Data from: DNA barcoding analyses and taxonomy reveal two new species of genus Inocybe from pine forests of Pakistan
Data files
Nov 04, 2025 version files 46.66 KB
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ITS_LSU.phy
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README.md
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Abstract
Here we describe two new species of Inocybe from the Pine forests of Pakistan: I. hazarensis and I. shimlaensis. Morphological and molecular data show that these species have not been described before, hence they need to be described as new. Both species are smooth-spored and pruinose only in the apical part of the stipe. Inocybe hazarensis is characterized by a rather small size, brown to dark brown, dense radial fibrils to rimose to glabrous pileus with prominent umbo, finely fibrillose at apex only becoming pruinose to strigose, along the rest of the length of stipe, slightly bulbous stipe base, narrow basidiospores spores and smaller clavate, oblong, ovoid, narrowly utriform cheilocystidia. Inocybe shimlaensis is characterized by a brown to golden pileus, a low and broad umbo, radially fibrillose, rimose to granulose, with brown appressed squalmulose, a stipe with only the apex pruinose, and a submarginate base. Anatomically, it has small (6.8 × 4.4 μm) basidiospores and smaller conical to fusiform cheilocystidia. 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 Inocybe.
Dataset DOI: 10.5061/dryad.pzgmsbd1v
Description of the data and file structure
Microscopic characters are based on free-hand 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.
Total Genomic DNA was extracted from basidioma gills following a modified CTAB extraction method (Bruns 1995). We targeted two DNA regions: (1) the internal transcribed spacer regions of nuclear ribosomal DNA (ITS); (2) partial large subunit of nuclear ribosomal DNA (LSU). The following primer pairs were used for the DNA amplifications: ITS1F–ITS4 for ITS (White et al. 1990; Gardes & Brun 1993), LR0R–LR5 for LSU (Vilgalys and Hester 1990). 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). After sequencing of Purified products, 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).
Files and variables
File: ITS_LSU.phy
Description: Alignement file for Maximum Likelihood Phylogram of Inocybe hazarensis & Inocybe shimlaensis, inferred from combine ITS & LSU dataset with Inosperma geminum (OR823936 & OR823937) used as out-group. Bootstrap support values (≥ 50 %) are indicated above the nodes. The sequence generated in this study are represented in boldface.
Code/software
BioEdit was used for the analysis.