Mycological surveys reveal two new species of genus Lepiota (Agaricaceae) from the semi‑arid climatic region of Punjab, Pakistan

Abstract

During our studies on the genus Lepiota in Pakistan, we collected two putatively new species from Punjab Province, with distinct morphology, ITS, and 28S of nrDNA profile. L. aurantiopilea is featured by orange to yellow-orange pileus with a reddish-brown umbo, tiny granules on the surface that are concolorous to pileus, absence of annulus, subglobose to oblong, ellipsoid or spurred basidiospores, clavate fusoid-ventricose, utriform cheilocystidia and hymeniderm made up clavate to subfusiform elements. Another new species, L. bahawalnagarensis has a light grayish-brown pileus with dark grayish-brown reddish-brown umbo, grayish-yellow brown zonation on the surface, pale yellow stipe, single-edged annulus, ellipsoid basidiospores, versiform cheilocystidia, hymeniderm pileipellis, and clavate to utriform caulocystidia. Photographs of fresh basidiomata, descriptions, and line drawings of key microscopic features are provided. Morphological characters and phylogenetic trees inferred from nrITS and 28S of nrDNA sequences show that both of our new species clustered within section Liliaceae.

Full text

Vol.:(0123456789)
Mycological Progress (2024) 23:20
https://doi.org/10.1007/s11557-024-01958-0
ORIGINAL ARTICLE
Mycological surveys reveal two new species ofgenus Lepiota
(Agaricaceae) fromthesemi‑arid climatic region ofPunjab, Pakistan
MuhammadAsif1,2 · AimanIzhar1 · AbdulRehmanNiazi1 · AbdulNasirKhalid1 · MalkaSaba2
Received: 4 May 2023 / Revised: 6 February 2024 / Accepted: 7 February 2024
© German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2024
Abstract
During our studies on the genus Lepiota in Pakistan, we collected two putatively new species from Punjab Province,
with distinct morphology, ITS, and 28S of nrDNA profile. L. aurantiopilea is featured by orange to yellow-orange pileus
with a reddish-brown umbo, tiny granules on the surface that are concolorous to pileus, absence of annulus, subglobose
to oblong, ellipsoid or spurred basidiospores, clavate fusoid-ventricose, utriform cheilocystidia and hymeniderm made
up clavate to subfusiform elements. Another new species, L. bahawalnagarensis has a light grayish-brown pileus with
dark grayish-brown reddish-brown umbo, grayish-yellow brown zonation on the surface, pale yellow stipe, single-edged
annulus, ellipsoid basidiospores, versiform cheilocystidia, hymeniderm pileipellis, and clavate to utriform caulocystidia.
Photographs of fresh basidiomata, descriptions, and line drawings of key microscopic features are provided. Morpho-
logical characters and phylogenetic trees inferred from nrITS and 28S of nrDNA sequences show that both of our new
species clustered within section Liliaceae.
Keywords Bahawalnagar· Molecular systematics· Saprotrophic· Taxonomy· Two new taxa
Introduction
Molecular systematic studies of macrofungi in Punjab,
Pakistan, are in progress (Asif etal. 2021, 2022a, b; Izhar
etal. 2022; Rehman etal. 2023). Southern Punjab has a
huge diversity of macrofungi, but unfortunately, it has not
received much attention despite recent taxonomic explo-
rations. This area lies in a semi-arid region with an aver-
age annual rainfall of 205mm with July and August being
the wettest months and an average annual temperature of
42.5°C with June being the hottest month (Amin etal.
2018). During the recent mycological surveys of the area,
some species of macrofungi have been reported (Bashir etal.
2020; Asif etal. 2021, 2022a, b; Niazi etal. 2021; Izhar etal.
2022; Haqnawaz etal. 2022; Khalid 2022).
Lepiota (Pers.) Gray is one of the largest genus in Agari-
caceae, with more than 500 species (Singer 1986; Kirk etal.
2008; Razaq etal. 2012, 2013a,b; Nawaz etal. 2013; Qasim
etal. 2015, 2016; Sysouphanthong etal. 2016; Bashir etal.
2020; Niazi etal. 2021; Asif etal. 2022b; Haqnawaz etal.
2022) distributed globally from tropical to temperate areas
and rarely in deserts and arctic-alpine habitats (Vellinga
2004), and is characterized by relatively small basidiomata,
a pileus having concentric rings of scales, a slender stipe,
free lamellae (Candusso and Lanzoni 1990; Vellinga 2003),
pale dextrinoid spores, absence of pleurocystidia, presence
of cheilocystidia, regular hymenophoral trama, and presence
of clamp connections. Two important features, i.e., pileus
covering and shape of basidiospores, are used to distinguish
the species of Lepiota (Candusso and Lanzoni 1990; Bon
1993; Vellinga 2001a, 2003).
The first species of Lepiota from Pakistan was reported
by Ahmad etal. (1997). Since then, 33 species of Lepiota
have been described from different areas of Pakistan (Ahmad
etal. 1997; Razaq etal. 2012, 2013a,b; Nawaz etal. 2013;
Qasim etal. 2015; 2016; Bashir etal. 2020; Niazi etal. 2021;
Asif etal. 2022b; Haqnawaz etal. 2022). In this paper, we
Section Editor: Zhu-Liang Yang
* Muhammad Asif
m.asif@bs.qau.edu.pk; asifgondal101@gmail.com
1 Fungal Biology andSystematics Research Laboratory,
Institute ofBotany, University ofthePunjab, Quaid-E-Azam
Campus, Lahore54590, Pakistan
2 Plant Mycology Research Laboratory, Department ofPlant
Sciences, Faculty ofBiological Sciences, Quaid-I-Azam
University, Islamabad45320, Pakistan

Mycological Progress (2024) 23:20 20 Page 2 of 18
report two new species, i.e., L. aurantiopilea and L. baha-
walnagarensis, based on morpho-anatomical and molecular
phylogenetic analyses of nrITS and 28S sequences, and both
belong to L. sect. Liliaceae.
Materials andmethods
Sampling site
The holotypes of both species were collected from Haroon-
abad, District Bahawalnagar, located in the vicinity of the
Cholistan desert (29°58′32″ N, 72°91′87″ E) during the hot
and humid rainy season of 2019. The temperature of the col-
lection site varies from a minimum of 11°C to a maximum of
50°C, and the average annual precipitation is 99mm (Ahmed
etal. 2014a, b). The main vegetation of the area includes Dal-
bergia sissoo Roxb., Vachellia nilotica (L.) P.J.H. Hurter &
Mabb., Eucalyptus camaldulensis Dehnh., Azadirachta indica
A. Juss., and Albizia lebbeck (L.) Benth (Asif etal. 2021).
Morphological study
Field photographs of the specimens were taken and habit,
habitat, soil type, and location were noted. Color codes fol-
lowed the Munsell Soil Color Chart (1975), and terminology
follows Vellinga (2001a). The specimens were brought to
the Fungal Biology and Systematics Research Laboratory,
at the Institute of Botany, University of the Punjab (Lahore,
Pakistan), and morphological and microscopic analyses were
carried out. Then, specimens were dried at 40–50 °C tem-
perature and deposited in the LAH Herbarium, Institute of
Botany, University of the Punjab, Lahore, Pakistan.
For microscopic analysis, free hand sections were made
of the dried material rehydrated in 5% KOH and stained with
Congo Red and Melzer’s reagent following the microscopic
standard protocol of Liang and Yang (2011), Cai etal. (2018),
and Liang etal. (2018). The microscopic characteristics, such
as the size and shape of basidiospores, cheilocystidia, basidia,
structure, and hyphae of pileus covering and stipe covering,
were observed with a light microscope (CXRII, Labomed
Labo America Inc., Fremont, CA, USA) through an HDCE-
X5 microscopic camera under 400 × and oil immersion
1000 × magnification. The measurements of 25 basidiospores,
basidia, and cheilocystidia were taken from the basidiomata
of each collection. The representations [n/b/p] describe those
measurements that were taken on “n” spores from “b” basidio-
mata of “p” collections. The following abbreviations are used:
“l” for length, “w” for width, “avl” for average length, “avw”
for average width, “Q” for the quotient of length and width,
and “Qav” for average quotient (Bas 1969; Yu etal. 2020).
DNA extraction, PCR, andsequencing
The DNA was extracted from the dried tissue of the speci-
mens using a modified 2% CTAB protocol (Zhao etal. 2011).
The primer combinations used were ITS1F/ITS4 for nrITS
and LR0R/LR5 for 28S (Vilgalys and Hester 1990; White
etal. 1990; Gardes and Bruns 1993). The PCR amplification
was performed in a thermal cycler (Perkin-Elmer, Applied
Biosystems). The PCR program followed as preliminary
denaturation for 5 min at 94 °C, then 35 cycles of the follow-
ing denaturation at 94 °C for 50 s, annealing at 52 ℃ for 50
s, and extension at 72 °C for 1 min. The PCR amplified prod-
ucts were purified and sequenced using the same primer com-
binations as used in PCR. The newly generated sequences
were then deposited in GenBank under accession numbers
OQ282886–OQ282891 (ITS) & OQ282882–OQ282885
(28S) (L. aurantiopilea) and OQ658766–OQ658768 (ITS)
& OQ658771–OQ658773 (28S) (L. bahawalnagarensis).
Sequence alignment andphylogenetic analyses
The final combined nrITS-28S dataset comprised 152
sequences including Melanophyllum haematospermum (Bull.)
Kreisel (ecv2249, ecv2517, DAOM197183) as outgroups
(Table1) (Vellinga 2001a, b, c; Moncalvo etal. 2002). For
the final analysis, sequences were retrieved from GenBank
based on maximum percentage identity from BLAST results
and from phylogenetic studies on Lepiota (Hosen etal. 2016;
Sysouphanthong etal. 2020; Bashir et al. 2020; Asif etal.
2022a, b). The combined nrITS-28S matrix was aligned
using MUSCLE v.3.8 (Edgar 2004), and manual adjustments
were done to cut out the leading and trailing gaps only 1
using BioEdit v 7.2.5. The final alignment was deposited in
TreeBASE (http:// www. treeb ase. org/) under the accession
numbers http:// purl. org/ phylo/ treeb ase/ phylo ws/ study/ TB2:
S30561. On the CIPRES Portal v. 3.1 (Miller etal. 2010),
maximum likelihood (ML) analyses of the combined nrITS-
28S dataset were carried out using RAxML-HPC2 v 8.1.11
(Stamatakis 2014). In the ML analysis, 1000 bootstrap iterations
were attained as statistical supports with rapid bootstrapping,
and significant support was considered to be ≥ 70%. FigTree v
1.4.3 (Rambaut 2014) was used for displaying the phylogenetic
trees and then exported to Adobe Illustrator.
Results
Phylogenetic analysis
In BLAST results, the nrITS sequences of L. auran-
tiopilea showed 82% maximum similarity with Lepiota

Mycological Progress (2024) 23:20 Page 3 of 18 20
Table 1 Taxa used in the phylogenetic analyses of combined nrITS-28S sequences. The new sequences are in bold
Taxon Voucher GenBank accession Origin References
ITS LSU
Lepiota albogranulosa T14 LK932284 No data Pakistan Qasim etal. 2015
Lepiota albogranulosa T19 LK932285 No data Pakistan Qasim etal. 2015
Lepiota alopochroa MFLU 090178 HQ647294 No data Thailand Sysouphanhong etal. 2012
Lepiota andegavensis MV13.9543 ON567747 No data Italy Unpublished
Lepiota andegavensis 8-X-1994, P.D.H. Roux 2121 AY176461 No data France Vellinga 2003
Lepiota angusticystidiata HKAS 50064 KP177192 No data China Liang etal. 2018
Lepiota angusticystidiata HKAS 50085 KP177193 No data China Liang etal. 2018
Lepiota angusticystidiata L. L. Zhou 46 KP177194 No data China Liang etal. 2018
Lepiota attenuate HKAS50110 EU681776 No data China Liang etal. 2011
Lepiota attenuate HKAS42320 EU681777 No data China Liang etal. 2011
Lepiota aurantiopilea LAH37660 OQ282886 OQ282882 Pakistan This study
Lepiota aurantiopilea LAH37662 OQ282888 OQ282884 Pakistan This study
Lepiota aurantiopilea LAH37663 OQ282889 No data Pakistan This study
Lepiota aurantiopilea LAH37664 OQ282890 OQ282885 Pakistan This study
Lepiota aurantiopilea LAH37665 OQ282891 No data Pakistan This study
Lepiota aurantiopilea (T) LAH37661 OQ282887 OQ282883 Pakistan This study
Lepiota aureofulvella MFLU 090183 HQ647293 No data Thailand Sysouphanhong etal. 2012
Lepiota bahawalnagarensis LAH37785 OQ658765 OQ658773 Pakistan This study
Lepiota bahawalnagarensis LAH37786 OQ658767 OQ658772 Pakistan This study
Lepiota bahawalnagarensis (T) LAH37787 OQ658766 OQ658771 Pakistan This study
Lepiota bengalensis Iqbal 825 KU563148 KU563150 Bangladesh Hosen etal. 2016
Lepiota bengalensis Iqbal 860 KU563149 No data Bangladesh Hosen etal. 2016
Lepiota boudieri MCVE:474 FJ998388 No data USA Unpublished
Lepiota boudieri HKAS 5803 EU416280 No data China Liang etal. 2010
Lepiota brunneogranulosa LAH36803 OL331705 OL331514 Pakistan Asif etal. 2022b
Lepiota brunneogranulosa LAH36804 OL331704 OL331515 Pakistan Asif etal. 2022b
Lepiota brunneogranulosa LAH36954 OL331703 OL331516 Pakistan Asif etal. 2022b
Lepiota brunneoincarnata xsd08106 FJ481017 No data China Unpublished
Lepiota brunneoincarnata MHHNU 31032 MK095190 No data China Long etal. 2020
Lepiota brunneosquamula HKAS 50142 KP177195 KP177200 China Liang etal. 2018
Lepiota brunneosquamula HKAS 50035 KP177196 MK651668 China Liang etal. 2018
Lepiota castaneidisca E.C. Vellinga 2312 AF391061 No data USA Vellinga 2001a
Lepiota castaneidisca E.C. Vellinga 2395 AF391062 No data USA Vellinga 2001a
Lepiota cholistanensis L14 MK087674 MK106095 Pakistan Bashir etal. 2020
Lepiota cholistanensis L98 MK087675 No data Pakistan Bashir etal. 2020

Mycological Progress (2024) 23:20 20 Page 4 of 18
Table 1 (continued)
Taxon Voucher GenBank accession Origin References
ITS LSU
Lepiota cingulum LAH36290 MN240457 No data Pakistan Razzaq etal. 2022
Lepiota cingulum 20-IX-1995, M. Enderle (L) AY176359 No data Germany Vellinga 2004
Lepiota clypeolaria KUN-HKAS 87248 MN810123 MN810080 China Hou and Ge 2020
Lepiota clypeolaria KUN-HKAS 106012 MN810122 No data China Hou and Ge 2020
Lepiota clypeolaria KUN-HKAS 75696 MN810127 MN810084 China Hou and Ge 2020
Lepiota coloratipes Zhu L. Yang 4951 KC819622 No data China Vizzini etal. 2014b
Lepiota coloratipes Zhu L. Yang 4790 KC819621 No data China Vizzini etal. 2014b
Lepiota coloratipes NL-5353 No data MK278270 USA Varga etal. 2019
Lepiota condylospora MFLU 090173 JN224823 No data Thailand Sysouphanhong etal. 2012
Lepiota condylospora MFLU 090048 JN224822 No data Thailand Sysouphanhong etal. 2012
Lepiota cortinarius MCVE:865 FJ998393 No data France Unpublished
Lepiota cortinarius MCVE:708 FJ998391 No data France Unpublished
Lepiota cortinarius HMAS144769 No data MK685367 China Unpublished
Lepiota cristata E.C. Vellinga 2714 GQ203807 No data USA Vellinga 2010
Lepiota cristata E.C. Vellinga 2515 AF391052 No data USA Vellinga 2001a
Lepiota cristata E.C. Vellinga 2611 AF391045 No data USA Vellinga 2001b
Lepiota cristata Z.L. Yang 2238 AF391044 No data China Vellinga 2001a
Lepiota cristata KUN: HKAS 45053 No data JN940283 China Unpublished
Lepiota cristata KUN: HKAS 61649 No data JN940284 China Unpublished
Lepiota cristatoides H.A. Huijser s.n. AY176363 No data Netherlands Vellinga 2004
Lepiota cystophoroides E.C. Vellinga 2142 AF391031 No data USA Vellinga 2001a
Lepiota echinacea KUN-HKAS 105582 MN810155 MN810104 China Hou & Ge 2020
Lepiota echinacea KUBOT-KRMK-2020–99 MW486623 No data India Unpublished
Lepiota echinella MCVE:2320 FJ998397 No data USA Unpublished
Lepiota echinella 4-X-1998, H.A. Huijser s.n. AY176366 No data Belgium Vellinga 2004
Lepiota elaiophylla S.D. Russell HRL2240 MH979467 No data Canada Unpublished
Lepiota elaiophylla E.C. Vellinga 1006, 15-IX-1986 AF391024 No data USA Vellinga 2001b
Lepiota elseae AH40487 KP640556 No data Spain Caballero etal. 2015
Lepiota elseae AC1456 KP640557 No data Spain Caballero etal. 2015
Lepiota ermine S38 OL527680 No data Germany Sarawi etal. 2022
Lepiota ermine E.C. Vellinga 2290 (L) AY176470 No data Netherlands Vellinga 2003
Lepiota ermine NL-3095 No data MK278263 Hungry Varga etal. 2019
Lepiota farinolens 21-X-1997, E.C. Vellinga 2146 (L) AY176368 No data France Vellinga 2004
Lepiota feline UBC F-27583 MF955074 No data Canada Unpublished
Lepiota feline UBC F-29655 MF955091 No data Canada Unpublished

Mycological Progress (2024) 23:20 Page 5 of 18 20
Table 1 (continued)
Taxon Voucher GenBank accession Origin References
ITS LSU
Lepiota forquignonii E.C. Vellinga 2284 (L) AY176370 No data USA Vellinga 2004
Lepiota grangei 4-X-1998, H.A. Huijser AY176471 No data Belgium Vellinga 2003
Lepiota griseovirens MCVE:13747 FJ998403 No data Unpublished
Lepiota haroonabadensis LAH36801 MZ172897 No data Pakistan Niazi etal. 2021
Lepiota haroonabadensis LAH36944 MZ172898 No data Pakistan Niazi etal. 2021
Lepiota haroonabadensis LAH36802 MZ172899 No data Pakistan Niazi etal. 2021
Lepiota helveola S.D. Russell HRL2181 MH979466 No data Canada Unpublished
Lepiota helveola PUL00030876 OM522689 No data USA Unpublished
Lepiota himalayensis KP-63 HE614898 No data Pakistan Razaq etal. 2013a, b
Lepiota himalayensis LAH230810 NR155308 No data Pakistan Razaq etal. 2012
Lepiota ignicolor 17-X1-999, H.A. Huijser AY176472 No data Netherlands Vellinga 2003
Lepiota ignicolor Recolte personnelle MT123903 No data France Unpublished
Lepiota ignivolvata RITF2419 MK651618 No data China Unpublished
Lepiota ignivolvata E.C. Vellinga 2127 (L) AY176473 No data France Vellinga 2004
Lepiota ignivolvata RITF2198 MK651619 No data China Unpublished
Lepiota jacobi KUN-HKAS 48802 MN810138 No data China Hou & Ge 2020
Lepiota jacobi RITF2423 MK651600 MK651661 China Unpublished
Lepiota lahoensis T18 KT182475 No data Pakistan Qasim etal. 2016
Lepiota lahoensis TQ15 KT186609 No data Pakistan Qasim etal. 2016
Lepiota lilacea M. Enderle (L) GQ203822 No data Italy Vellinga 2010
Lepiota lilacea E.C. Vellinga 2451 AY176379 AY176380 USA Vellinga 2004
Lepiota lilacea MEL:2305323 No data OL652960 Australia Unpublished
Lepiota magnispora M. Enderle, 2-IX-1994 AF391005 No data Germany Vellinga 2001a
Lepiota magnispora Z.L. Yang 2521 AF391006 No data China Vellinga 2001a
Lepiota magnispora HKAS46369 No data MK651672 China Unpublished
Lepiota magnispora HKAS52426 No data MK685368 China Unpublished
Lepiota metulispora HMAS61859 EU681778 MK651673 Hong Kong Unpublished
Lepiota metulispora HMGID25584 MK651632 MK651674 Hong Kong Unpublished
Lepiota neophana rh24 08/27/07 (ISC) GQ375546 No data USA Vellinga 2010
Lepiota neophana rh39 08/11/07 (ISC) GQ375547 No data USA Vellinga 2010
Lepiota neophana RITF2402 No data MK651661 China Unpublished
Lepiota ochraceofulva M.M. Nauta (coll. E.C. Vellinga 2267) AF391032 AY176387 Netherlands Vellinga 2001b
Lepiota ochraceofulva E.C. Vellinga 2273 AY176386 No data Netherlands Vellinga 2004
Lepiota ochraceofulva NL-2973 No data MK278267 USA Varga etal. 2019
Lepiota omninoflava KUN-HKAS 106734 MN810157 MN810092 China Hou & Ge 2020

Mycological Progress (2024) 23:20 20 Page 6 of 18
Table 1 (continued)
Taxon Voucher GenBank accession Origin References
ITS LSU
Lepiota oreadiformis MSM#0016 KJ906506 No data Pakistan Razzaq etal. 2022
Lepiota oreadiformis PAM14110915 KT315651 No data France Vidal etal. 2015
Lepiota pilodes E.C. Vellinga 3234 (UC) EF080865 No data USA Vellinga 2006
Lepiota pilodes SDR-MM1578 MF461043 No data USA Unpublished
Lepiota pilodes 19-IX-1998, H.A. Huijser AY176476 No data Netherlands Vellinga 2003
Lepiota psalion WU 5152, Holotype No data MG581699 Austria Vizzini etal. 2019
Lepiota psalion CAG P.11_9/7.68b No data MG581700 Italy Vizzini etal. 2019
Lepiota psalion HMJAU3799 GU199362 No data China Liang etal. 2011
Lepiota psalion 15-IX-1999, H.A. Huijser AY176390 No data Netherlands Vellinga 2004
Lepiota pseudolilacea E.C. Vellinga 2278 (L) AY176392 No data Netherlands Vellinga 2004
Lepiota pseudolilacea HKAS 8288 EU416304 No data China Liang etal. 2010
Lepiota pyrochroa E.C. Vellinga 2006 (L) AY176477 No data Netherlands Vellinga 2003
Lepiota revelata HKAS50115 GU199359 No data China Liang etal. 2011
Lepiota revelata HKAS50111 MK651616 MK651669 China Unpublished
Lepiota rhodophylla E.C. Vellinga 3026 (UC) EF080864 No data USA Vellinga 2006
Lepiota rhodophylla 8-XII-2000, E.C. Vellinga 2610 (UCB) AY176480 No data USA Vellinga 2003
Lepiota sanguineofracta TO-HG2916 KF879620 MG581701 Italy Vizzini etal. 2014a, b
Lepiota sanguineofracta TO-HG2917 KF879621 MG581702 Italy Vizzini etal. 2014a
Lepiota scaberula E.C. Vellinga 2595 (holotype) AF391030 No data USA Vellinga 2001a
Lepiota scaberula UC1999143 No data MK278271 USA Varga etal. 2019
Lepiota scaberula E.C. Vellinga 2307 (UCB) No data AY176396 USA Vellinga 2004
Lepiota sindhudeltana LAH37025 OM987446 No data Pakistan Haqnawaz etal. 2022
Lepiota sindhudeltana LAH37026 OM987447 No data Pakistan Haqnawaz etal. 2022
Lepiota sosuensis ANGE 259 (JBSD) MN250948 No data Dominican Republic Unpublished
Lepiota sosuensis CA3 KR022005 No data Dominican Republic Justo etal. 2015
Lepiota sosuensis ANGE 519 (JBSD) MN250947 No data Dominican Republic Unpublished
Lepiota sp. MCVE:480 FJ998390 No data Italy Unpublished
Lepiota sp. KUN-HKAS 106733 MN810156 MN810105 China Hou & Ge 2020
Lepiota sp. MFLU 090142; P. Sysouphanthong PS068 JN224821 No data Thailand Unpublished
Lepiota sp. KUN-HKAS 106781 MN810132 MN810086 China Hou & Ge 2020
Lepiota spheniscispora E.C. Vellinga 2298 AF391000 No data USA Vellinga 2001a
Lepiota spheniscispora E.C. Vellinga 2429 AF391002 No data USA Vellinga 2001a
Lepiota squamulodiffracta CA21 KR022006 No data Dominican Republic Justo etal. 2015
Lepiota subalba CWU(MYC)8416 OK041522 No data Ukraine Unpublished
Lepiota subalba 19-IX-1998, E.C. Vellinga 2242 (L) AY176489 No data Netherlands Vellinga 2003

Mycological Progress (2024) 23:20 Page 7 of 18 20
Table 1 (continued)
Taxon Voucher GenBank accession Origin References
ITS LSU
Lepiota subgracilis E.C. Vellinga 1783 (L) AY176490 No data Netherlands Vellinga 2004
Lepiota subincarnata 19-IX-1998, E.C. Vellinga 2234 (L) AY176491 No data Netherlands Vellinga 2003
Lepiota subincarnata CUP-070736 MW417401 No data USA Unpublished
Lepiota thiersii E.C. Vellinga 2602 AY176492 No data USA Vellinga 2003
Lepiota thiersii E.C. Vellinga 2589 GQ203817 No data USA Vellinga 2010
Lepiota thrombophora HKAS41003 EU681780 No data China Liang etal. 2011
Lepiota thrombophora HKAS48451 EU681779 No data China Liang etal. 2011
Lepiota thrombophora RITF548 No data MK685365 China Unpublished
Lepiota thrombophora RITF541 No data MK685364 China Unpublished
Lepiota tomentella RITF570 MK651653 MK651689 China Unpublished
Lepiota tomentella H.A. Huijser (L) EF080868 No data Netherlands Vellinga 2006
Lepiota vellingana MCR09 HE974764 No data Pakistan Nawaz etal. 2013
Lepiota vellingana MCR52 HE974765 No data Pakistan Nawaz etal. 2013
Lepiota xanthophylla E.C. Vellinga 2240 (L) AY176405 No data Netherlands Vellinga 2004
Lepiota xanthophylla TUB 011553 No data DQ071788 Germany Garnica etal. 2007
Outgroup
Melanophyllum haematospermum ecv2249 AF391038 No data Netherlands Vellinga 2001a
Melanophyllum haematospermum ecv2517 AF391039 No data Netherlands Vellinga 2001a
Melanophyllum haematospermum DAOM197183 No data AF261476 USA Moncalvo etal. 2002

Mycological Progress (2024) 23:20 20 Page 8 of 18
sp. (OM473870) reported from Indiana, USA, and 28S
sequences showed 89% identity with L. brunneogranulosa
M. Asif, A. Izhar, Haqnawaz, Niazi & Khalid (OL331703,
OL331704 & OL331705), reported from Pakistan, while
the nrITS sequences of L. bahawalnagarensis showed
92% similarity with L. cholistanensis H. Bashir, Usman
& Khalid (MK087674), reported from Pakistan, and
28S sequences showed 90% maximum similarity with L.
cholistanensis (MK106095). The combined nrITS-28S
dataset has 1888 nucleotide sites (820 for nrITS and 1080
for 28S, gaps included) for each sample, of which 790
were conserved and 1098 were variable. In this phyloge-
netic analysis, L. aurantiopilea is sister to L. brunneogran-
ulosa (LAH36803, LAH36804, LAH36954) on a distinct
branch with a strong bootstrap value (BS = 100%), while L.
bahawalnagarensis is sister to L. cholistanensis forming a
separate branch with a strong bootstrap value (BS = 100%)
and lies within sect. Liliaceae (Fig.1).
Taxonomy
Lepiota aurantiopilea M. Asif, A. Izhar, Niazi & Khalid,
sp. nov. (Figs.2 and 3).
MycoBank: MB847217.
Diagnosis: Differs from Lepiota brunneogranulosa due
to its orange to yellow-orange pileus with reddish-brown
umbo, tiny granules on the surface that are concolorous to
pileus, stipe becoming yellowish upon handling, absence of
annulus, subglobose basidiospores, and hymeniderm pileus
covering made up of clavate to subfusiform elements.
Type: Pakistan. Punjab Prov.: Village 37/3R, Haroonabad,
District Bahawalnagar, (29°58′32″ N, 72°91′87″ E), 163
m alt., on nutrient-rich soil, 31 July 2019, M. Asif BWN-
36 (Holotype-LAH37661) GenBank: ITS = OQ282887;
28S = OQ282883.
Etymology: The specific epithet is derived from the Latin
adjectives “aurantio” which means bright orange and “pilea”
which means pileus of the fruiting bodies.
Basidiomata very small. Pileus 0.8–2.1cm, plane to con-
vex at younger stage, becoming subumbonate at maturity,
orange (5YR6/8) at young stage becoming yellow-orange
(7.5YR7/8) with age, umbonate with distinct reddish-brown
(5YR4/8) umbo, surface uneven or rugulose, covered with
minute granules that are concolorous to pileus, aerolate
or radially cracked around the umbo at maturity, dull and
dry; smooth to striate margins at younger stage becoming
uplifting or undulating with age, rarely splitting. Lamellae
free and approximate, subdistant to close, broad, rarely ven-
tricose, white (2.5YR5/12) at young stage becoming light
yellow (10YR8/3) with age, edges smooth at young stage
becoming eroded at maturity, margins entire and wavy, three
tiers of regularly arranged lamellulae, concolorous. Stipe
1.5–4.2 × 0.2–0.4cm, reddish-brown (2.5YR4/6) at young
stage becoming light yellowish-orange (10YR8/4) with
age and dull reddish-brown (5YR4/4) to yellowish upon
handling, central, cylindrical, equal, rarely curved or bent,
slightly pruinose, context thin, surface pubescent near the
cap becoming dull towards margins, slightly bulbous base.
Annulus absent. Volva absent. Smell acrid. Taste mild.
Basidiospores [150/6/6] (3.7–) 4.2–4.8 (–5.2) × (2.6–)
2.–3.2 (–3.5) µm, avl × avw = 4.5 × 3.1µm, Q = 1.2–1.3,
Qav = 1.25, thick-walled, slightly brownish KOH, sub-
globose, oblong to ellipsoid or spurred, non-guttulate,
smooth with a prominent apiculus, multinucleate. Basidia
17–20.5 × 5.8–7 µm, avl × avw = 18.8 × 6.4 µm, thick
walled, clavate, frequently 4-spored, rarely 2-spored,
clamped, hyaline in KOH, non-guttulate. Cheilocystidia
17–22.8 × 6–7.4µm, avl × avw = 11 × 6.6µm, thick-walled,
clavate, fusoid-ventricose, utriform, smooth, hyaline in
KOH, non-guttulate, present in clusters, basal cells sep-
tate, frequently clamped. Pleurocystidia absent. Pileipel-
lis a hymeniderm (20–36 × 8–13µm), composed of clavate
sometimes subfusiform to narrowed, few broadly clavate,
with smooth thin-, slightly thick-walled, hyaline in KOH,
terminal elements slightly swollen with a round apex, not
incrusted, clamp connections frequent. Central disc a cutis
of threads with globose to subglobose elements, 5–15µm
wide, with uplifted elements at the terminal, thinner hyphae
towards pileus margin. Pileocystidia absent. Stipitipel-
lis a cutis made up of thick-walled 7–17µm in diameter,
avw = 11.3µm, hyphae, septate, parallel arrangement, rarely
branched, hollow and cylindrical, terminal cells not modi-
fied, clamp connections frequent. Caulocystidia absent.
Habitat: On nutrient-rich loamy soil, scattered or in small
groups, under Vachellia nilotica in agricultural fields and
along the canal bank.
Geographical distribution range: Known only from the
type locality, Haroonabad, District Bahawalnagar and Dis-
trict Sheikhupura Punjab, Pakistan.
Additional specimens examined: Pakistan: Punjab
Prov.: Haroonabad, District Bahawalnagar, (30°33′02″ N,
73°23′26″ E), 163m alt., 04 Oct. 2019, M. Asif BWN-80
(LAH37664) GenBank: ITS = OQ282890; 28S = OQ282885.
Pakistan: Punjab Prov.: Haroonabad, District Bahawalnagar,
(30°33′02″ N, 73°23′26″ E), 14 Aug. 2020, M. Asif AG-19
(LAH37662) GenBank: ITS = OQ282888; 28S = OQ282884.
Pakistan. Punjab Prov.: Dahranwala, District Bahawalna-
gar, (29°59′67″ N, 72°84′56″ E), 178m alt., 25 Aug. 2020,
M. Asif AG-76 (LAH37665) GenBank: ITS = OQ282891.
Pakistan. Punjab Prov.: Dahranwala, District Bahawalna-
gar, (29°59′67″ N, 72°84′56″ E), 178m alt., 14 Aug. 2021,
M. Asif MA-20 (LAH37663) GenBank: ITS = OQ282889.
Pakistan. Punjab Prov.: District Sheikhupura (31°42′47″ N;
73°58′41″ E), 236m alt., 08 Sep. 2020, A. Izhar SKP-126
(LAH37660) GenBank: ITS = OQ282886; 28S = OQ282882.

Mycological Progress (2024) 23:20 Page 9 of 18 20
Fig. 1 Molecular phylogenetic
placement of Lepiota based
on maximum likelihood (ML)
method of combined nrITS-28S
sequence dataset

Mycological Progress (2024) 23:20 20 Page 10 of 18
Fig. 2 a–e Basidiomata of Lepiota aurantiopilea (a, b holotype, LAH37661). Scale bar a–e 1cm. Photos by Muhammad Asif

Mycological Progress (2024) 23:20 Page 11 of 18 20
Lepiota bahawalnagarensis M. Asif, Niazi & Khalid, sp.
nov. (Figs.4 and 5).
MycoBank: MB848302.
Diagnosis: Differs from Lepiota cholistanensis due to its
light grayish-brown pileus with dark grayish-brown reddish-
brown umbo, grayish-yellow brown zonation on surface,
pale yellow stipe, single-edged annulus, ellipsoid to amyg-
daliform basidiospores, versiform cheilocystidia, and clavate
to utriform caulocystidia.
Type: Pakistan. Punjab Prov.: Haroonabad, District
Bahawalnagar, (29°58′32″ N, 72°91′87″ E), 163 m alt.,
on nutrient-rich soil, 04 Oct. 2019, M. Asif BWN-92
(Holotype-LAH37787) GenBank: ITS = OQ658766;
28S = OQ658771.
Etymology: The specific epithet, “bahawalnagarensis”
in Latin, refers to the district Bahawalnagar from where the
type specimen was collected.
Basidiomata very small. Pileus 1.4–2.6cm, plane to
convex at younger stage, becoming applanate at maturity,
light grayish brown (5Y8/2), umbonate with dark grayish-
brown (10YR4/2) umbo at younger stage becoming brown
(7.5YR4/3) at maturity, surface granulose at younger stage
becoming cracked at maturity, covered with grayish-yellow
brown (5YR5/2) aerolate or zonate squamules which are
Fig. 3 a–f Microscopic
structures of Lepiota aurantio-
pilea (holotype, LAH37661).
a Basidia, b basidiospores, c
cheilocystidia, d central disc, e
pileipellis elements, f stipitipel-
lis hyphae. Scale bar a 5μm, b
3μm, c–f 5μm. Drawings by
Aiman Izhar

Mycological Progress (2024) 23:20 20 Page 12 of 18
Fig. 4 a–e Basidiomata of Lepiota bahawalnagarensis (a–d holotype, LAH37787). Scale bar a–e 1cm. Photos by Muhammad Asif

Mycological Progress (2024) 23:20 Page 13 of 18 20
denser in central disc, becoming spares towards margins,
dull and dry; margins smooth at younger stage becoming
undulating with age, rarely split radially. Lamellae free and
even, subdistant, narrow to broad, pale-yellow (2.5Y8/2),
edges smooth, rarely eroded at maturity, margins entire,
rarely wavy and eroded, two tires of regularly arranged,
lamellulae, concolorous. Stipe 1.5–2.6 × 0.3–0.5cm, pale-
yellow (2.5Y8/2) from upper half and light brown (6YR7/1)
from lower half, central, cylindrical rarely compressed,
equal, surface fibrous, shiny near the cap becoming dull
towards base, dry, context thin. Annulus present, light-brown
(6YR7/1), single-edged, superior, maiden in position. Volva
absent. Smell acrid. Taste mild.
Basidiospores (6–) 6.5–7.5 (–8.6) × (3.5–) 4–5 (–5.5)
µm, avl × avw = 7 × 4.5µm, Q = 1.5–1.7, Qav = 1.6, ellip-
soid to ovoid in front view, ellipsoid to amygdaliform in side
view, thin-walled, hyaline in KOH, globose, non-guttulate,
smooth with a prominent apiculus, without germ pore.
Fig. 5 a–e Microscopic structures of Lepiota bahawalnagarensis (Holotype; LAH37787) a = Basidia, b = Basidiospores, c = Cheilocystidia, d
= Pileipellis, e = Caulocystidia. Scale Bar a = 5 μm, b = 3 μm, c–e = 5 Drawings by Aiman Izhar

Mycological Progress (2024) 23:20 20 Page 14 of 18
Basidia (21.8–) 22–25 (–25.8) × (6.5–) 6.9–8.8 (–9) µm,
avl × avw = 23.8 × 7.7 µm, clavate, thin walled, 4-spored,
rarely 2-spored, guttulate, sterigmata up to 2 µm long,
basal cells 1–2 septate, clamp connections present. Cheilo-
cystidia (17.9–) 19.6–23 (–24.8) × (6–) 6.5–8 (–8.5) µm,
avl × avw = 21 × 7.3µm, variable in shape, cylindrical, clavate,
cylindro-clavate, utriform or broadly clavate, thin walled, hya-
line, guttulate, 1–2 olivish colored oil droplets, clamp con-
nections present at the base. Pleurocystidia absent. Pileipellis
a hymeniderm consisting of outer layer with inflated tightly
packed mostly clavate elements 83–200 × 11–15µm, occa-
sionally with pale yellowish brown fine incrustation, pileus
hyphae parallel in the innermost layer, 6–9µm wide, hya-
line, some with slightly yellowish content, clamp connec-
tions frequent. Stipitipellis a cutis with 4–11µm in diameter,
avw = 7.5µm, hyaline in KOH, septate, parallel, branched,
closely septate, clamp connections frequent. Caulocystidia
11–55 × 4–13µm, numerous, often in tufts near stipe apex,
broadly clavate, clavate to utriform, slightly thick-walled, few
having light brown intracellular pigments.
Habitat: On nutrient-rich loamy soil, scattered or in small
groups, under Vachellia nilotica along the bank of the 3R
irrigation canal.
Geographical distribution range: So far known only from
the type locality, District Bahawalnagar Punjab, Pakistan.
Additional specimens examined: Pakistan: Punjab Prov.:
Haroonabad, District Bahawalnagar, (30°33′02″ N, 73°23′26″
E), 163m alt., 04 Oct. 2019, M. Asif BWN-93 (LAH37786)
GenBank: ITS = OQ658767; 28S = OQ658772. 04 Oct. 2019,
M. Asif BWN-83 (LAH37785) GenBank: ITS = OQ658768;
28S = OQ658773.
Discussion
In combined nrITS-28S-based phylogeny, i.e., Lepiota
aurantiopilea and L. bahawalnagarensis clustered within
L. sect. Liliaceae in accordance with Vellinga 2003, Lepi-
ota sect. Liliaceae appears to be polyphyletic because the
species of the section are dispersed in three different sub-
clades (I, II, and III) following Vellinga 2003. Lepiota
sect. Liliaceae is characterized by a hymeniderm pileus
covering which is made up of tightly packed clavate to
narrowly clavate elements and by subglobose to ellipsoid
or spurred basidiospores (Vellinga 2001a, b, c) (Fig.6).
Candusso and Lanzoni (1990) put Lepiota cristata (species
with spurred spores and hymenodermal pileus covering)
into section Stenosporae, but Vellinga (2001c) put every
species with hymenodermal pileus covering into section
Liliaceae; this is supported by phylogenetic studies based
on molecular characters (Vellinga 2003).
Lepiota aurantiopilea clustered within L. sect. Liliaceae
along with L. lilacea in subclade II. The clustering of L.
aurantiopilea along with L. lilacea is in accordance with the
studies of Vellinga 2003. Besides the molecular support, the
shape of basidiospores and pileus covering also confirmed that
L. aurantiopilea is a member of the L. sect. Liliaceae (Vel-
linga 2001c). The other new species, i.e., L. bahawalnagaren-
sis, also followed the studies of Vellinga (2003) and clustered
within the sect. Liliaceae (subclade III) and its allied species
from Pakistan, i.e., L. cholistanesis (Bashir etal. 2020) and L.
albogranulosa (Qasim etal. 2015) (both species have hymeni-
derm pileus and ellipsoid spores) that were previously in sect.
Cristatae must also be transferred here to sect. Liliaceae based
on morphological as well as molecular data which is also in
accordance to Vellinga (2003).
Lepiota aurantiopilea is characterized by small basidi-
omata, orange to yellow-orange pileus with reddish-brown
umbo, tiny granules on the surface that are concolorous to
pileus, stipe turns yellowish upon handling, absence of annu-
lus, subglobose or oblong to ellipsoid basidiospores, clavate
cheilocystidia, and hymeniderm to epithelium pileus covering
made up of globose to subglobose elements. L. aurantiopilea
lies in subclade II, close to L. brunneogranulosa, a species
of the same section recently reported from the same local-
ity. Morphologically, L. brunneogranulosa can be separated
by its very pale brown, plano-convex pileus with yellowish-
brown central disc and unequally distributed brown granules
(Asif etal. 2022b) in contrast to L. aurantiopilea that has
orange to yellow-orange pileus with a reddish-brown umbo
and tiny concolorous granules on the surface. The basidi-
ospores of L. brunneogranulosa are ellipsoid to oblong, while
L. aurantiopilea has ellipsoid to oblong basidiospores. The
cheilocystidia of the former species are narrowly clavate and
larger cheilocystidia with an average size of 19.6 × 7.02µm
(Asif etal. 2022b), whereas in the latter, cheilocystidia are
clavate, fusoid-ventricose, utriform, with an average size
of 11 × 6.6µm. An important similar character is the pileus
covering, which is a hymeniderm with obovoid, clavate to
broadly clavate, utriform or subfusiform in both species (Asif
etal. 2022b), but the central disc of L. aurantiopilea consists
of threads with globose to subglobose elements, with uplifted
elements at the terminal, which is a unique character of this
species. Another phylogenetically closely related species, L.
lilacea, differs from L. aurantiopilea due to its plano-convex
to applanate pileus with a dark violet central disc that has
radially arranged scales and the presence of distinct annulus
(Vellinga 2001b; Naseer etal. 2022).
Lepiota ochraceofulva P.D. Orton and L. bengalensis
Hosen & T.H. Li also lie in clade I of L. sect. Liliaceae. L.
ochraceofulva reported from Europe differs from L. auran-
tiopilea by its crowded lamellae, long and broad stipe
(5.2–7 × 0.6–0.9 cm) with clavate bulbous base, tinged
ochraceous-cream color at stipe apex, larger, and wider basidi-
ospores 5.5–7 × 3.5–4.5µm (Or ton 1960). L. bengalensis can
be differentiated from our new species due to its pastel red to

Mycological Progress (2024) 23:20 Page 15 of 18 20
brownish red, conico-convex to hemispherical pileus with the
dark ochraceous or reddish-brown central disc, and brownish
red to reddish-orange appressed or radially arranged squam-
ules on the pileus (Hosen etal. 2016).
Lepiota bahawalnagarensis is featured by very small
basidiomata, light gray-brown pileus with dark gray-brown
central disc, surface cracked with brown zonation at matu-
rity, pale yellow cylindrical stipe, single-edged and superior
annulus, ellipsoid to amygdaliform basidiospores, versiform
cheilocystidia, and pileipellis consisting of long, subcylin-
drical, or clavate to attenuate terminal elements.
Lepiota cholistanensis and L. coloratipes are found to be
close relatives of L. bahawalnagarensis in nrITS-28S phylo-
genetic analysis and L. cholistanensis differs from L. baha-
walnagarensis due to a combination of morpho-anatomical
characteristics, i.e., convex pileus with brown furfuraceous
scales, with appendiculate margins, equal to unequal creamy
stipe with a scabrous surface that changes color to dark
brown upon touch and bruising, and single edged annulus,
subglobose to broadly ellipsoid basidiospores and clavate
cheilocystidia. Both species have the same pileipellis with
a compact hymeniderm comprised of hyphae with clavate
elements (Bashir etal. 2020). Lepiota coloratipes is clearly
distinguished from L. bahawalnagarensis by having a
peculiar combination of characteristics including a usually
smooth pileus surface with cream to pale ochraceous tinges,
a fugacious partial veil not forming an annulus but leaving
fibrillose remnants on stipe surface, a stipe with brownish
reddish tinges at base, the hymeniderm pileus covering con-
sisting of a tightly arranged palisade of more or less clavate
Fig. 6 a, b Basidiospores of Lepiota aurantiopilea (holotype,
LAH37661). a In Congo red. b In 5% KOH. c, d Basidiospores of
Lepiota bahawalnagarensis (holotype, LAH37787). c In Congo red.
d In 5% KOH. Scale bar a, b 4μm; c, d 5μm. Photos by Muhammad
Asif. Spores were arranged manually from different pictures

Mycological Progress (2024) 23:20 20 Page 16 of 18
to sphaeropedunculate elements, and ellipsoid basidiospores
(Vizzini etal. 2014b).
Phylogenetically, L. sanguineofracta and L. psalion are also
closely related to L. bahawalnagarensis and L. sanguineofracta
can be differentiated from L. bahawalnagarensis by having a
micaceous, not a squamulose pileus surface with conspicuous
green spots at maturity, context with rosy smell and blood red
coloration upon bruising (Vizzini etal. 2014a), and L. psalion
possesses an evident well-formed annulus, a looser pileipellis
structure, absence of oil-like droplets in tissues and broadly
ellipsoid to ellipsoid basidiospores (Vellinga & Huijser 1999;
Vellinga 2001a; Vizzini etal. 2019). A Pakistani species lies
in subclade III; L. albogranulosa differs from L. bahawalna-
garensis due to its predominantly white color, stipe with white
scales, and broadly ellipsoid basidiospores (Qasim etal. 2015).
From all the discussion and comparisons of morpho-
anatomical characteristics of all the phylogenetically closely
related species, it is evident that both of our new species, i.e.,
L. aurantiopilea and L. bahawalnagarensis, are distinct from
all the closely related taxa by not only their morpho-anatomi-
cal characters but also their unique nrITS and 28S sequences.
Acknowledgements We are sincerely thankful to Dr. Shaun Pennycook
(Mnaaki Whenua Landcare Research, Auckland, New Zealand) and Dr.
Konstanze Bensch (Mycobank curator at Westerdijk Fungal Biodiver-
sity Institute, Netherlands) for their help with the name of taxa. We are
also thankful to Dr. Genevieve Gates (Honorary Associate, Mycology
and Forest Ecology, Tasmanian Institute of Agriculture, University of
Tasmania, Tasmania, Australia) for her linguistic as well as scientific
review of this manuscript which helped us to improve the paper. The
first author is thankful to Mr. Ali Hassan and Mr. Ali Hamza and the
second author to Mr. Izhar-ul-Haque for their help at the sampling sites.
Author contribution All authors contributed to this study’s concep-
tion and design. Material preparation, data collection, and phyloge-
netic analyses were performed by Muhammad Asif and Aiman Izhar.
The first draft of the manuscript was written by Muhammad Asif and
Aiman Izhar, and all the authors commented on the previous versions
of the manuscript. Prof. Dr. Abdul Nasir Khalid, Dr. Abdul Rehman
Niazi, and Dr. Malka Saba supervised and validated the manuscript.
All authors read and approved the final manuscript.
Funding The authors declare that no funds, grants, or other support
was received during the preparation of this manuscript.
Data availability DNA sequence data supporting this study’s findings
have been deposited in the GenBank repository. Other data generated
or analyzed during this study are included in this article.
Code availability Not applicable.
Declarations
Ethics approval Not applicable.
Consent to participate Not applicable.
Consent for publication Not applicable.
Conflict of interest The authors declare no competing interests.
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