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.

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 Inocybe demetris and 87% with Inocybe comis. 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 I. demetris. 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.