The genus Parasola in Pakistan with the description of two new species

Abstract Parasola is a genus of small, veil-less coprinoid mushrooms in the family Psathyrellaceae (Agaricales). The genus is not well documented in Asia, specifically in Pakistan. In this study we describe two new species Parasola glabra and P. pseudolactea from Pakistan, based on morphological and molecular data. Phylogeny based on three DNA regions: nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with the 5.8S rDNA (ITS), nuc 28S rDNA D1-D2 domains (28S) and translation elongation factor 1α gene (TEF1α) show that the new taxa are clustered in a clade formed by the members of section Parasola of genus Parasola. Parasola glabra with grayish pileus, slightly depressed pileal disc, lamellae separated from the stipe by pseudocollarium, basidiospores 14.5–16.5 × 9.5–11.5 × 8.0–10.5 µm, in front view broadly ovoid to oblong, some with rhomboidal outline, in side view ellipsoid, with eccentric germ-pore of 1.5 µm diameter. Parasola pseudolactea with yellowish brown to dull brown pileus, disc indistinctly umbonate, lamellae free, pseudocollarium absent, basidiospores 13.5–14.5 × 10.5–12.0 × 9.5–10.5 µm, in face view rounded triangular to heart shaped, rarely ovoid to subglobose, in side view ellipsoid to oblong, with eccentric germ-pore of 1.5 µm diam. In addition to these new species, P. auricoma and P. lilatincta were also studied. Morphological descriptions for the new species and comparison with known Parasola species are provided. Our observations highlight the diversity of Parasola in northern Pakistan and further document the need for additional systematic focus on the region’s fungi.

Species of Parasola are divided into section Auricomi (Singer) D.J. Schaf. and section Parasola Redhead, Vilgalys & Hopple (previous references to Parasola section Glabri (Lange) D.J. Schaf. -see Schafer (2010) -should be replaced by Parasola section Parasola to conform with the International Code of Nomenclature for Algae, Fungi and Plants (Schafer, D.J., personal communication). The sections are distinguished on the basis of presence or absence of hair-like, golden-to dark brown, thick walled sclerocystidia in the pileipellis (Schafer 2010). In mature fruitbodies during basidiospore discharge, the gill cystidia of Parasola lose turgor and collapse, a characteristic feature of the genus (Nagy et al. 2009).
Basidiospore shape and size are the main descriptive features for species identification in Parasola (Nagy et al. 2009, 2010, Schafer 2014. Previously, five species of this genus (Parasola auricoma (Pat.) Redhead (Ahmad 1980, Hussain et al. 2016. In this study, we describe two new species P. glabra and P. pseudolactea, based on morphological characters and phylogenetic analyses of nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS), nuc 28S rDNA D1-D2 domains (28S) and translation elongation factor 1α gene (TEF1α). In addition to these new species we also studied P. auricoma and P. lilatincta.

Sampling and morphological characterization
Specimens were collected from Malakand, Shangla and Swat districts of Khyber Pakhtunkhwa, Pakistan in summer seasons, 2013-2017. Basidiomata were photographed, tagged and field notes were made. Munsell (1975) was used for determination of color. The specimens were air-dried and kept in zip-lock bags. Specimens examined in this study are deposited in the Herbaria of Hazara University Mansehra, Pakistan (HUP), University of the Punjab, Lahore, Pakistan (LAH) and University of Swat, Pakistan (SWAT).
For anatomical studies slides were prepared in 5% aqueous KOH (w/v). Microscopic features such as size and shape of basidiospores, basidia, cheilocystidia, pleurocystidia and pileipellis were studied under a light microscope (MX4300H, Meiji Techo Co., Ltd., Japan) with at least 20 structures measured in each instance. Cheilocystidia and pleurocystidia were observed and measured by cutting the gill edge from the rest of gill to avoid confusion between the two types of cystidia. In the case of basidiospores, 50 spores were measured in face view and/or side view through 1000× magnification with a calibrated optical micrometer and measurements were rounded to the nearest 0.5 µm. Basidiospores measurements are presented as follows: length range × breadth range × width range. Q values were calculated as: Q 1 = length divided by breadth; Q 2 = length divided by width (Nagy et al. 2010).

DNA extraction, PCR and sequencing
We extracted genomic DNA using the DNeasy Plant Mini Kit (Qiagen, Redwood City, California, USA.). We amplified nuc rDNA internal transcribed spacer (ITS) and 28S loci and translation elongation factor 1α gene (TEF1α) using the primer combinations ITS1F/ITS4; LR0R/LR5 and EF1-983F/EF1-1567R, respectively (White et al. 1990, Gardes and Bruns 1993, Rehner and Buckley 2005. For PCR amplification, we followed Hussain et al. (2017). PCR products were purified using the QIAquick PCR Purification kit (Qiagen). Sequencing was performed with the same PCR primers using the Big Dye Sequencing Kit v.3.1 on an ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, California, USA). Sequences produced for this study have been deposited in GenBank (Table 1).

Alignments and phylogenetic inference
ITS, 28S and TEF1α sequences were aligned using BIOEDIT v 7.2.5 (Hall 1999) and CLUSTAL X 2.1 (Larkin et al. 2007). The ITS, 28S and TEF1α alignments were concatenated into a supermatrix. Psathyrella candolleana (Fr.) Maire was selected as outgroup. Alignments are submitted to TreeBase (Treebase ID 21639). Phylogenetic inference was conducted using Bayesian and Maximum Likelihood (ML) methods. For Bayesian inference, we used BEAST 1.6.2 (Drummond and Rambaut 2007) with a Markov chain Monte Carlo (MCMC) coalescent approach. A Yule tree prior (Gernhard 2008) was used in all simulations, and the starting tree was randomly generated. Four independent runs were undertaken. Chain length was 10 million generations, with a sampling frequency of 1000. TRACER 1.6 (Rambaut et al. 2014) was used to check the effective sample size (ESS), and burn in values were adjusted to achieve an overall ESS (Effective Sample Size) of ≥ 200. Maximum clade credibility tree (20% burn-in) was generated using TREEANNOTATOR 1.6.2 (Drummond and Rambaut 2007). Maximum Likelihood analyses were run in RAXML-VI-HPC (Stamatakis 2006). Rapid bootstrap analysis/search for best-scoring ML tree (-f a) was configured. For the bootstrapping phase, the GTRCAT model was selected. One thousand rapid bootstrap replicates were run. Nodes were considered strongly supported when maximum likelihood bootstrap (MLB) were ≥ 70% and Bayesian posterior probability (BPP) were ≥ 0.95.

Phylogenetic analyses
Sequence length varied from 631 bp (SHP-8) to 644 bp (SHP-11) for our 10 new ITS (ITS1-5.8S-ITS2) sequences and 1042 bp (SHP-12) to 1144 bp (SHP-8) for 10 28S sequences. The 7 TEF1a sequences generated for this study varied from 402 bp (SHP-5) to 502 bp (SU-412). The ITS dataset contained 52 taxa and 631 characters long after being trimmed (Trimming was done manually in BIOEDIT v 7.2.5). The combined ITS-28S dataset represented 47 taxa and 1892 characters long after being trimmed. Similarly, the combined ITS-28S-TEF1a dataset comprised 20 species and with 2890 nucleotides, after being trimmed. The results of phylogenetic analyses of ITS, ITS-28S and combined ITS-28S-TEF1a datasets are summarized in Figures 1, 2 and 3, respectively. Each tree represents ML phylogeny produced by RAXML analysis. Maximum likelihood bootstrap (MLB) percentages > 70% are given above or below the branch node, followed by Bayesian posterior probabilities (BPP) > 0.95. The novel sequences in this study are represented in boldface (Figures 1, 2 and 3), their Genbank accessions are provided in Table 1.
Using Bayesian and ML methods, P. auricoma, P. conopilus, P. setulosa and P. malakandensis were recovered as basal groups with strong support, collectively forming section Auricomi, whereas species of section Parasola fall in a single clade represented as gray highlighted, called 'the crown Parasola' clade (Nagy et al. 2009). Statistical support for the specimens that represent P. pseudolactea was strong in ITS dataset (MLB 98% and BPP 1), and excellent in combined ITS-28S and ITS-28S-TEF1a datasets, respectively (MLB 100% and BPP 1). Similarly, statistical support for P. glabra in both ITS and combined ITS-28S datasets was maximal (MLB 100% and BPP 1). In combined ITS-28S-TEF1a dataset P. glabra was represented by a single specimen and poorly recovered ( Figure 3).
Habitat and distribution. Saprotrophic, scattered under herbaceous plants on grass land. So far only known from the lowland of northern Pakistan. This species is, however, common in lowland northwest Pakistan.
Etymology. Specific epithet 'glabra' refers to the glabrous cap found in species of section Parasola of the genus Parasola, where this species belongs.
Comments. The new species belongs to Parasola section Parasola due to the absence of sclerocystida in the pileipellis. This species resembles Parasola lactea and is close to that species in the molecular phylograms. However, its spores are substantially larger, closer to P. schroeteri or P. hercules in size. The spores of P. pseudolactea are mostly rounded triangular, rarely ovoid to subglobose in face view and larger (14.0 × 11.3 × 9.7 µm), whereas those of P. lactea are mostly broadly ovoid to subglobose, rarely rounded triangular in face view, and comparatively smaller (10.73 × 8.81 × 6.73 µm). Other species similar to the new taxon are P. megasperma and P. plicatilis. Both these species share pileus color with P. pseudolactea. Lamellae of P. megasperma and P. plicatilis are separated from the stipe by a pseudocollarium, whereas in P. pseudolactea, a pseudocollarium is generally absent. Basidiospores are more ellipsoid rarely ovoid in face view and on average 16.5 × 10.66 × 8.5 µm in P. megasperma. Basidiospore shape is quite variable in P. plicatilis, in face view mostly limoniform-subhexagonal, rarely ovoid, in side view broadly ellipsoid, on average 12.41 × 8.21 × 7.14 µm (Nagy et al. 2010). Comparison of morpho-anatomical features of P. pseudolactea with regards to other species of the genus Parasola are set out in Table 2 Description. Pileus 15-30 mm diam, convex to broadly convex, deeply plicate towards the margin, light grayish-brown (2.5YR 5/2) to grayish reddish-brown (2.5YR 3/2); disc indistinctly umbonate to umbilicate, dark reddish orange (7.5R 4/8) to grayish reddish orange (2.5YR 5/6). Lamellae free and remote, pseudocollarium absent, closed, initially concolorous with pileus, later on dark black, finally losing turgor and collapsing. Stipe 40-65 × 2-5 mm, equal, smooth, central, hollow, without annulus.

Discussion
The incorporation of molecular phylogenetics has significantly benefited the systematic and taxonomic studies of coprinoid mushrooms. These mushrooms are deliquescent or, at least, have morphological characters like gill cystidia, coloration and surface features that are quickly changed during basidioma maturation. So morphology based taxonomy of coprinoid mushrooms is always a difficult task for mushroom biologists. In the present study two new species of mushroom genus Parasola are described from Pakistan, based on morphological and molecular data.
On account of absence of sclerocystidia in the pileipellis, both the new species P. glabra and P. pseudolactea belong to section Parasola of genus Parasola. Parasola glabra with light gray to moderate gray pileus was collected in Malakand region of Pakistan. This region is rich in diversity of Parasola species (Hussain et al. 2016. The new species P. glabra with broadly ovoid to oblong, some with rhomboidal basdiospore is closely related to P. hercules. Morphological features of P. glabra are discussed with other species of section Parasola genus Parasola, set out in Table 2. Phylogenetic infer-ence of P. glabra based on ITS and combined ITS-28S datasets was strongly supported (MLB 100% and BPP 1). While in combined ITS-28S-TEF1a dataset, P. glabra was represented by single specimen and was poorly recovered.
Similarly, the second new species P. pseudolactea in this study was collected in Shangla district, Khyber Pakhtunkhwa province of Pakistan. This species with yellow brown to dull brown pileus, basidiospores mostly rounded triangular to heart shape, was found in a Quercus forest. The species most closely related to P. pseudolactea on the basis of basidiospore morphology is P. lactea. Basidiospores are mostly rounded triangular to heart shape, rarely ovoid to subglobose in face view in P. pseudolactea; while spores are ovoid to subglobose, rarely rounded triangular in face view in P. lactea. A poorly described species P. subprona (Cleland) J.A. Simpson & Grgur. with elliptical basidiospores (15 × 8 µm) can be differentiated from both the new species on account of central germ-pore (Grgurinovic 1997). Phylogenetic analyses recovered P. pseudolactea well supported in ITS, combined ITS-28S and combined ITS-28S-TEF1a datasets (Figures 1, 2 and 3), respectively. Along with these new species, collections of P. auricoma and P. lilatincta from Pakistan were also documented in this study. The phylogenetic separation of P. auricoma collected in Pakistan from European collections (albeit into adjacent clades) suggests that the taxon from Pakistan may be a distinct, previously undescribed species. However, morphological features do not yet provide a basis for distinguishing separate species.

Conclusion
It is concluded form this study that low altitude mountains of northern Khyber Pakhtunkhwa Province of Pakistan are rich in the diversity of Parasola and other coprinoid mushrooms.