamura et al. 2013) for additional manual adjustments.The ends of the alignment were trimmed to create a data set of 658 bp in length.The appropriate model of evolution for phylogenetic analysis was determined using jModeltest 2.1.6v20140903 (Darriba et al. 2012) in the CIPRES web portal (Miller et al. 2009).Based on the Bayesian information criterion (BIC), the GTR+I+G (6808.186355)model was selected for the ITS data.
Maximum likelihood (ML) analyses was performed with RAxML 8.2.9 (Stamatakis 2014) on the CIPRES NSF XSEDE resource, using the parameters specified by jModeltest 2.1.6v20140903 with bootstrap statistics calculated from 1000 bootstrap replicates.Bayesian phylogenetic analyses were carried out using Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) methods with MrBayes v. 3.2.2(Ronquist et al. 2012), under a GTR+I+G model.For a given data set, the General time reversible (GTR) model was employed with gamma-distributed substitution rates.Markov chains were run for one million generations, saving a tree every 100 th generation.Default settings in MrBayes were used for the incremental heating scheme for the chains (3 heated and 1 cold chain), unconstrained branch length (unconstrained: exponential (10.0)), and uninformative topology (uniform) priors.MrBayes was used to compute a 50% majority rule consensus of the remaining trees to obtain estimates of the posterior probabilities (PPs) of the groups.Bayesian posterior probabilities values ≥ 0.95 are shown in the resulting trees as thickened line.
Habit and habitat: Gregarious to scattered, on dead and decomposed wood.

Diagnostic features of Gymnopilus purpureosquamulosus
includes a medium-sized to large pileus coloured whitish The morphological features of our Indian collection nicely match with that of the type description made by Høiland (1998), but concordant with Guzmán-Dávalos et al. ( 2008), the squammules on the pileus centre was erect to suberect and appressed towards margin.
The newly generated sequence was deposited in GenBank (http://www.ncbi.nlm.nih.gov) with the accession number KU302712.Bayesian analyses reached a standard deviation of split frequencies of 0.003 after one million generations.In the phylogenetic tree (Figure 4 In the clade, the ITS sequence of the Indian collection of G. purpureosquamulosus cluster together with other sequences of the same species, collected and subsequently reported from a wide range of phytogeographical zones (Guzmán-Dávalos et al. 2008) with weak ML bootstrap (50% BS) but with significant posterior probability support (0.95 PP).The Mexican collection of G. cyanopalmicola Guzm.-Dáv.cluster with the nrITS sequence of the Indian G. purpureosquamulosus with moderate to strong statistical support (63% BS, 0.99 PP) that could be attributed to reason of identical or high similarity of the ITS sequence (Guzmán-Dávalos et al. 2008).
Based on overall morphology, G. cyanopalmicola differs from G. purpureosquamulosus by having a stipe surface that turns purple to dark reddish or dark brown when bruised, the absence of pileocystidia, and the presence of considerably larger cheilocystidia (Guzmán-Dávalos 2006).However, these differences might not be enough to differentiate between these two species as pointed out by Guzmán-Dávalos (2006).But for the present moment, we believe that more collections of G. cyanopalmicola from other regions and additional DNA sequence data are necessary to resolve whether G. cyanopalmicola is an independent species or a synonym of G. purpureosquamulosus.

Figure 3 .
Figure 3. Gymnopilus purpureosquamulosus. A. World distribution, countries marked with green.B. India map, showing the state in green from where the specimen was collected.C. Map of West Bengal showing the collection place.

Figure 4 .
Figure 4. Maximum likelihood tree (-lnL = 3160.453779)generated using a GTR+I+G model of nucleotide evolution for the ITS sequence data.Numbers above the branch lengths refer to ML bootstrap percentages (≥50 %).Bayesian posterior probabilities (PP) ≥0.95 are indicated as black coloured thickened lines and the scale bar represents the expected changes per site.The newly generated sequence of Gymnopilus purpureosquaulosus is placed in bold font to highlight its phylogenetic position in the tree.Demarcation of the clades follows Guzmán-Dávalos et al. (2003).