Research Article |
Corresponding author: Shah Hussain ( shahpk85@gmail.com ) Academic editor: Bryn Dentinger
© 2018 Shah Hussain, Muhammad Usman, Najam-ul-Sehar Afshan, Habib Ahmad, Junaid Khan, Abdul Nasir Khalid.
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.
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: 111-126. https://doi.org/10.3897/mycokeys.39.26743
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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.
Coprinellus section Domestici, Coprinellus sect. Micacei, coprinoid fungi, taxonomy
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 (
The genus Coprinellus, with approximately 80 described species, represents an independent lineage in Psathyrellaceae (
Previously, only 18 species of coprinoid mushrooms have been reported from Pakistan (
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.
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
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 (
Consensus sequences were generated from both forward and reverse primer reads in BioEdit sequence alignment editor version 7.2.5 (
Phylogenetic inference was conducted using Bayesian and Maximum Likelihood (ML) methods. For Bayesian inference, we used BEAST 1.6.2 (
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
The diagnostic features of Coprinellus campanulatus are: campanulate pileus with greyish-olive tinge, surface with glistening clusters of micaceous veil at maturity, 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.
PAKISTAN: Khyber Pakhtunkhwa, Qaldara, Dargai, Malakand, 480 m alt., gregarious on wood chip, 14 Aug 2014, S. Hussain, SH144 (
The epithet “campanulatus” (Latin) refers to the campanulate shape of the pileus of this species.
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, narrow, 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.
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. Pleurocystidia 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 composed of globose to subglobose cells, 50–90 µm diam., slightly thick-walled, yellowish-brown in KOH. Caulocystidia absent. Clamp connections rarely present.
Gregarious on woody litter under Morus alba, so far only known from lowland northern Pakistan.
PAKISTAN: Khyber Pakhtunkhwa, Malakand, Qaldara, on woody pasture, 14 August 2014, S. Hussain, SH144 (SWAT SHP144).
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 (
The most important features of Co. disseminatus-similis are: pileus parabolic to campanulate, greyish-brown, with umbonate centre; surface pruinose to pulverulent, with sparse micaceous-glistening veil, bright white, deeply plicate from centre 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.
PAKISTAN: Khyber Pakhtunkhwa, Malakand, Sarogai, 450 m alt., gregarious on wood chips, 23 Sept 2014, S. Hussain, SHCr3w (SWAT-SHCr3w, holotype); GenBank accession ITS: MH753670.
“Similis” (Latin) meaning like, referring to the similarity of the new species to Coprinellus disseminatus.
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 margin; 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 membranous. 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.
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, ellipsoid 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 narrowly utriform to utriform pileocystidia, 118−165 × 23−28 µm, light-brownish to hyaline, smooth. Veil elements 20–40 µm, globose to subglobose, greyish-brown, smooth. Clamp connection not observed.
Gregarious on leaf litter under Populus alba and Morus alba, so far only known from lowland northern Pakistan.
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).
The new species would be placed in sect. Setulosi because of its pileocystidia. 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 section Micacei that lack such pileocystidia, underlining the need to update the formal description of the sections. Both these species share basidiospore morphology. However, 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 golden brown incrustations (
The distinguishing features of Coprinellus pakistanicus are: light yellowish-green to greyish-yellow pileus, surface smooth with sub-membranous context, basidiospores 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.
PAKISTAN: Punjab, Pabbi Forest Park, 286 m alt., 11 Aug 2016, M. Usman and Abdul N. Khalid, MU37 (Holotype LAH35323); GenBank accession ITS: MH366736.
The specific epithet “pakistanicus” refers to the holotype locality of this species.
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 margin; centre depressed to slightly papillate, orange yellow (7.5YR 6/8); context sub-membranous, light greyish (10Y 5/2). Lamellae free, crowded, regular, dark brown to blackish, 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.
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 = 1.3; in face view, broadly ellipsoid, obovoid to phaseoliform, in side view, ovoid, ellipsoid 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 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.
Scattered on moist soil, under trees of Acacia nilotica and A. modesta, so far only known from lowland northern Pakistan.
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).
In phylogenetic analyses, Coprinellus pakistanicus forms Clade III, adjacent to the Sabulicola and Eurysporoid clades of
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 (
The new species Coprinellus tenuis can be recognised by its thin and membranous 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.
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.
“tenuis” (Latin) meaning thin, referring to the membranous pileus of the new species.
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 reddish-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.
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. Pileocystidia 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.
Scattered on leaf litter under Acacia modesta, so far only known from lowland northern Pakistan.
PAKISTAN. Khyber Pakhtunkhwa: Malakand, Qaldara, on leaf litter under Acacia modesta, 10 July 2014, S. Hussain, SH10 (SWAT SH-10).
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 (
The genus Coprinellus is one of the most species-rich genera in Psathyrellaceae, with approximately 80 described species (
In the phylogeny we present here, based on ITS sequences, the genus is recovered in seven clades (Figure
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 species are represented with bold fonts and T represents the holotype collection.
Clade V includes species of sect. Micacei, along with Co. disseminatus and our new species Co. disseminatus-similis, reflecting the Micacei clade of
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, yellow-brown chains of inflated, ellipsoid or globose cells (thin-walled and hyaline in Co. flocculosus) and correspond to section Domestici. "Coprinus maysodisporus" in
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.
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 International Research Support Initiative Program (IRSIP).