First records of Bolbitius coprophilus (Agaricales, Bolbitiaceae) from Pakistan

In efforts to record fungal biodiversity of Pakistan, we collected specimens of the genus Bolbitius Fr. from two arid to semi-arid localities in Punjab Province. On the basis of morphological evidence and ITS-nrDNA sequence data, our materials are identified as B. coprophilus (Peck) Hongo. These specimens were found to be identical in morphological characteristics to other B. coprophilus collections from throughout the world. Based on a phylogenetic analysis, sequences from our material clustered within the B. coprophilus clade, which is consistent with our morphological findings. Our three records of B. coprophilus from Pakistan are the first from the country. The distribution of B. coprophilus is discussed and a morphological comparison with closely related taxa is provided.


Sampling sites and morphological characterization.
We collected Bolbitius coprophilus from alluvial plains in three locations in Punjab province, Pakistan: two in Khanewal and one in Nankana Sahib (Fig. 1). These locations were ideal for agricultural activities. The vegetation in these areas is similar to that of a dry desert. Specimens were labelled, photographed, and field notes were prepared. The specimens were dried in a fan heater at >35 °C before preserved in cellophane paper packages. Macromorphological characterization of basidiomata (color, size, and shape), pileus (color, size, and shape), lamellae (type and color), stipe (color, size, and shape), rhizomorphs, and spore print were based on fresh specimens, according to Thomas et al. (2001), Manimohan et al. (2007), and Malysheva et al. (2015).
For microscopic characterization of dried mushroom samples, we prepared fungal slides in 5% aqueous KOH, lactic acid, and trypan blue as the mounting reagent. Details of the basidiospores (shape, size, and position of the germ pore), pileal hyphae, stipe hyphae, cystidia, and basidia were recorded using labomed LX300 binocular microscope at 40× and 100× magnifications. Congo red was used to stain some microscopic characters such as basidiospores, tramal elements, cheilocystidia, caulocystidia, cells/hyphae of pileipellis, and stipitipellis. At least 30 measurements of basidiospores, and 10 measurements of other microscopic characters were recorded per collection. Average and Q values (ratio of length to width) for basidiospores were calculated. Drawings were made with the aid of camera lucida fitted to a light microscope at 40× magnification. The terminology of Pegler (1977), Singer (1986), Atri (2005), and Malysheva et al. (2015) was used to describe the microscopic morphology of our specimens. Specimens are deposited in LAH Herbarium Department of Botany, University of the Punjab, Lahore, Pakistan.

Molecular characterization and phylogenetical analy sis.
For molecular studies, genomic DNA was extracted from the dried lamellae using modified CTAB method of Gardes and Bruns (1993). Internal transcribed spacer region (ITS) of nrDNA was amplified using the ITS1F/ITS4 primers (White et al. 1990). PCR conditions for ITS-rDNA included: initial denaturation at 95 °C for 2 min, 95 °C for 1 min, 52 °C for 1 min, 72 °C for 45 s, and 72 °C for 10 min, followed by a 4 °C soak. Amplifications were verified in 1.5% agarose gel prepared with 1× TAE buffer and dyed with ethidium bromide. Bands were visualized in a Gel Documentation System. PCR products were then purified and sequenced at Macrogen in Korea. Consensus sequences were generated using the BioEdit sequence alignment editor version 7.2.5 using forward and reverse primers (Hall 1999). Initial BLAST search was performed at the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). Following the results of a BLAST search and published literature (Malysheva et al. 2015), closely related sequences were downloaded from GenBank, and multiple alignment was performed using Clustal W in MUSCLE alignment software (Edgar 2004). Maximum likelihood and the Tamura-Nei model of sequence evolution were used to build a phylogenetic tree using 1000 bootstrap replicates. Phylogenetic analysis was conducted in MEGA X (Kumar et al. 2018). All newly generated ITS sequences were deposited to the GenBank. ( Taxonomic description. Basidiomata 7.0-20 cm high, bright. Pileus 2-7 cm in diameter, 1-3 cm high, convex to campanulate when young, flat at maturity, umbonate; umbo extended, milky white to pale brown, with distinct pinkish tinge when young, light brownish grey with age; surface viscid, fragile; margins wavy, split at maturity, flesh thin, non-deliquescent; pileal veil absent. Lamellae 0.2-0.35 cm broad, free, crowded, unequal, somewhat narrow to moderately broad, white when young, pink to brown with age, fragile, deliquescent, gill edges curled with age. Stipe 7.0-18.5 cm long, 0.6-0.8 mm broad, tubular, hollow, fragile, pale yellow, unchanging with time, silky, shiny, fibrillose, thin towards the top, broader towards the base, centrally attached with the pileus. Base equal. Taste and odor not quite distinct. Spore print reddish brown. Rhizomorphs present.
Phylogenetic results. ITS dataset contains 34 nucleotide sequences used in molecular phylogenetic analysis, including 32 ingroup sequences of Bolbitius and two outgroup sequences (Conocybe apala (Fr.) Arnolds JX968209 and Galeropsis desertorum Velen. & Dvorák AY194534). Thirty-three sequences, based on the dataset of Malysheva et al. (2015), were retrieved from Gen-Bank, and we added three new generated sequences. In our maximum-likelihood analysis of 779 positions, 393 positions were constant, 169 parsimony-uninformative, and 317 were variable. A phylogenetic tree with a superior log likelihood value (−3716.39) is shown in Figure 5. The genus has been shown to be monophyletic in almost all phylogenetic studies (Moncalvo 2002;Rees et al. 2003;Golden et al. 2005;Matheny et al. 2006). Based on morphological and phylogenetic analyses, our specimens were identified as B. coprophilus. In the B. coprophilus clade, the sequences from our collections clustered with those from Italy and Russia. This relationship is strongly supported by a significant bootstrap value of 99.

Morphological comparison with closely related taxa.
Bolbitius coprophilus is characterized by a broad pileus (4.0-7.0 cm), which is pale with a distinct pinkish tinge and a shape that usually varies from convex or campanulate when young and flat at maturity (Watling 1994;Amandeep et al. 2013); the gills are free and non-deliquescent, and the basidiospores are ellipsoid to ovoid (11.4-17.1 × 9.9-11.4 µm) (Szczepkowski et al. 2009;Malysheva et al. 2015). This species prefers to grow on organic substrates that are rich in nutrients, such as dung or compost.
The macroscopic and microscopic characteristics of our specimen are consistent with the taxonomic characteristics of B. coprophilus (Singer 1979;Watling 1982;Hausknecht and Krisai-Greilhuber 2003;Amandeep et al. 2013). A morphological comparison of B. coprophilus reported from different regions of the world is given in Table 1.