Descolea quercina (Bolbitiaceae), a new species from moist temperate forests in Pakistan

Abstract A new species, Descolea quercina, is described and illustrated from Northern parts of Khyber Pakhtunkhwa, Pakistan. It is characterized by medium to large basidiomata, squamose to squamose-granulose hygrophanous pileus, and limoniform, verrucose basidiospores with partly concrescent verrucae. Phylogenetic analyses of nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains (28S) also confirmed it as a new species. A comparison with similar taxa is provided.


Introduction
The genus Descolea Singer was based on D. antarctica Singer, which has agaricoid basidiomata with an annulus, thus resembling Rozites or Pholiotina spp. (Horak 1971). Descolea is currently placed in the Bolbitiaceae (Kirk et al. 2008) and is characterized by dry to viscid pileus with or without squamules, central stipe with striated annulus, ochraceous spore deposit, amygdaliform to limoniform, verrucose basidiospores with a smooth apiculus, and a hymeniform pileipellis (Horak 1971). Descolea was once considered to be restricted to the southern hemisphere, however, the known 15 species (Sharma and Kumar 2011) have a wide geographical distribution (Australia, India, Japan, Korea, New Guinae, New Zealand, Pakistan, Siberia, South America) (Horak 1971;Bougher and Malajczuk 1985;Niazi et al. 2007). From Pakistan, only D. flavoannulata (Lj.N. Vassiljeva) E. Horak was reported to date. During our macrofungal surveys, we collected a rare and interesting species of Descolea from two locations in Northern areas of Khyber Pakhtunkhwa, Pakistan. The species appeared unique and based on discrete morphological characteristics and sequences derived from nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains (28S), it is described here as new to science.

Collection and morphological characterization
Collections were made on routine mycological visits to the moist temperate Quercus dominated mixed forests of Malam Jabba (Swat district) and Toa valley (Shangla district), Khyber Pakhtunkhwa province, Pakistan. Basidiomata were collected following Lodge et al. (2004) and photographed in their natural habitats. Descriptions of the macro-characters are based on fresh collections and colored photographs. Color codes follow Munsell soil color charts (1975) and are presented in parenthesis after common color names.
Microscopic characters are based on free hand sections from fresh and dried specimens mounted in 5% (w/v) aqueous Potassium Hydroxide (KOH) solution. Measurements of anatomical structures are based on calibrated computer based software "PIXIMÈTRE version 5.9" connected to a compound microscope (BOECO, Model: BM120) and visualized through a microscopic camera (MVV 3000). A total of twenty basidiospores, basidia, cystidia and hyphae were measured from all the collections. For measurements; Q is the range of length/width (L/W) ratio of the total measured basidiospores; Qe is the average L/W ratio of all the measured basidiospores; Me is the average L × W of all the measured basidiospores. Surface of the basidiospores was studied both in 5% KOH solution and scanning electron microscopy (SEM).

DNA extraction
DNA from herbarium specimens was extracted following the procedure mentioned in Peintner et al. (2001). A primer pair ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) was used to amplify the ITS region and primer pair LR5 and LR0R (Vilgaly's lab http://sites.biology.duke.edu/fungi/mycolab/primers.htm) was used to amplify the 28S region. Polymerase chain reactions (PCR) were performed in 25 µL volume per reaction. PCR procedure for ITS region consisted of initial 4 minutes denaturation at 94°C, 40 cycles of 1 minute at 94°C, 1 min at 55°C, 1 min at 72°C, and a final extension of 10 minutes at 72°C. PCR procedure for 28S region consisted of initial denaturation at 94°C for 2 minutes, 35 cycles of 94°C for 1 minute, 52°C for 1 minute, 72°C for 1 minute, and final extension at 72°C for 7 minutes. Visualization of PCR products were accomplished using 1% agarose gel added with 3 µL ethidium bromide and a UV illuminator. Sequencing of the amplified products was accomplished through outsourcing (BGI, Beijing Genomic Institute, Hong Kong).
The 28S region yielded a 958 bp fragment for MJ-1590 and AST33, while the third collection (MJ-1590a) yielded a noisy sequence which was not included in the final analyses. The query sequences on blast showed 99% similarity with Descolea recedens (Sacc.) Singer (HQ827174), Descolea maculata Bougher (DQ457664) and Descolea gunnii (Berk. ex Massee) E. Horak (AF261523) from USA. Based on high similarity with query sequences, some unknown Descomyces species were also included in the phylogenetic analyses. Hebeloma fastibile (AY033139) and H. affine Smith, Evenson & Mitchel (FJ436324) were used as outgroup taxa.
DNA Sequences were aligned using online webPRANK tool at http://www.ebi. ac.uk/goldman-srv/webprank/ (Löytynoja and Goldman 2010). 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/search for 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. Maximum parsimony (MP) analyses were performed using PAUP* 4.0b (Swofford 2002), with all characters of type unordered and equally weighted. Gaps were treated as missing data. Heuristic searches were performed with 1000 replicates with random taxon addition. MAX-TREES was set to 5000 with MulTrees option in effect and TBR branch swapping. All characters were of type 'unord' and equally weighted.

Molecular phylogenetic analyses
The ITS based analysis involved 27 nucleotide sequences. There were a total of 694 characters in the alignment file of which 345 characters were constant, 45 variable characters were parsimony-uninformative while 304 variable characters were parsimony-informative. The tree resulting from the ITS based ML analysis (Fig. 1) was similar to the MP. The distribution of Descolea species among different clades is in conformity with Peintner et al. (2001). The sequences from the Pakistani collections (MJ-1590, MJ-1590a and AST33) formed a separate clade with robust bootstrap support (ML 100% and MP 71%), supporting its independent position.
The 28S based analysis involved 17 nucleotide sequences with a total of 941 characters, out of which 867 characters were constant, 16 variable characters were parsimony-uninformative and 58 variable characters were parsimony-informative. The ML phylogram (Fig. 2) was found congruent with MP phylogram (not shown). The sequences from Pakistani collections (MJ-1590 and AST33) formed a separate clade (Fig. 2), with was poorly supported by bootstrap values (ML 71% and MP 73 %), but tree topologies further support its unique position. Diagnosis. Basidiomata medium to large, pileus convex to convex-campanulate with a broad umbo in young stages, light yellowish brown to deep yellowish brown, surface dry, hygrophanous, squamose to squamose-granulose with striate margin; basidiospores limoniform, coarsely verrucose with partly concrescent verrucae.
Ecology. Associated with Quercus species. Season July-August Etymology. The epithet "quercina" refers to association of this taxon with Quercus species.
Based on phylogenetic evidence, D. quercina is sister to a clade circumscribing D. maculata, D. gunnii and D. recedens. Descolea maculata also has a pileus with appressed squamulae, similar colored basidiomata, and basidiospores of almost the same size (10-13 × 6-7.5 µm). But D. maculata has a rippled or wrinkled pileus surface and amygdaliform to sublimoniform basidiospores, which are minutely verrucose (Bougher and Malajczuk 1985). Comparison of D. quercina with other closely related species is given in Table 1.
Descolea quercina is a striking new species associated with Quercus in temperate areas of Pakistan. The ecology and biogeography of this species are particularly significant since most Descolea species associated with Fagaceae are native to the Southern hemisphere (New-Zealand, Australia, South America). The only known Descolea species associated with Quercus or Castanopsis and occurring in the Northern hemisphere are now D. flavoannulata and D. quercina.