The genus Coprinellus (Basidiomycota; Agaricales) in Pakistan with the description of four new species

Abstract Mushrooms with a thin-fleshed pileus that becomes plicate on opening, deliquescent lamellae and dark brown to blackish basidiospores are commonly called coprinoid mushrooms. The genus Coprinellus is one of the important lineages of coprinoid mushroom in the family Psathyrellaceae. Species-level taxonomy in Coprinellus is based mainly on the presence or absence and the structure of veil and cystidia on the pileus, of cystidia on the lamellae and on basidiospore morphology. In this study, four new species of Coprinellus (Co.campanulatus, Co.disseminatus-similis, Co.pakistanicus and Co.tenuis) are described from Pakistan. Species descriptions are based on morphological and molecular data. Phylogenetic analyses based on nuc rDNA ITS region show that the new species Co.campanulatus and Co.disseminatus-similis are clustered in a clade including members of section Micacei; Co.tenuis falls in a clade with members of section Domestici; and Co.pakistanicus recovered in a separate clade adjacent to other recently described clades of genus Coprinellus. Morpho-anatomical descriptions of the new species and comparison with closely allied taxa are provided. With this study, the number of known species of Coprinellus in Pakistan has reached eight.


Introduction
Coprinoid fungi form an important group of macrofungi and are striking in the field because of their deliquescent lamellae. Coprinoid mushrooms have generally a thinfleshed pileus that becomes plicate on opening with deliquescent lamellae and dark brown to blackish basidiospores with germ-pore (Schafer 2010). The evolutionary lineages of coprinoid taxa are set amongst those that are not, or not fully coprinoid. Fully coprinoid genera include: Coprinus Pers. in Agaricaceae; Coprinellus P. Karst., Coprinopsis P. Karst. and Parasola Redhead, Vilgalys & Hopple in Psathyrellaceae. Certain species of Leucocoprinus Pat. (L. birnbaumii, L. brebissonii, L. fragilissimus) in Agaricaceae have a coprinoid combination of characters (Nagy 2011). Within the Bolbitiaceae, coprinoid taxa include: species of Conocybe Fayod belonging to section Candidae Watling, few Bolbitius Fr. species (B. coprophilus, B. elegans, B. lacteus, B. reticulatus, B. subvolvatus, B. titubans) and two species of Galerella Earle (G. floriformis, G. nigeriensis). Nevertheless, taken together, at least eight independent lineages with coprinoid fruiting bodies have hitherto been identified in the Psathyrellaceae (3), Bolbitiaceae (3) and Agaricaceae (2) (Matheny et al. 2006, Nagy 2011, Tóth et al. 2013. The genus Coprinellus, with approximately 80 described species, represents an independent lineage in Psathyrellaceae (Redhead et al. 2001, Walther et al. 2005, Vašutová et al. 2008, Padamsee et al. 2008, 2012, Örstadius et al. 2015. These mushrooms are common saprotrophs of, for example, wood chip, leaf-litter and herbivore dung (Schafer 2010). Species of this genus are divided into three sections on the basis of veil anatomy and the presence or absence of cap pileocystidia. Section Domestici (Singer) D.J. Schaf. has a veil on the pileus in the form of floccose scales, consisting of chains of fusiform or subglobose cells, often with encrusted walls. In Micacei (Fr.) D.J. Schaf., veil remnants are present in the form of scattered, granulose flocks, often disappearing and consisting of globose cells arising from a matrix of narrow branched hyphae. In Setulosi (J.Lange) D.J. Schaf., the veil may be present or absent, but the pileus and stipe are covered with thin-walled pileocystidia and caulocystidia, respectively (Schafer 2010). However, Nagy et al. (2012) showed that these sections were not entirely consistent with the molecular phylogeny, in particular because clades corresponding to sections Micacei and Domestici each included some setulose species.
Previously, only 18 species of coprinoid mushrooms have been reported from Pakistan (Ahmad 1980, Hussain et al. 2016, 2017, 2018 During explorations of basidiomycetous fungi in Pakistan in 2014-2017, some interesting collections of Coprinellus were encountered. Upon further examination, it was discovered that these collections represent four new species. The current report provides species descriptions based on morphological characters and molecular phylogenetic analyses of nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2 = ITS). With this study, the number of known species in Coprinellus in Pakistan increases to eight.

Sampling and morphology
Samples were collected in August-September 2014-2017, in the Malakand district of Khyber Pakhtunkhwa and Pabbi district of Punjab, Pakistan. Specimens were photographed, tagged and morphological features including size, shape and colour of basidiomata were noted. For colour designations, the Munsell (1975) colour system was followed. For anatomical study, slides were prepared in 5% aqueous KOH (w/v). Anatomical features, including size and shape of basidiospores, basidia, cheilocystidia, pileipellis and position of germ-pore in basidiospores, were studied using a light microscope (MX4300H, Meiji Techo Co., Ltd., Japan). Data of morpho-anatomical features were recorded from at least 20 measurements. In case of basidiospores, at least 50 spores were measured in face view and side view at a magnification of 1000× and measurements were rounded to the nearest 0.5 µm. Basidiospore measurements are presented as: length range × breadth range × width range. Q values were calculated as: Q 1 = length divided by breadth; Q 2 = length divided by width (Nagy et al. 2010). Specimens studied during this work are deposited in the Herbarium of University of the Punjab, Lahore (LAH) and the Herbarium of University of Swat, Swat, Pakistan (SWAT).

DNA extraction, PCR amplification and sequencing
For DNA extraction, we used the DNeasy Plant Mini Kit (Qiagen, Redwood City, California, USA). We amplified nuc rDNA internal transcribed spacer region (ITS) using the primer combination ITS1F/ITS4 (White et al. 1990). The polymerase chain reaction (PCR) was performed in a 25 µl reaction volume: containing 2.5 µl 10× Econo Taq Buffer (Lucigen, Middleton, Wisconsin, USA), 0.5 µl dNTPs, 1.25 µl of each primer (10 µM/µl), 0.125 µl of Econo Taq® DNA Polymerase (Lucigen), 14.375 µl H 2 O and 5 µl DNA template. PCR amplification were performed with 4 min initial denaturation at 95°C, followed by 34 cycles of 50 s at 94°C, 40 s at 54°C, 50 s at 72°C  and a final extension of 7 min at 72°C followed the last cycle. The PCR products were purified using a QIAquick PCR purification kit (Qiagen Inc., Valencia, California, USA). Sequencing was performed using a Bigdye terminator cycle sequencing kit (Applied Biosystems, Foster City, California, USA). Sequencing reactions were purified using Pellet Paint (Novagen, Madison, Wisconsin, USA) and were run on an Applied Biosystems 377 XL automated DNA sequencer. Sequence chromatograms were compiled with Sequencher 4.1 software (GeneCodes Corporation, Ann Arbor, Michigan, USA). Sequences generated for this study are deposited in GenBank (MH366735-MH366737, MH753663-MH753670).

Alignment and phylogenetic analyses
Consensus sequences were generated from both forward and reverse primer reads in Bi-oEdit sequence alignment editor version 7.2.5 (Hall 1999) and then homology searches were performed at the National Center for Biotechnology Information (NCBI) Web site using BLAST. These BLAST results, along with the sequences recently employed in the phylogeny of Coprinellus (Nagy et al. 2012), were used in the phylogenetic analyses. DNA sequences were aligned in Clustal X 2.1 (Larkin et al. 2007). Psathyrella candolleana (Fr.) Maire was used as outgroup. Sequence alignment was deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S23199).
Phylogenetic inference was conducted using Bayesian and Maximum Likelihood (ML) methods. For Bayesian inference, we used BEAST 1.6.2 (Drummond and Rambaut 2007) with a Markov chain Monte Carlo (MCMC) coalescent approach. For tree prior, a Yule-type speciation model (Gernhard 2008) was used in all simulations and the starting tree was randomly generated. Four independent runs were undertaken. Chain length was 20 million generations, with a sampling frequency of 1000. Tracer 1.6 (Rambaut et al. 2014) was used to check the effective sample size (ESS) and burn-in values were adjusted to achieve an overall ESS of ≥200. A Maximum Clade Credibility Tree (MCCT) with 20% burn-in was generated using TreeAnnotator 1.6.2 (Drummond and Rambaut 2007). Maximum Likelihood analyses were run in RAXML-VI-HPC (Stamatakis 2006) under the GTRCAT model. Branch support was calculated by 1000 bootstrap replicates. Nodes were considered strongly supported when the maximum likelihood bootstrap (MLB) values were ≥ 70% and Bayesian posterior probability (BPP) values were ≥ 0.95.

Phylogenetic analyses
The ITS dataset comprises 97 sequences and the resulting alignment was 708 bp in length. Phylogenetic trees reconstructed using both Bayesian and ML methods were mostly congruent with each other. Taxa of Coprinellus were recovered in seven clades ( Figure 3). Clades I-IV consisted of species of section Setulosi, three corresponding to clades described in Nagy et al. (2012). Clade I, corresponding to core Setulosi clade, was recovered with strong statistical support (BPP/ML 1/98   Etymology. The epithet "campanulatus" (Latin) refers to the campanulate shape of the pileus of this species.
Habitat and distribution. Gregarious on woody litter under Morus alba, so far only known from lowland northern Pakistan.
Comments. The main distinguishing features of Coprinellus campanulatus are: campanulate pileus with greyish-olive tinge, dark yellowish-brown centre, veil on pileus in the form of micaceous-glistening clusters which are composed of globose to subglobose cells and basidiospores 8.0-10.5 × 5.5-6.5 × 4.5-5.5 µm, spores mitriform in face view and cylindrical to amygdaliform in side view. Based on veil anatomy, Co. campanulatus belongs in sect. Micacei. Coprinellus micaceus and Co. truncorum are most closely related to Co. campanulatus amongst the species sampled for our phylogenetic analyses. The new species Co. campanulatus with pulvinate to campanulate pileus can be differentiated from Co. micaceus and Co. truncorum, which have broadly convex pilei. At maturity, the pileus is light brown in Co. micaceus and Co. truncorum when compared to Co. campanulatus with greyish-olive pileus. On basis of spore morphology, Co. campanulatus can be differentiated from Co. micaceus. Basidiospores in Co. micaceus are slightly smaller (6.5-10.0 × 4.5-7 µm), lacrimiform to submitriform or mitriform in face view, conical towards base (Keirle et al. 2004, Uljé 2005. In Co. micaceus, voluminous, broadly clavate, (sub)globose to ellipsoid pleurocystidia up to 150 × 70 µm are present, in Co. campanulatus pleurocystidia are absent. Also, in C. micaceus, caulocystidia are abundant, in Co. campanulatus absent. Spores of Co. truncorum are 8.5-9.0 × 5.5-6 µm, ellipsoid in all views, not distinctly lentiform, with very broad central to slightly eccentric germ pore, broadly rounded apex, not truncate, smooth, dark grey to grey brown or black (Keirle et al. 2004, Uljé 2005.
Etymology. "Similis" (Latin) meaning like, referring to the similarity of the new species to Coprinellus disseminatus.
Type Etymology. The specific epithet "pakistanicus" refers to the holotype locality of this species.
Habitat and distribution. Scattered on moist soil, under trees of Acacia nilotica and A. modesta, so far only known from lowland northern Pakistan.
Habitat and distribution. Scattered on leaf litter under Acacia modesta, so far only known from lowland northern Pakistan.
Comments. Coprinellus tenuis with thin membranous pileus, shows similarities with Co. curtus. Both these species can be differentiated on (i) pileus morphology (ii) basidiospore shape and (iii) habitat. Pileus is deeply plicate in both these species, in Co. tenuis pileus is glabrous and furred; however, there is no furcation in the pileus of Co. curtus. Spores in Co. curtus are substantially smaller (8.0-10.0 × 5.5-7.0 µm), ellipsoid to ovoid in face view, narrowly ellipsoid or phaseoliform in side view, apiculus often not visible, with a distinct central to slightly eccentric germ-pore, not truncate. Basidiospores in Co. tenuis are larger (10.5-14.5 × 8.0-9.5 × 6.5-8.5 µm), in face view broadly ellipsoid to ovoid, in side view slightly pyriform to ellipsoid, usually with truncate base, apiculus mostly not visible, with eccentric germ-pore of 1.5-2 µm diam. Coprinellus curtus has a substrate preference and is most commonly collected from herbivores' dung as opposed to Co. tenuis basidioma on leaf litter (Uljé and Bas 1991).

Discussion
The genus Coprinellus is one of the most species-rich genera in Psathyrellaceae, with approximately 80 described species (Kirk et al. 2008, Nagy et al. 2012, Gomes and Wartchow 2014. Species of Coprinellus have been classified in three sections, reflecting earlier sub-sections of Coprinus sensu lato, primarily based on veil anatomy and the presence or absence of cap pileocystidia (Schafer 2010). The most recent phylogenetic study of this genus by Nagy et al. (2012), does not provide evidence for the monophyly of morphologically based sections of previous classifications (Orton and Watling 1979, Uljé 2005, Schafer 2010).
In the phylogeny we present here, based on ITS sequences, the genus is recovered in seven clades (Figure 3). In morphology-based taxonomy, species in section Setulosi have setules on their pilei and the majority of such species recovered as a non-monophyletic lineage consisting of four clades in this study. Clade I, corresponding to core Setulosi clade in the Nagy et al. (2012) phylogeny, is a large group of species with the characteristic setules on the pileus. Clade II corresponds to Sabulicola clade with a single species Co. sabulicola L. Nagy, Házi, Papp & Vágvölgyi. This species bears some unique features compared with other Coprinellus species; amongst these are relatively large basidiospores (15-22 × 10-13 µm), lack of a pedicel on the cystidia, habitat in dry, sandy sites and short, capitate pileocystidia with incrusted base (Nagy et al. 2012). Clade III represents the new species Coprinellus pakistanicus. This species has ellipsoid to phaseoliform basidiospores, cylindrical to lageniform cheilocystidia, pileocystidia lageniform to cylindrical with tapering neck and obtuse apex, veil with rounded to globose cells, slightly thick-walled, clamp connections present amongst most tissues. Clade IV, corresponding to the Eurysporoid clade ( fig. 1 of Nagy et al. 2012), was inferred with strong statistical support (1/100) and consisted of some well-studied species, forming a basal group in this phylogeny. Amongst the species, there are Clade V includes species of sect. Micacei, along with Co. disseminatus and our new species Co. disseminatus-similis, reflecting the Micacei clade of Nagy et al. 2012. It also includes Co. verrucispermus and Co. deliquescens (=Co. silvaticus), which were placed in the Domestici clade in that study, although data would allow a plausible phylogenetic position for those two species in the Micacei clade (Nagy et al. 2012, p.256). Taxa in section Micacei have a veil in the form of glistening mica-like granules, consisting of thin-walled globose cells in a matrix of narrow branched hyphae. The granules can be easily washed off by rain drops, causing difficulties in differentiation (Schafer 2010). Rich veil coverage on the pileus was suggested as a character linking the non-setulose and setulose species in both the Domestici and Micacei clades, the key feature for the Micacei clade being mitriform shaped basidiospores (Nagy et al. 2012).
Clade VI and VII, if taken together, would collectively correspond to the Domestici clade, inferred as a non-monophyletic group in Coprinellus. Species in clade VI have a veil consisting of floccose scales, made up of generally thick-walled, yellowbrown chains of inflated, ellipsoid or globose cells (thin-walled and hyaline in Co. flocculosus) and correspond to section Domestici. "Coprinus maysodisporus" in Nagy et al. 2012 ("Coprinus maysoidisporus" in GenBank) appears to refer to collection FVDB1743 and appears to relate to a collection of a provisionally named species "Coprinus maydisiformis", close to Co. xanthothrix, from Washington State, USA in 1972(Van de Bogart 1975. Clade VII is entirely comprised of species containing thick-walled, encrusted veil cells as well as pileal setules with capitate or swollen apex (Coprinellus curtus, Co. tenuis). These differences between the clades found in our study and those in Nagy 2012 might therefore provide DNA phylogenic support for the morphologically defined section Domestici, but still leave the remaining sections in need of updating, clade VII being a separate Curtus clade.
In the present study, we demonstrated that low-altitude mountains and grasslands of Pakistan are rich in species of Coprienllus. The climatic conditions of these areas of the country are favourable for growth of coprinoid mushrooms. With the description of these four new species, the number of know species of Coprinellus from Pakistan increases to eight.