DOI: 10.5061/dryad.bcc2fqzjb
title: A new species of smooth-spored Inocybe from Coniferous forests of Pakistan

description: Inocybe bhurbanensis is described and illustrated as a new species from
 Himalayan Moist Temperate forests of Pakistan. It is characterized by
 fibrillose, conical to convex, umbonate, brown to dark brown pileus,
 non-pruinose, fibrillose stipe with whitish tomentum at the base and
 smooth basidiospores that are larger (9 × 5.2 µm) and thicker
 caulocystidia (up to 21 m) as compared to the sister species Inocybe
 demetris. Phylogenetic analyses of a nuclear rDNA region encompassing the
 internal transcribed spacers 1 and 2 along with 5.8S rDNA (ITS) and the
 28S rDNA D1–D2 domains (28S) also confirmed its novelty.
description: Collection and morphological characterization
 Collections were made on routine mycological visits to the
 Himalayan Forests of Pakistan during 2020–2022. Basidiomata were collected
 following Lodge et al. (2004) and photographed in their natural habitats.
 Descriptions of macromorphological characters were made based on fresh
 collections and color photographs. Colors were designated with reference
 to Munsell Soil Color Charts (1975). Microscopic characters were described
 based on free-hand sections from fresh and dried specimens mounted in 5%
 (w/v) aqueous Potassium Hydroxide (KOH) solution. Measurements of
 anatomical structures were taken with the 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 sixty basidiospores, basidia, different types of
 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 while av.Q is the average L × average W of all the
 measured basidiospores. DNA extraction
 DNA from herbarium specimens was extracted following the
 procedure of (Peintner et al. 2001). The primer pair ITS1F (Gardes and
 Bruns 1993) and ITS4 (White et al. 1990) were used to amplify the ITS
 region and the 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 the ITS region consisted of an 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. The PCR procedure for the 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 was 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). Phylogenetic analyses The ITS region of the voucher collections GB19, BR19 and AN-87 yielded 730, 752 and 780 bp fragments, respectively. Sequences of all three specimens were used as a reference to BLAST against GenBank. All the query sequences matched 91% with <em>Inocybe</em> <em>demetris</em> and 87% with <em>Inocybe</em> <em>comis</em>. Other closely related sequences were downloaded for high similarity with query sequences and used in the subsequent phylogenetic analyses. The 28S region yielded a 988 bp fragment for GB-19 and ANK-87. The query sequences (BLAST) showed 99% similarity and 94% query cover with <em>I. demetris</em>. DNA sequences were aligned using the 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.