MycoKeys

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MycoKeys 39: 41–61 (2018)
The genus Coprinellus in Pakistan 41
doi: 10.3897/mycokeys.39.26743 RESEARCH ARTICLE
http://mycokeys.pensoft.net Launched to accelerate biodiversity research

The genus Coprinellus (Basidiomycota; Agaricales) in

Pakistan with the description of four new species

Shah Hussain1, Muhammad Usman2, Najam-ul-Sehar Afshan3, Habib Ahmad4,

Junaid Khan1, Abdul Nasir Khalid2

1 Centre for Plant Sciences and Biodiversity, University of Swat, Swat, Pakistan 2 Department of Botany, Uni-
versity of the Punjab, Lahore 54590, Pakistan 3 Centre for Undergraduate Studies, University of the Punjab,
Lahore 54590, Pakistan 4 Islamia College Peshawar, Pakistan

Corresponding author: Shah Hussain ([email protected])

Academic editor: Bryn Dentinger | Received 17 May 2018 | Accepted 28 August 2018 | Published 11 September 2018

Citation: Hussain S, Usman M, Afshan N-ul-S, Ahmad H, Khan J, Khalid AN (2018) The genus Coprinellus
(Basidiomycota; Agaricales) in Pakistan with the description of four new species. MycoKeys 39: 41–61. https://doi.
org/10.3897/mycokeys.39.26743

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 Domes-
tici; 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.

Keywords
Coprinellus section Domestici, Coprinellus sect. Micacei, coprinoid fungi, taxonomy

Copyright Shah Hussain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
42 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

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 thin-
fleshed 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), Bol-
bitiaceae (3) and Agaricaceae (2) (Matheny et al. 2006, Nagy 2011, Nagy et al. 2011,
Tóth et al. 2013).
The genus Coprinellus, with approximately 80 described species, represents an inde-
pendent lineage in Psathyrellaceae (Redhead et al. 2001, Walther et al. 2005, Vašutová
et al. 2008, Padamsee et al. 2008, Nagy et al. 2011, 2012, 2013, 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 Do-
mestici (Singer) D.J. Schaf. has a veil on the pileus in the form of floccose scales, con-
sisting 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 Pa-
kistan (Ahmad 1980, Hussain et al. 2016, 2017, 2018). These include two species
of Coprinus (C. comatus (O.F. Müll.) Pers., C. hookeri Berk.); four of Coprinellus (Co.
disseminatus (Pers.) J.E. Lange, Co. marculentus (Britzelm.) Redhead, Vilgalys & Mon-
calvo, Co. micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson, Co. radians (Desm.)
Vilgalys, Hopple & Jacq. Johnson); five of Coprinopsis (Cop. atramentaria (Bull.) Red-
head, Vilgalys & Moncalvo, Cop. jonesii (Peck) Redhead, Vilgalys & Moncalvo, Cop.
lagopus (Fr.) Redhead, Vilgalys & Moncalvo, Cop. macropus (Berk. & Broome) Red-
head, Vilgalys & Moncalvo, Cop. patouillardii (Quél.) G. Moreno); and seven of Para-
sola (P. auricoma (Pat.) Redhead, Vilgalys & Hopple, P. glabra Hussain, Afshan, Ahmad
 The genus Coprinellus in Pakistan 43

& Khalid, P. lilatincta (Bender & Uljé) Redhead, Vilgalys & Hopple, P. malakandensis
Hussain, Afshan & Ahmad, P. plicatilis (Curtis) Redhead, Vilgalys & Hopple, P. pseu-
dolactea Sadiqullah, Hussain & Khalid, P. setulosa (Berk. & Broome) Redhead, Vilgalys
& Hopple).
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 pro-
vides species descriptions based on morphological characters and molecular phyloge-
netic 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.

Materials and methods

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 pho-
tographed, 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 micro-
scope (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 measure-
ments were rounded to the nearest 0.5 µm. Basidiospore measurements are presented
as: length range × breadth range × width range. Q values were calculated as: Q1 =
length divided by breadth; Q2 = 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 H2O 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
44 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Figure 1. Basidiomata of species of Coprinellus. A–B Coprinellus disseminates-similis (holotype SHCr3W)

C–D Coprinellus tenuis (holotype SHP10) E Coprinellus campanulatus (holotype SH144). The arrow
shows remnants of membranous annulus. Scale bars: 20 mm.
 The genus Coprinellus in Pakistan 45

Figure 2. Basidiomata of Coprinellus pakistanicus Holotype (MU37). Scale bar: 20 mm.

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 (Ap-
plied 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 com-
piled 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 search-
es 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 Ram-
baut 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
46 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

and the starting tree was randomly generated. Four independent runs were under-
taken. 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 TreeAnnota-
tor 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 pos-
terior probability (BPP) values were ≥ 0.95.

Results
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). Clade II corresponded
to Sabulicola clade with a single species Co. sabulicola L. Nagy, Házi, Papp & Vágvölgyi
with strong statistical support (1/100). Clade III was the new species Coprinellus pa-
kistanicus, forming an independent lineage (1/100). Clade IV corresponded to Eu-
rysporoid clade with strong support (1/100). Clade V consisted of species of the Micacei
clade of Nagy et al. (2012), including Co. disseminatus (morphologically placed in sec-
tion Setulosi) along with species of morphological section Micacei and recovered with
strong statistical support (1/99). The two new species Coprinellus campanulatus and
Co. disseminatus-similis fall in this clade. Coprinellus campanulatus formed a sister clade
(weak statistical support) with Co. micaceus (Bull.) Vilgalys, Hopple & Jacq. Johnson
and Co. truncorum (Scop.) Redhead, Vilgalys & Moncalvo and would be placed in
morphological section Micacei. Coprinellus disseminatus-similis (1/100) formed a sister
clade with Co. disseminatus (Pers.) J.E. Lange, adding a further setulose species to this
group. Clades VI and VII collectively consisted of species of the Domestici clade of
Nagy et al. (2012), including species that would be placed morphologically in section
Setulosi. The fourth new species, Co. tenuis, formed a sister clade (1/100) with Co. cur-
tus (Kalchbr.) Vilgalys, Hopple & Jacq. Johnson.

Figure 3. Phylogenetic inference of Coprinellus species inferred from 97 ITS sequences, with species
names following GenBank accessions, specimen voucher numbers and country. Values above branch node
represent Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap (MLP), the new spe-
cies are represented with bold fonts and T represents the holotype collection.
 The genus Coprinellus in Pakistan 47

Co. pallidus JN159522 NL4218 Hungary

1/100
Co. pallidus JN159521 NL1556 Hungary
Co. pallidus HQ846989 NL0625 Hungary
Co. callinus FN396105 NL1931 Hungary
Co. callinus JN159518 Ulje1204 Netherlands
Co. congregatus JN159554 NL2138 Hungary
Co. congregatus FM878013 NL0588 Sweden
1/100
Co. congregatus JN159550 NL1221 Hungary
Co. congregatus JN159552 NL1433 Hungary
Co. hiascens FM878018 NL2536 Norway
1/96
Co. hiascens JN159526 NL2598 Hungary
0.95/-
Co. hiascens GU227720 NL1350 Hungary
Co. hiascens JN159525 NL1349 Hungary
1/100 Co. cinereopallidus HQ847001 NL0177 Hungary
Co. cinereopallidus JN159524 Ulje1163 Netherlands
0.96/86
Co. bisporiger HQ846974 WU7403 Hungary
Co. bisporiger JN159520 Daams71-98 Hungary
1/99 Co. impatiens JN159511 NL0568 Hungary
Co. impatiens FM163177 NL1164 Hungary
1/99 Co. fuscocystidiatus HQ846977 NL2720 Norway
Co. fuscocystidiatus JN159514 NL3622 Sweden
1/100
Co. sassii JN159509 NL1237 Hungary Clade I
Co. sassii FN396101 NL1495 Hungary

Section Setulosi
Co. uljei JN159513 NL2492 Sweden
0.97/98
Co. uljei JN159515 NL3985 Slovakia
0.96/91
Co. uljei HQ846982 NL0157 Sweden
0.95/- Co. plagioporus HQ846981 NL1365 Hungay
0.95/85 Co. plagioporus JN159512 NL1086 Hungary
Co. radicellus GU227718 NL0957 Norway
Co. radicellus GU227717 NL2121 Sweden
1/99
Co. radicellus GU227716 NL0594 Sweden
1/98 0.99/94 Co. radicellus GU227719 NL3168 Sweden
1/100 Co. pellucidus GU227714 NL2928 Slovakia
1/100
Co. pellucidus GU227713 NL1076 Hungary
-/87
Co. pellucidus FM878023 NL2344 Hungary
Co. brevisetulosus GU227709 NL1965 Hungary
0.95/- 1/100 Co. bisporus FN396107 NL2512 Sweden
Co. bisporus JN159517 WU6011 Sweden
1/97
Co. bisporus GU227705 NL0158 Sweden
Co. sabulicola JN159558 NL2906 Hungary
Co. sabulicola JN159559 NL1027 Hungary
1/100 Clade II
Co. sabulicola JN159557 NL1560 Hungary
Co. sabulicola HQ847007 NL1763 Hungary
1/- Co. pakistanicus sp. nov. MU37 Pak T
1/100
Co. pakistanicus sp. nov. MU39 Pak Clade III
Co. pakistanicus sp. nov. MU07 Pak
Co. sclerocystidiosus JN159539 NL0797 Hungary
Co. sclerocystidiosus JN159538 NL1022 Hungary
1/96
Co. sclerocystidiosus JN159537 NL1444 Hungary
0.96/85
Co. sclerocystidiosus HQ846991 NL1018 Hungary
Co. aff. eurysporus HQ846995 Uljé1191 Netherlands
1/100Co. eurysporus JN159534 Arnolds4845 Netherlands
Clade IV

Co. eurysporus HQ846992 NL1761 Hungary

0.97/93 Co. aff. eurysporus JN159540 NL3418 Hungary
Co. angulatus JN159535 NL0906 Hungary
1/100 Co. angulatus JN159536 Arnolds99-22 Netherlands
0.95/-
Co. angulatus HQ846994 NL1934 Hungary
1/100 Co. subimpatiens JN159542 NL0162 Hungary
Co. subimpatiens HQ846993 ToK 27/8-08 Hungary
Co. micaceus JN159565 NL2744 Hungary
48 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Co. subimpatiens HQ846993 ToK 27/8-08 Hungary

Co. micaceus JN159565 NL2744 Hungary
Co. micaceus JN159564 NL2739 Hungary
Co. micaceus JN159566 NL4253 Hungary
0.95/87 Co. xylophilus JN159563 FVBD1726 Hungary
Co. micaceus JN159567 NL3656 Hungary

Section Micacei
Co. micaceus FN396104 NL1939 Hungary
1/100 Co. campanulatus sp. nov. SH144 Pak T
Co. campanulatus sp. nov. SHP144 Pak
-/88 Co. micaceus GU227721 NL3888 Hungary

Clade V
1/100 Co. truncorum JN159562 NL1101 Hungary
1/99
Co. truncorum FM878007 NL1294 Sweden
1/99 Co. disseminatus JN159561 NL3401 Hungary
0.99/92
Co. disseminatus JN159560 NL0786 Hungary
1/100 Co. disseminatus FM87801 NL2337 Sweden
1/100 Co. disseminatus-similis sp.nov. SHCr3w Pak T
1/100
Co. disseminatus-similis sp.nov. SHCr3b Pak
Co. disseminatus-similis sp.nov. SH15 Pak
Co. verrucispermus JN159577 NL2146 Hungary
Co. silvaticus HQ846986 NL3035 Hungary
Co. xanthothrix JN159578 TOK12808 Netherlands
0.99/96
Co. xanthothrix HQ847044 NL3417 Hungary
0.98/91
Co. radians HQ847045 NL3896 Sweden
Co. radians JN159579 NL1373 Sweden

Section Domistici
-/81
1/100 Co. domesticus JN159580 TOK21808 Netherlands

Clade VI
-/90
Co. domesticus HQ847043 Cervenka286 Hungary
0.95/92
Co. sp. SH23 Pak
Co. aff. xanthothrix FM878008 NL1292 Sweden
0.96/-
Co. flocculosus JN159573 NL0838 Hungary
Co. flocculosus JN159575 NL1567 Hungary
0.97/95
Co. flocculosus FN396138 NL1661 Norway
C. maysoidisporus JN159574 FVDB1743 Hungary
1/100 Co. curtus JN159569 NL1490 Hungary
1/100
Co. curtus JN159568 NL1023 Hungary
Clade VII

1/100 Co. curtus FM878016 NL2339 Sweden

1/100 Co. tenuis sp. nov. SHP10 Pak
Co. tenuis sp. nov. SH10 Pak T
Co. deminutus JN159572 NL0761 Hungary
Psathyrella candolleana SH163 Pak
Outgroup

0.04

Figure 3. Continued.

Taxonomy

Coprinellus campanulatus Hussain & Ahmad, sp. nov.

MycoBank: MB825477
Figures 1E and 4

Diagnosis. The diagnostic features of Coprinellus campanulatus are: campanulate pi-

leus with greyish-olive tinge, surface with glistening clusters of micaceous veil at ma-
turity, dark yellowish-brown centre, 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.
 The genus Coprinellus in Pakistan 49

Type. PAKISTAN: Khyber Pakhtunkhwa, Qaldara, Dargai, Malakand, 480 m

alt., gregarious on wood chip, 14 Aug 2014, S. Hussain, SH144 (LAH-SH-144, holo-
type); GenBank accession ITS: MH753667.
Etymology. The epithet “campanulatus” (Latin) refers to the campanulate shape of
the pileus of this species.
Macroscopic characters. Pileus at young stage 3–8 × 3–7 mm, ovoid to parabolic,
light orange-yellow (7.5YR 9/8) to pale orange-yellow (7.5YR 9/4), surface pruinose;
at mature stage 25–40 × 10–15 mm, pulvinate to campanulate, light greyish-olive
(10Y 5/2) to greyish-olive (5Y 3/2), centre slightly campanulate, strong yellowish-
brown (10YR 4/8) to dark yellowish-brown (10YR 1/2); surface finely furfuraceous to
granulose, with clusters of micaceous-glistening veil, bright white, plicate from near
centre to margin; context membranous to submembranous. Lamellae adnexed, nar-
row, with fimbriate edge, crowded with 1–4 series of lamellulae, pale orange-yellow
(7.5YR 9/4) at young stage, dark yellowish-brown at maturity (10YR 2/2). Stipe 70–
100 × 3–7 mm, equal, white, surface smooth, context hollow. Annulus absent with a
membranous layer at the base. Odour pungent. Not tasted.
Microscopic characters. Basidiospores (7.0–)8.0–10.5(–11.5) × (5.0–)5.5–6.5(–
7.0) × (4.0–)4.5–5.5(–6.0) µm, on average 9.4 × 5.7 × 5.1 µm, Q1 = 1.6, Q2 = 1.8,
av. Q = 1.7; in face view mitrifrom, triangular to ellipsoid; in side view cylindrical,
amygdaliform to ellipsoid; dark brown to blackish in KOH, smooth, thick-walled, with
truncate base, apiculus visible, germ-pore 1.5–2.5 µm wide, central, prominent, pale to
hyaline. Basidia 19–29 × 7–10 µm, cylindrical, clavate to subclavate, hyaline, 4-spored.
Cheilocystidia 36–47 × 35–45 µm, globose to subglobose, hyaline, abundant. Pleuro-
cystidia absent. Pileipellis an epithelium of loosely arranged globose to subglobose or
ellipsoid, hyaline to light olive, thin-walled elements, 30–80 × 25–60 µm. Veil com-
posed of globose to subglobose cells, 50–90 µm diam., slightly thick-walled, yellowish-
brown in KOH. Caulocystidia absent. Clamp connections rarely present.
Habitat and distribution. Gregarious on woody litter under Morus alba, so far
only known from lowland northern Pakistan.
Additional specimens examined. PAKISTAN: Khyber Pakhtunkhwa, Malakand,
Qaldara, on woody pasture, 14 August 2014, S. Hussain, SH144 (SWAT SHP144).
Comments. The main distinguishing features of Coprinellus campanulatus are:
campanulate pileus with greyish-olive tinge, dark yellowish-brown centre, veil on pi-
leus 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 phy-
logenetic 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 mor-
phology, Co. campanulatus can be differentiated from Co. micaceus. Basidiospores in
50 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Figure 4. Line drawing of anatomical characters of Coprinellus campanulatus A Basidiospores B Basidia

C Cheilocystidia D Pileipellis E Veil elements. Scale bars: 10 µm (A), 20 µm (B–E).

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. trun-
corum 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).
 The genus Coprinellus in Pakistan 51

Coprinellus disseminatus-similis Hussain, sp. nov.

MycoBank: MB825478
Figures 1A–B and 5

Diagnosis. The most important features of Co. disseminatus-similis are: pileus para-
bolic to campanulate, greyish-brown, with umbonate centre; surface pruinose to pul-
verulent, with sparse micaceous-glistening veil, bright white, deeply plicate from cen-
tre to margin; basidiospores 8.0–9.0 × 5.0–5.5 × 4.5–5.5 µm, in face view ellipsoid to
cylindrical or obovoid, in side view ellipsoid to amygdaliform, smooth, thick-walled,
with truncate base, germ-pore central, 0.5–1.0 µm wide.
Type. PAKISTAN: Khyber Pakhtunkhwa, Malakand, Sarogai, 450 m alt., gregari-
ous on wood chips, 23 Sept 2014, S. Hussain, SHCr3w (SWAT-SHCr3w, holotype);
GenBank accession ITS: MH753670.
Etymology. “Similis” (Latin) meaning like, referring to the similarity of the new
species to Coprinellus disseminatus.
Macroscopic characters. Pileus at young stage cylindrical and closed, 3–5 × 3–7
mm, whitish to light greyish (2.5Y 7/4), surface pruinose, slightly plicate toward mar-
gin; at mature stage 15−20 × 20 mm, parabolic to campanulate to umbonate, light
greyish-brown (7.5YR 6/2) to greyish-yellowish-brown (7.5YR 6/2); with umbonate
centre, in old specimens centre papillate, centre moderate orange (2.5YR 6/8) to
brownish-orange (2.5YR 5/8); surface pruinose to pulverulent, with sparse micaceous-
glistening veil, bright white, deeply plicate from centre to margin; context membra-
nous. Lamellae sinuate to uncinate, distant with 0–2 lamellulae, initially white, fading
with age and dark greyish-brown at maturity. Stipe 20−40 × 1 mm, equal, central,
white, surface pruinose to pulverulent with sparse micaceous-glistening veil, context
hollow, annulus absent. Odour pungent, not tasted.
Microscopic characters. Basidiospores (7.5–)8.0–9.0(–9.5) × (4.5–)5.0–5.5(–
6.0) × (4.0–)4.5–5.5(–6.0) µm, on average 8.5 × 5.2 × 4.9 µm, Q1 = 1.53–1.7, Q2 =
1.7–1.9, av. Q = 1.6; in face view, ellipsoid to cylindrical or obovoid, in side view, el-
lipsoid to amygdaliform, dark brown to blackish in KOH, smooth, thick-walled, with
truncate base, germ-pore central, 0.5–1.0 µm wide. Basidia 26−30 × 7−10 µm, clavate
to cylindrical, 2 to 4−spored, hyaline. Cheilocystidia 70−165 × 11−15 µm, cylindrical,
narrowly clavate to narrowly utriform, some with subcapitate apex, abundant, smooth,
hyaline. Pleurocystidia absent. Pileipellis a loosely arranged euhymeniderm with nar-
rowly utriform to utriform pileocystidia, 118−165 × 23−28 µm, light-brownish to hya-
line, smooth. Veil elements 20–40 µm, globose to subglobose, greyish-brown, smooth.
Clamp connection not observed.
Habitat and distribution. Gregarious on leaf litter under Populus alba and Morus
alba, so far only known from lowland northern Pakistan.
Additional specimens examined. PAKISTAN. Khyber Pakhtunkhwa: Malakand,
Sarogai, on leaf litter under Populus alba and Morus alba, 22 Sept 2014, S. Hussain,
SH-Cr3-b (SWAT SH-Cr3-b).
52 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Figure 5. Line drawing of anatomical characters of Coprinellus disseminatus-similis A Basidiospores B Ba-

sidia C Cheilocystidia D Pileipellis with pileocystidia E Veil elements. Scale bars: 10 µm (A), 20 µm (B–E).

Comments. The new species would be placed in sect. Setulosi because of its pileo-
cystidia. However, as with Co. disseminatus, which it resembles and is close to in the
molecular phylogram, Co. disseminatus-similis falls in a clade along with members of
 The genus Coprinellus in Pakistan 53

section Micacei that lack such pileocystidia, underlining the need to update the formal
description of the sections. Both these species share basidiospore morphology. How-
ever, they differ on the basis of: (i) pileus shape and colour, (ii) cheilocystidia and (iii)
pileocystidia and veil anatomy. In Co. disseminatus, initially the pileus is (sub)globose
or ovoid, then hemispherical or obtusely conical to convex, rarely flat, the fruit bodies
often form in very large groups and are initially very pale, almost white, darkening as
the spores mature; cheilocystidia are absent along most of the gill edge; pileocystidia
are lageniform with cylindrical neck and rounded, rarely subcapitate, apex and large
50–200 × 15–24 µm; and veil elements are globose to subglobose, generally with gold-
en brown incrustations (Uljé and Bas 1991, Uljé 2005). In Co. disseminatus-similis, at
young stage, the pileus is cylindrical and closed, parabolic to campanulate to umbonate
at mature stage, with papillate centre in some old specimens; cheilocystidia are large
(70−165 × 11−15 µm), narrowly clavate to narrowly utriform, some with subcapitate
apex; pileocystidia are narrowly utriform to utriform; and veil elements are globose
to subglobose and smooth. Using ML and Bayesian analyses, Coprinellus verrucisper-
mus (Joss. & Enderle) Redhead, Vilgalys & Moncalvo is another species close to Co.
disseminatus-similis. Spores in Co. verrucispermus are substantially larger (11.0–14.5 ×
7.0–9.0 µm), ellipsoid to slightly amygdaliform, chestnut brown, apiculus slight, warty
with perisporial sac and central germ pore (Uljé and Bas 1991, Keirle et al. 2004).

Coprinellus pakistanicus Usman & Khalid, sp. nov.

MycoBank: MB825483
Figures 2 and 6

Diagnosis. The distinguishing features of Coprinellus pakistanicus are: light yellowish-

green to greyish-yellow pileus, surface smooth with sub-membranous context, basidi-
ospores 8.5–11.5 × 6.5–8.0 × 5.5–6.5 µm, on average 10 × 7.4 × 6.2 µm, in face view
broadly ellipsoid, obovoid to phaseoliform, in side view ovoid, ellipsoid to obovoid,
base not truncate, apiculus visible in side view, germ-pore central.
Type. PAKISTAN: Punjab, Pabbi Forest Park, 286 m alt., 11 Aug 2016, M. Us-
man and Abdul N. Khalid, MU37 (Holotype LAH35323); GenBank accession ITS:
MH366736.
Etymology. The specific epithet “pakistanicus” refers to the holotype locality of
this species.
Macroscopic characters. Pileus 25–35 mm diam, convex to plan, with depressed
centre, light yellow green (2.5GY 8/6) to greyish-greenish-yellow (7.5Y 7/4); surface
smooth with sparsely pulverulent to granulose, deeply plicate from centre towards mar-
gin; centre depressed to slightly papillate, orange yellow (7.5YR 6/8); context sub-mem-
branous, light greyish (10Y 5/2). Lamellae free, crowded, regular, dark brown to black-
ish, with 0–2 series of lamellulae. Stipe 27–50 × 1 mm, central, hollow, smooth, white,
with slightly bulbous base. Annulus and volva absent. Odour and taste not recorded.
Microscopic characters. Basidiospores (7–)8.5–11.5(–12) × (6.0–)6.5–8.0(–8.5)
× (–5.0)5.5–6.5(–7.0) µm, on average 10 × 7.4 × 6.2 µm, Q1 = 1.4, Q2 = 1.6, av. Q =
54 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Figure 6. Line drawing of anatomical characters of Coprinellus pakistanicus A Basidiospores B Basidia

C Pileocystidia D Cheilocystidia E Pileal hyphae F Veil elements. Scale bars: 10 µm (A), 20 µm (B–F).

1.3; in face view, broadly ellipsoid, obovoid to phaseoliform, in side view, ovoid, ellip-
soid to obovoid, base not truncate, apiculus slightly visible, germ-pore central, smooth,
slightly thin-walled, dark brown to blackish in KOH. Basidia 13.5–32 × 8.5–12 µm,
clavate to narrowly clavate, hyaline, smooth, 2- to 4-spored, sterigmata up to 4 µm
in length. Cheilocystidia 42–75 × 14–25 µm, cylindrical to lageniform, hyaline with
crystals usually at the apex of cystidium. Pleurocystidia absent. Pileipellis irregular
epithelium, 3.5–7.5 µm diam., pale to hyaline in KOH. Pileocystidia 30–90 × 9–24
 The genus Coprinellus in Pakistan 55

µm, lageniform to cylindrical with tapering neck and obtuse apex, pale to hyaline in
KOH. Veil rounded to globose cells, 15–25 µm diam., slightly thick-walled, yellowish
in KOH. Clamp connection present.
Habitat and distribution. Scattered on moist soil, under trees of Acacia nilotica
and A. modesta, so far only known from lowland northern Pakistan.
Additional specimens examined. PAKISTAN. Punjab: Pabbi Forest Park, 286
m alt., 20 Aug 2016 & 2017, M. Usman, Abdul N. Khalid and A. Hameed, MU07,
MU39 (LAH35324 and LAH35325).
Comments. In phylogenetic analyses, Coprinellus pakistanicus forms Clade III, ad-
jacent to the Sabulicola and Eurysporoid clades of Nagy et al. (2012) and morphologi-
cally would be placed in sect. Setulosi. The new species is compared with the following
species of sect. Setulosi: Co. bisporus (J.E. Lange) Vilgalys, Hopple & Jacq. Johnson, Co.
cinereopallidus L. Nagy, Házi, Papp & Vágvölgyi, Co. congregatus (Bull.) P. Karst., Co.
pellucidus (P. Karst.) Redhead, Vilgalys & Moncalvo, Co. radicellus Házi, L. Nagy, Papp
& Vágvölgyi and Co. sabulicola L. Nagy, Házi, Papp & Vágvölgyi.
In Co. bisporus, the pileus is small, up to 20 mm diam., ochre or pale brown; with
dark red-brown basidiospores; cheilocysticdia subglobose, ovoid, ellipsoid to broadly
utriform and smaller in size (24–40 × 16–23 µm) when compared to Co. pakistanicus
(Prydiuk 2010). In Co. cinereopallidus, basidiospores are larger 12.1 × 6.5 µm, ellipsoid
to subamygdaloid, not lentiform (Nagy et al. 2012). Similarly, Co. congregatus with
pileus up to 20 mm in diam., cream-coloured, at centre ochre-brown to light brown,
cheilocystidia subglobose, ovoid to ellipsoid, sometimes utriform, 22–50 × 15–36 µm
in size (Prydiuk 2010). Coprinellus pellucidus with substantially small pileus (7 mm
diam.), basidiospores 9.25 × 4.75 µm, elongate-ellipsoid to cylindrical-ellipsoid, with
subglobose cheilocystidia, 20–25 × 14–22 µm (Prydiuk 2010). Pileus in Co. radicellus
up to 10 mm diam., cream coloured to dark melleous-brown, expanding to convex
applanate with uprolled margin, basidiospores on average 9.48 × 4.91 µm, reddish-
brown, ellipsoid to subcylindrical, with globose to subglobose or clavate cheilocys-
tidia, 9–20 × 8–14 µm in size (Házi et al. 2011). Co. sabuilcola has concave, warm
reddish-brown pileus, basidiospores on average 17.3 × 10.9 µm, cheilocystidia 17–32
× 12.5–27 µm, globose to vesiculose or broadly ellipsoid (Nagy et al. 2012).

Coprinellus tenuis Hussain, sp. nov.

MycoBank: MB825479
Figures 1C–D and 7

Diagnosis. The new species Coprinellus tenuis can be recognised by its thin and mem-
branous pileus, surface glabrous and furred, deeply plicate towards margin; lamellae
sinuate to uncinate; basidiospores 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, 1.5–2 µm wide.
56 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

Figure 7. Anatomical features of Coprinellus tenuis A Basidiospores B Basidia C Pileocystidia D Caulo-

cystidia E Cheilocystidia F Veil cells. Scale bars: 10 µm (A), 20 µm (B–F).

Type. PAKISTAN: Khyber Pakhtunkhwa, Malakand, Qaldara, 430 m alt., solitary

on leaf litter, 7 July 2014, S. Hussain, SHP10 (SWAT-SH-P10, holotype); GenBank
accession ITS: MH753663.
Etymology. “tenuis” (Latin) meaning thin, referring to the membranous pileus of
the new species.
Macroscopic characters. Pileus 15–20 mm diam, pulvinate to convex to plane,
light greyish-brown (7.5YR 5/2) to light brown (5YR 6/4); surface glabrous, furred,
deeply plicate from centre towards margin; centre truncately conical, moderate red-
 The genus Coprinellus in Pakistan 57

dish-orange (10R 5/8) to greyish-reddish-orange (2.5YR 5/6); context membranous.

Lamellae sinuate to uncinate, distant, with 0–2 series of lamelullae, light greyish-brown
(7.5YR 5/2) to light brown (5YR 6/4), lamellae edge blackish and fimbriate to eroded.
Stipe 40–60 × 1 mm, equal, cylindrical, surface scabrous, white, translucent, fragile,
context hollow.
Microscopic characters. Basidiospores (9.0–)10.5–14.5(–15.5) × (7.5–)8.0–9.5(–
10.5) × (5.0–)6.5–8.5(–9.0) µm, on average 13.1 × 9.0 × 7.8 µm; Q1 = 1.25–1.49, Q2
= 1.57–1.63, av. Q = 1.45; in face view, broadly ellipsoid to ovoid, in side view, slightly
pyriform to ellipsoid, usually with truncate base, apiculus mostly not visible, germ-
pore eccentric, 1.5–2 µm wide, wall 1.5 µm thick, dark brown to almost black. Basidia
22–24 × 9–12 µm, clavate, 2- to 4-spored, hyaline in KOH. Cheilocystidia 22–30 ×
19–28 µm, rounded to globose, abundant, hyaline. Pleurocystidia absent. Pileocyst-
idia 78–94 × 10–12 µm, lageniform to cylindrical with rounded apex, elongated rod
shape neck with rounded enlarged base, hyaline in KOH. Caulocystidia 50–67 × 9–11
µm, narrowly clavate to clavate, with rounded to obtuse apex, cylindrical base. Veil
comprised of rounded to subglobose cells, arranged in short chain, thick-walled with
encrusted walls, dark brown, with terminal cell 17–23 × 12–15 µm.
Habitat and distribution. Scattered on leaf litter under Acacia modesta, so far only
known from lowland northern Pakistan.
Additional specimens examined. PAKISTAN. Khyber Pakhtunkhwa: Mala-
kand, Qaldara, on leaf litter under Acacia modesta, 10 July 2014, S. Hussain, SH10
(SWAT SH-10).
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), el-
lipsoid 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
58 Shah Hussain et al. / MycoKeys 39: 41–61 (2018)

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-mono-
phyletic 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 spe-
cies, forming a basal group in this phylogeny. Amongst the species, there are Coprinel-
lus eurysporus (M. Lange & A.H. Sm.) Redhead, Vilgalys & Moncalvo, Co. sclerocystid-
iosus (M. Lange & A.H. Sm.) Vilgalys, Hopple & Jacq. Johnson, Co. subimpatiens (M.
Lange & A.H. Sm.) Redhead, Vilgalys & Moncalvo.
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 Domes-
tici 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, yellow-
brown 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
 The genus Coprinellus in Pakistan 59

(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.

Acknowledgments
We greatly thank Dr Mykola Prydiuk (M.G. Kholodny Institute of Botany, Ukraine)
for review of the manuscript. S.H. thanks all members of Farlow Herbarium (Harvard
University) for facilitating his stay during this study. The authors also thank Derek J.
Schafer (UK) for the critical review of the manuscript. Financial support for this study
was provided by the Higher Education Commission of Pakistan under the Interna-
tional Research Support Initiative Program (IRSIP).

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