Two new species of Hygrophorus from temperate Himalayan Oak forests of Pakistan

Abstract The genus Hygrophorus is poorly studied from Asia. From Pakistan, only one species has been reported so far. Two new species in the genus have been collected from Himalayan oak forests of Pakistan. Hygrophorusalboflavescens (section Pudorini, subgenus Colorati) is characterised by its pure white, centrally depressed pileus, occurrence of white stipe with yellow patches at lower half and broader (4.98 μm) basidiospores. Hygrophorusscabrellus (section Hygrophorus, subgenus Hygrophorus) is characterised by its yellowish-green stipe with white apex that has fine scales on the entire stipe, an off-white pileus with dark green and greyish fibrils, ovoid to ellipsoid basidiospores and clavate 4-spored basidia. Macro- and micromorphological descriptions have revealed that both these taxa are not yet described. Phylogenetic estimation based on DNA sequences from the internal transcribed spacer (ITS) region and large subunit (LSU) of the nuclear ribosomal DNA (rDNA) genes, is congruent with the morphological characters that help to delimit these as new species of Hygrophorus. Allied taxa are also compared.


Introduction
The genus Hygrophorus Fr. (Hygrophoraceae, Agaricales) is one of the ectomycorrhizal (ECM) genera in Agaricales. The genus name Hygrophorus Fr. (Hygrophoraceae, Agaricales) comes from hygro meaning moisture and phorus meaning bearer. This may refer to the glutinous to viscid pileus character that many of these fungi have due to a layer of gel that makes them sticky to touch when moist. The genus is characterised by diverse basidiomata colours, basidiomata which are tricholomatoid, collybioid, clitocyboid or omphalinoid, lamellae that are subdecurrent, spores that are smooth and hyaline and a hymenium without cystidia. Basidiomata in this group vary from small to large; thin to fleshy; dry to very glutinous or viscid pileus; with a dry to glutinous, glabrous or fibrillose, generally pruinose or granulose stipe (Singer 1986;Bas et al. 1990;Boertmann 1995;Young 2005;Kovalenko 2012). Colour of the pileus is a characteristic feature in the classification of Hygrophorus especially at the level of subsection (Hesler and Smith 1963). Sect. Hygrophorus has white to cream basidiomata while taxa with colourful basidiomata are in different sections and subsections (Fries 1874;Singer 1943;Candusso 1997).
The family Hygrophoraceae Lotsy was revised by Lodge et al. (2014) on the basis of integrated molecular phylogeny, morphological analyses, pigment chemistry and ecology. They classified the family with three new subfamilies, eight tribes, eight subgenera, 26 sections and 14 subsections. Subgenus Colorati of genus Hygrophorus contain coloured mushrooms. In the new classification, the subg. Colorati (Bataille) E. Larss. has been divided into three sections: Olivaceoumbrini (Bataille) Konrad & Maubl., Pudorini (Bataille) Konrad & Maubl. and Aurei (Bataille) E. Larss. In addition, the section Pudorini is divided into two subsections: Clitocyboides and Pudorini. The subgenus Hygrophorus is divided into two sections: Hygrophorus and Fulventes.
Hygrophorus species are globally distributed and mostly occur in woodlands and forests with pines or with ectomycorrhizal (ECM) angiosperms (Bas et al. 1990). Hygrophorus are essential components of ECM communities of temperate regions in the Northern Hemisphere (Tedersoo et al. 2010). Recently, a new edible species, H. parvirussula has been described from south-western China (Huang et al. 2018) and it belongs to Hygrophorus section Pudorini. A few studies on the genus have been performed in Pakistan. Only one species, Hygrophorus chrysodon, was reported as a new record by Razaq et al. (2014), from the western Himalayan forests of Pakistan. Here we present two new species of Hygrophorus based on both morphology and molecular phylogeny.

Morpho-anatomical analyses
Collections were made during field investigations for ECM communities associated with the oaks of Swat and Dir districts, Khyber Pakhtunkhwa province, Pakistan dur-ing 2014-2016. Basidiomata were found in a pure Quercus forest from Shawar Valley, Swat that is representative of the western Himalayan Province (Naseer et al. 2017b) and from Toa, Alpurai forests (Naseer et al. 2017), Swat, KP, Pakistan. The ECM roots were collected from the same forests as well as Biar, Upper Dir, KP, Pakistan. Biar is located in moist parts of dry temperate zones and it has Q. baloot as the leading species (89.44%) with Q. dilatata (10.46%). Basidiomata were collected following Lodge et al. (2014) and photographed in their natural habitats using a Nikon D70S camera. Morphological characters were recorded from fresh specimens. Colours were designated with reference to mColorMeter application (Yanmei He, Mac App Store). Specimens were deposited in the Herbarium, Department of Botany, University of the Punjab, Lahore, Pakistan (LAH) and the University of Florida Fungal Herbarium, Gainesville FL, USA (FLAS).
Microscopic characters are based on freehand sections from fresh and dried specimens mounted in 5% (w/v) aqueous potassium hydroxide (KOH) solution. Tissues from lamellae, pileipellis and stipitipellis were mounted in phloxine (1%) for better contrast and examined using a Meiji Techno MX4300H compound microscope.
A total of thirty basidiospores, basidia, cystidia and hyphae were measured from each collection. For basidiospores, the abbreviation "n/m/p" indicates n basidiospores measured from m fruit bodies of p collections. Dimensions for basidiospores are given using length × width (L × W) and extreme values are given in parentheses. The range contains a minimum of 90% of the values. Measurements include arithmetic mean of spore length and width for all spores measured, Q means spore length divided by spore width and avQ indicates average Q of all spores ± standard deviation.

Molecular analyses
Genomic DNA was extracted from basidioma gills following a modified CTAB extraction method (Bruns 1995) and from ectomycorrhizal roots by Extract-N-AmpTM Kit (Sigma-Aldrich, St Louis, MO, USA). ITS and LSU regions of nuclear rDNA were amplified using the pairs of primers ITS1F-ITS4B and LR0R-LR5 (Vilgalys and Hester 1990, White et al. 1990, Gardes and Bruns 1993. Polymerase chain reactions (PCR) were performed in 25 μl volume reactions. Visualisation of PCR products were accomplished using SYBR Green and 1.5% agarose gels with TAE buffer for gel electrophoresis. Successful amplicons were purified by enzymatic purification using Exonuclease I and Shrimp Alkaline Phosphatase enzymes (Werle et al. 1994). Purified products were sequenced by the University of Florida's Interdisciplinary Center for Biotechnology Research (http://www.biotech.ufl.edu/). Sequence chromatograms were trimmed, edited and assembled using Sequencher 4.1 (GeneCodes, Ann Arbor, MI). Once sequences were assembled and edited, they were deposited in GenBank (http://www.ncbi.nlm.nih.gov).
Consensus sequences for ITS and LSU were used to query GenBank and the UNITE database using BLAST searches. Representative sequences from across the genus Hygro-phorus were downloaded and imported into an alignment in Bioedit (Hall 1999). Sequences from ECM roots were also included in the ITS alignment. Cantharocybe gruberi (JN006422, DQ200927) sequences were chosen to root the phylogenetic trees following Razaq et al. (2014). Sequences were aligned with the programme MUSCLE (Edgar 2004). Maximum likelihood analyses for individual gene regions were performed via CIPRES Science Gateway (Miller et al. 2010) employing RAxML-HPC v.8. Rapid bootstrap analysis for the best-scoring ML tree was configured for each dataset. For the bootstrapping phase, the GTRCAT model was selected. One thousand rapid bootstrap replicates were run. A bootstrap proportion of ≥ 70% was considered significant.

Molecular phylogenetic analyses
Consensus sequences for the ITS region of H. alboflavescens were 601-638 bp after trimming. BLAST searches in NCBI and UNITE revealed 91% similarity to Hygrophorus penarioides Jacobsson & Larss. (EF395370, EF395371, EF395372 & UDBO1556) from Sweden (99% query cover, 0.0 E value). The two ITS sequences from ECM root tips of Q. incana from same forest (Shawar Valley) matched with Hygrophorus alboflavescens fruiting body sequences and these are depicted in the phylogenetic tree (Fig. 5) Etymology. The species epithet refers to the white pileus with yellow dots and white stipe with yellow patches.
Habit and distribution. Solitary and in pairs on soil under Quercus incana, at 2100 m a.s.l., in thick moist temperate forest of the western Himalaya. Notes. Hygrophorus alboflavescens nom. prov. can be distinguished from closely related species by the following combination of characters: a white, plane, centrally depressed pileus having straight margins; stipe that is white above and yellow below; and broadly ellipsoid spores. The closely related species Hygrophorus penarioides is also an oak-specific species (Table 1). However, they differ morphologically. Hygrophorus penarioides can easily be distinguished by its convex pileus with broad umbo and its involute margins (Jacobsson and Larsson 2007), whereas H. alboflavescens has centrally depressed pileus (without umbo) and straight margins. Hygrophorus penarioides has a pure white pileus and stipe which become cream or slightly pinkish with age, whereas H. alboflavescens has a white stipe and pileus with yellow colouration on both. Hygrophorus alboflavescens has a longer stipe (8-12.5 cm) and broader spores (3.9-6.7 μm) as compared to H. penarioides. Hygrophorus alboflavescens is further differentiated from closely related taxa, H. sordidus, which has a convex, expanded to plane pileus that is larger (8-20 cm broad) compared with the smaller (7-10.5 cm broad), centrally depressed pileus of H. alboflavescens. Hygrophorus alboflavescens has even, smooth and straight margins that differ from involute and subnoccose margins of H. sordidus. Molecular analyses based on ITS and LSU regions also support H. alboflavescens as a distinct taxon and demonstrate its ECM relationship with oak in Pakistan. Figures 3, 4 Diagnosis. Hygrophorus scabrellus is characterised by off-white, plano-convex pileus with greyish, dark green fibrils; yellowish-green, longer (2.1-2.4 cm) stipe with white apex and fine scales along the whole stipe; ovoid to ellipsoid, smooth and smaller (6.5 × 3.8 μm) basidiospores.

Comments
Hygrophorus scabrellus is characterised by a yellowish-green stipe with a white apex that has fine scales on the entire stipe, planoconvex pileus which is off-white with dark green and greyish fibrils.
Hygrophorus scabrellus differs morphologically from the phylogenetically related species H. eburneus. Hygrophorus eburneus has fine scales only at the stipe apex (Table 1), whereas H. scabrellus has scales along the entire length of the stipe. Hygrophorus eburneus has a white stipe (yellowish-green stipe with a white apex in H. scabrellus). Hygrophorus eburneus also differs in having a pure white cap. Our new species H. scabrellus is similar to Hygrophorus cossus (Sow. ex Berk.) Fr. commonly known as Goat Moth Wax Cap, as both share plano-convex pileus. However, H. cossus has greyish white, broader pileus (3-9 cm) and smaller stipe (0.6-2 cm long) (Larsson and Jacobsson 2004) as compared to H. scabrellus that is distinguished by off-white pileus with dark green and greyish fibrils (2.4-2.8 cm) having longer stipe (2.1-2.4 cm). Anatomically, H. cossus has larger basidiospores (7-9 × 4-5 μm) (Larsson and Jacobsson 2004). Molecular phylogenetic analyses based on ITS and LSU sequences also support Hygrophorus scabrellus as a distinct species with strong bootstrap support.

Discussion
In this paper, two new species of Hygrophorus were studied morphologically and sequences of two DNA regions were analysed for each species. These studies revealed that H. alboflavescens falls into section Pudorini of subgenus Colorati and differs from other species in the section by having yellow dots or patches rather than having entirely white basdiomata. We also confirmed, based on ITS sequences from roots, that this new species forms ECM associations with Q. incana. Hygrophorus scabrellus clusters within section Hygrophorus of subgenus Hygrophorus and differs in colour and stipe scaliness from others in that subgenus. These two new species provide evidence that further research is needed to collect and identify the fungal diversity of Asia, which appears to be a global hotspot of fungal diversity.