Corresponding author: Shah Hussain (
Academic editor: M. Neves
Hussain S, Ahmad H, Ullah S, Afshan N, Pfister DH, Sher H, Ali H, Khalid AN (2018) The genus
Species of
Basidiospore shape and size are the main descriptive features for species identification in
Previously, five species of this genus (
Specimens were collected from Malakand, Shangla and Swat districts of Khyber Pakhtunkhwa, Pakistan in summer seasons, 2013–2017. Basidiomata were photographed, tagged and field notes were made.
For anatomical studies slides were prepared in 5% aqueous KOH (w/v). Microscopic features such as size and shape of basidiospores, basidia, cheilocystidia, pleurocystidia and pileipellis were studied under a light microscope (MX4300H, Meiji Techo Co., Ltd., Japan) with at least 20 structures measured in each instance. Cheilocystidia and pleurocystidia were observed and measured by cutting the gill edge from the rest of gill to avoid confusion between the two types of cystidia. In the case of basidiospores, 50 spores were measured in face view and/or side view through 1000× magnification with a calibrated optical micrometer and measurements were rounded to the nearest 0.5 µm. Basidiospores measurements are presented as follows: length range × breadth range × width range. Q values were calculated as: Q1 = length divided by breadth; Q2 = length divided by width (
We extracted genomic DNA using the DNeasy Plant Mini Kit (Qiagen, Redwood City, California, USA.). We amplified nuc rDNA internal transcribed spacer (
Voucher numbers, geographic origins and GenBank Accession numbers for the specimens included, in boldface are sequences produced in this study.
Species | Geographic origin | Voucher number | GenBank Accessions | ||
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28S | ||||
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Hungary | NL0268 |
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Hungary | NL0087 |
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Hungary | NL0465 |
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Hungary | NL0286 |
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Hungary | NL0285 |
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Netherlands | Uljé 1269 (L) |
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Netherlands | L146 holotype |
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Netherlands | Uljé 904 (L) |
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Hungary | NL0466 |
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Sweden | NL0095 |
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Germany | NL0283 |
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Sweden | NL0288 |
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Hungary | NL6601 |
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USA | MICH232885 |
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Latvia | KuP6.2.2.1 |
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Hungary | NL0683 |
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Hungary | NL0660 |
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Hungary | NL0472 |
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Hungary | NL0667 |
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Pakistan | SH4 |
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Pakistan | SHP2 |
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Pakistan | SHP9 |
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Hungary | NL0086 |
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Sweden | NL0096 |
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Denmark | C 19683 |
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Sweden | NL1924 |
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Pakistan |
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Pakistan |
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Hungary | NL0677 |
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Hungary | NL0280 |
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Hungary | NL0490 |
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Sweden | NL0477 |
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Hungary | NL0295 |
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Sweden | NL0097 |
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Hungary | NL0075 |
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Hungary | NL0284 |
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Netherlands | LBrier:1051999 |
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Hungary | L32 |
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Norway | NL3167 |
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Norway | NL3621 |
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Hungary | NL4175 |
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Hungary | NL0287 |
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Hungary | NL2952 |
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Hungary | NL2937 |
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Sequence length varied from 631 bp (SHP-8) to 644 bp (SHP-11) for our 10 new
The results of phylogenetic analyses of
Phylogeny of
Phylogeny of
Using Bayesian and
Phylogeny of
The diagnostic features of
PAKISTAN. Khyber Pakhtunkhwa Province, Malakand, Qaldara, scattered under herbaceous plants, 480 m alt., 15 August 2014, S. Hussain SHP5 (holotype:
Pileus 20–30 mm diam, initially subglobose, later convex to hemispheric; at first smooth, without veil, the center glabrous at maturity, becoming deeply plicate towards the margin; light gray (2.5R 6/2) to moderate gray (7.5R 6/2); disc slightly depressed, strong reddish orange (7.5R 5/12). Lamellae free, fairly crowded, separated from the stipe by pseudocollarium, 0–2 lamellulae, regular, initially whitish, then dark brown becoming black at maturity, finally losing turgor and collapsing. Stipe 30–60 × 2–3 mm, central, equal, smooth, slightly sub-bulbous at the base, hollow, white, fragile, without annulus.
Basidiomata of
Anatomical features of
Basidiospores (13)14.5–16.5(18) × (7.5)9.5–11.5(15) × (9)8.0–10.5(11.5) µm, on average 15.8 × 10.9 × 10.1 µm, Q1 = 1.3–1.5, Q2 = 1.4–1.6, avQ = 1.4; in face view broadly ovoid to oblong, some with rhomboidal outline, in side view ellipsoid, germ-pore eccentric and upto 1.5 µm diam; wall upto 1.5 µm thick, dark brown to blackish in KOH. Basidia 28–41 × 10–13 µm, clavate to cylindrical, 4-spored, hyaline in KOH. Cheilocystidia 50–63 × 17–23 µm, oblong, ellipsoid, narrowly to broadly utriform, hyaline. Pleurocystidia 60–75 × 22–38 µm, clavate to broadly lageniform, hyaline. Pileipellis hymeniform, consisting of clavate cells 47–60 × 13–16 µm, bright yellow at the base in KOH. Clamp connections present mostly in the pileipellis and at the base of basidia. Sclerocystidia absent.
Saprotrophic, scattered under herbaceous plants on grass land. So far only known from the lowland of northern Pakistan. This species is, however, common in lowland northwest Pakistan.
Specific epithet ‘
PAKISTAN, Khyber Pakhtunkhwa Province, Malakand, Qaldara, 480 m alt., 28 May 2015, S. Hussain SHP23 (
The distinguishing features of the new species
Characteristics distinguishing
Taxa | Pileus diam; and pileus color | Stipe size | Basidiospores size, length/breadth (Q1), length/width (Q2) ratios | Basidiopores shape and germ-pore position | References |
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20–30 mm diam, light-gray to moderate-gray | 30–60 × 2–3 mm | 15.8 × 10.9 × 10.1 µm; Q1 = 1.3–1.5, Q2 = 1.4–1.6, avQ = 1.4 | In face view broadly ovoid to oblong, some with rhomboidal outline, in side view ellipsoid; germ-pore eccentric, upto 1.5 µm diam. | Observed during this study. |
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15–20 mm diam, orange-brown to red-brown | 75 × 1.5 mm | 15.83 × 15.42 × 10.63 µm;Q1 = 1–1.15, Q2 = 1.4–1.5 | In face view rounded triangular to quadrangular, rarely subglobose to ovoid, in side view ellipsoid to amygdaliform; germ-pore eccentric, upto 2.7µm diam. | |
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35 mm diam, dark light grayish-brown | 100 × 3 mm | 9.36 × 7.85 × 5.9 µm;Q1 = 1.1–1.2, Q2 = 1.4–1.6 | In face view ovoid to rounded triangular, rhomboid to mitriform, in side view amygdaliform; germ- pore eccentric, 1.5 µm diam. | |
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15–23 mm diam, yellow-brown to dull red-brown | 140 × 3 mm | 10.73 × 8.81 × 6.73 μm; Q1 = 1.02–1.25, Q2 = 1.66–2.10 | In face view mostly broadly ovoid to subglobose, rarely angular to rounded triangular, in side view broadly ellipsoid to ellipsoid; germ-pore eccentric, upto 1.8 μm diam. | |
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15–25 mm diam, initially yellow-brown to dull-brown, moderate gray at maturity | 30–50 × 1 mm | 14.0 × 11.3 × 9.7 µm; Q1 = 1.3–1.5, Q2 = 1.4–1.5, avQ = 1.4 | In face view mostly rounded triangular to heart shape, rarely ovoid to subglobose, in side view ellipsoid to oblong, germ-pore eccentric, upto 1.5 µm diam. | Observed during this study |
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30–50 mm diam, dark reddish brown, not plicate | 70–100 ×2–4 mm | 14.4 × 10.8 × 9.2 µm; Q1 = 1.3–1.4, Q2 = 1.3–1.5 | In face view rounded triangular to quadrangular, in side view ellipsoid to amygdaliform; germ-pore eccentric, upto 2.5 µm diam. | |
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35 mm diam, chestnut-brown to red-brown or ochre-tawny | 50–100 × 1.5–3 mm | 16.5 × 10.66 × 8.5 μm; Q1 = 1.40–1.78, Q2 = 1.83–1.95 | In face view ellipsoid to broadly ellipsoid, rarely ovoid, in side view ellipsoid to subamygdaliform; germ-pore slightly eccentric, upto 2.3 µm diam. | |
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2–5 × 1–3 mm, tawny-orange to cinnamon-brown | 50 × 0.5 mm | 7.0–10.6 × 6.5–10.0 × 5.9–6.6 μm | In face view heart-shape to rounded triangular, irregularly globose, in side view ellipsoid; sometimes broader than long; germ-pore eccentric. | |
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35 mm diam, yellow-brown to dull pinkish-brown | 30–70 × 0.5–3 mm | 12.41 × 8.21 × 7.14 μm; Q1 = 1.34–1.67, Q2 = 1.61–1.86 | In face view mostly leminiform-subhexagonal, rarely ovoid, in side view ellipsoid to subamygdaliform; germ-pore eccentric, 2.3 µm diam. | |
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20–30 mm diam, yellow-brown to grayish red-brown | 40–60 × 1 mm | 14.44 × 11.83 × 9.72 μm, Q1 = 1.16–1.27, Q2 = 1.46–1.68 | In the face view rounded triangular to subglobose, in side view ovoid to amygdaliform; germ-pore eccentric, upto 2.5 μm diam. |
Pileus yellowish brown to dull brown, deeply plicate towards margin; disc subumbilicate, deep orange yellow; lamellae free, pseudocollarium absent; basidiospores 13.5–14.5 × 10.5–12.0 × 9.5–10.5 µm, in face view rounded triangular to heart shape, rarely ovoid to subglobose, in side view ellipsoid to oblong, with eccentric germ-pore of 1.5 µm diam; sclerocystidia absent.
PAKISTAN, Khyber Pakhtunkhwa Province, Shangla, solitary to scattered under
Pileus 15–25 mm diam, initially obtusely conical, later becoming applanate and deeply plicate towards margin; yellowish brown to dull brown (10YR 6/4) when young, moderate gray (7.5R 6/2) on maturity; disk subumbilicate, deep orange-yellow (7.5YR 6/12). Lamellae free, 0–2 lamellulae, distant, pseudocollarium absent, initially dark gray, becoming blackish at maturity and finally losing turgor and collapsing. Stipe 30–50 × 1 mm, equal, smooth, grayish-brown, translucent, hollow, without annulus.
Basidiomata of
Anatomical features of
Basidiospores (12.0)13.5–15.0(16.0) × (9.5)10.5–12.0(13.0) × (7.5)9.5–10.5(12.0) µm, on average 14.0 × 11.3 × 9.7 µm, Q1 = 1.3–1.5, Q2 = 1.4–1.5, avQ = 1.4; in face view mostly rounded triangular to heart shaped, rarely ovoid to subglobose, in side view ellipsoid to oblong, with eccentric germ pore of 1–1.5 µm diam, dark to blackish in KOH. Basidia 24–31 × 8–12 µm, clavate to cylindrical, 4-spored. Cheilocystidia 55–70 × 22–29 µm, clavate, broadly clavate to broadly cylindrical. Pleurocystidia 44–67 × 19–23 µm, utriform to lageniform. Pileipellis hymeniform, consisting of clavate cells, 33–38 × 17–22 µm. Clamp connections present. Sclerocystidia absent.
Solitary to scattered on humus rich loamy soil, under
The prefix “
PAKISTAN, Khyber Pakhtunkhwa Province, Shangla, 1480 m alt., 9 July 2014, Sadiq Ullah SU413 (
The new species belongs to
Pileus 15–30 mm diam, convex to broadly convex, deeply plicate towards the margin, light grayish-brown (2.5YR 5/2) to grayish reddish-brown (2.5YR 3/2); disc indistinctly umbonate to umbilicate, dark reddish orange (7.5R 4/8) to grayish reddish orange (2.5YR 5/6). Lamellae free and remote, pseudocollarium absent, closed, initially concolorous with pileus, later on dark black, finally losing turgor and collapsing. Stipe 40–65 × 2–5 mm, equal, smooth, central, hollow, without annulus.
Anatomical features of
Basidiospores (10.5)12.5–13.5(15.0) × (8.0)8.5–9.5(10.0) × (7.0)8.0–9.0(10.0) μm, on average 12.9 × 9.0 × 8.5 μm, Q1 = 1.5–1.6, Q2 = 1.3–1.4, avQ = 1.5; in face view subcylindrical to ellipsoid or ovoid, in side view ellipsoidal to elliptical; with central germ-pore, 2–2.5 μm diam, wall 1.5 µm thick, strong reddish-brown to blackish in KOH. Basidia 30–38 × 7–11 μm, clavate to subcylindrical, 2- or 4-spored. Cheliocystidia 33–45 × 12–25 μm, subclavate to subglobose, abundant. Pleurocystidia 30–40 × 11–15 μm, cylindrical to clavate, pale brown at the base, rare. Sclerocystidia 90–170 × 4–7 μm, dark brown, with acute apex and bulbous base, wall 1.5–2 μm thick. Clamp connection present.
Pakistan, Khyber Pakhtunkhwa Province, Malakand, Kharkai, alt. 460 m, scattered in grassland under herbaceous plants, 10 August 2014, S. Hussain SHP6 (
Pileus 20–30 mm diam, hemispheric to pulvinate, smooth, deeply plicate towards margin, yellow brown (2.5R 9/2–5R 9/2) to grayish red brown (2.5R 7/2–5R 7/2); disc slightly depressed, brilliant orange (2.5YR 8/12 – 5YR 8/12) to strong orange (2.5YR 6/12–5YR 6/12). Lamellae free, separated from the stipe by pseudocollarium, distant, lamellae edge blackish while faces initially concolorous with the pileus but later on black and finally losing turgor and collapsing. Stipe 40–60 × 1 mm, equal, smooth, white, fragile, without annulus with slightly sub-bulbous base.
Anatomical features of
Basidiospores (12)13–14.5(15.5) × (11.5)12–12.5(13.5) × (6.0)8.5–11(13.5) μm, on average 14.5 × 12.5 × 9.9 μm, Q1 = 1.1–1.2, Q2 = 1.2–1.5, avQ = 1.3; in the face view rounded triangular to subglobose, in side view ovoid to amygdaliform, with eccentric germ-pore of 2–2.5 μm diam; wall upto 2 µm thick, dark brown in KOH. Basidia 17–22 × 6–9 μm, 4-spored, cylindrical to clavate, hyaline in KOH. Cheilocystidia 25–29 × 23–26 μm, rounded to globose, rare. Pleurocystidia 34–40 × 11–14 μm, cylindrical to subclavate. Pileipellis of clavate cells, 33–37 × 9–12 μm, with rounded apex, bright yellow at the base. Clamp connections present in most of the tissues. Sclerocystidia absent.
PAKISTAN, Khyber Pakhtunkhwa Province, Malakand, Qaldara, alt. 430 m, scattered under herbaceous plants, 11 August 2014, S. Hussain SHP-8, SHP-31, SHP-12 (
The incorporation of molecular phylogenetics has significantly benefited the systematic and taxonomic studies of coprinoid mushrooms. These mushrooms are deliquescent or, at least, have morphological characters like gill cystidia, coloration and surface features that are quickly changed during basidioma maturation. So morphology based taxonomy of coprinoid mushrooms is always a difficult task for mushroom biologists. In the present study two new species of mushroom genus
On account of absence of sclerocystidia in the pileipellis, both the new species
Similarly, the second new species
It is concluded form this study that low altitude mountains of northern Khyber Pakhtunkhwa Province of Pakistan are rich in the diversity of
We greatly acknowledge Derek J. Schafer (UK) and Laszlo G. Nagy (Synthetic and Systems Biology Unit, Institute of Biochemistry, BRC, Szeged, Hungary) for the critical review of the manuscript. Financial support for this study was provided by the Higher Education Commission of Pakistan under International Research Support Initiative Program (IRSIP). Molecular work was carried out in Molecular Lab, Department of Organismic and Evolutionary Biology, Harvard University.