First record of Leucoagaricus nivalis from Pakistan

Leucoagaricus Locq. ex Singer is represented by more than 150 species of agaricoid, saprotrophic fungi distributed all over the world (Kirk et al., 2008; Kumari and Atri, 2013; Yuan and Liang, 2014; Nabe et al., 2014; Ge et al., 2017; Justo et al., 2015; Qasim et al., 2015; Yu et al., 2016; Hussain et al., 2018; Usman and Khalid 2018; Verma and Vimal, 2018; Sysouphanthong et al., 2018; Yang et al., 2019; Ullah et al., 2020). Only 11 Leucoagaricus species have been reported from Pakistan so far (Ahmad et al., 1997; Qasim et al., 2015; Ge et al., 2017; Hussain et al., 2018; Usman and Khalid, 2018; Ullah et al., 2020). Leucoagaricus is characterized by small to medium, fleshy or thin basidiomata, ranging in stature from slender to sturdy; a pileus surface that is radially fibrillose, floccose, squamulose to fibrillose-scaly or granulose (rarely); entire or very short striate margins; a central, equal to bulbous stipe with a membranous, sometimes moveable annulus; metachromatic basidiospores generally lack a welldefined germ pore and are thin-walled and smooth; and the pileipellis is either a trichoderm or a cutis of repent and radially arranged hyphae lacking sphaerocysts. Pleurocystidia are absent in most species. Clamp connections are absent (Singer 1986; Vellinga 2001). The present study focuses on morphological and molecular characterization of a Leucoagaricus species collected in the Changa Manga forest, Kasur district, Punjab, Pakistan. This research is an effort to establish the fungal diversity of this forest. During field survey in 2019 for the collection of macrofungi to explore the diversity of these fungi from Changa Manga. A number of basidiomata of Leucoagaricus were collected. Field notes were recorded and the samples were air dried and preserved for future analysis. Macroscopic descriptions were based on the fresh material. Significant characters involve size, shape and color of the pileus; attachment and color of lamellae; presence of annulus on stipe. Color codes were given using Munsell (1975) color system. For micro-morphology, dried samples were examined using standard microscopic techniques. Different chemicals were used as mounting media according to requirements. For rehydration, 5% KOH was used, and for staining the walls of hyaline hyphae, Congo red was used. The anatomical features were observed under microscope Xsz 107BN adjusting at 100× objective lens. Measurements were noted using calibrated Motic Images Plus 2.0 software. For basidiospores, [n/m/p] represents n number of spores, measured from m basidiomata and p collections, l × w represents spore dimensions, extreme values are given in parenthesis. Q values are given as l × w ratio while definitions of the Q values for spores are given following Bas (1969). Drawings were made from the laptop screen. The examined specimens are deposited in the herbarium (LAH), Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan. For DNA extraction, the Extract-N-AmpTM kit (SigmaAldrich, St Louis, MO, USA) was used following the manufacturer's protocol. PCR amplification and sequencing was carried