Lepiota brunneogranulosa (Agaricaceae): a new species from Punjab, Pakistan, based on integrated taxonomy

ABSTRACT

During our surveys of mushrooms in 2019 and 2020, an interesting species of the genus Lepiota was collected from three different areas of Punjab (Bahawalnagar, Sheikhupura, and Muzaffarghar), Pakistan. These areas vary in their altitude and somewhat climatic conditions, nevertheless, morpho-anatomical comparisons and molecular analyses proved that these collections represented the same taxon, described here as Lepiota brunneogranulosa. This new species is characterized by a pileus with a yellowish-brown central disc, with brown colored granules on a very pale brown background, dextrinoid, ellipsoid basidiospores, frequently bi-sporic basidia, and narrowly clavate cheilocystidia. Because of the hymeniform pileus covering, and the placement in the phylogeny, based on Internal Transcribed Spacer (nrITS) and Larger Subunit (LSU), it belongs to the Lepiota sect. Lilaceae.

Keywords:
Bahawalnagar; Lepiotaceous fungi; litter-inhabiting; Phylogeny; taxonomy

Introduction

Species of the genus Lepiota (Pers.) Gray (1821: 601) are saprobes in nature (Singer 1986; Vellinga 2004) and globally distributed from tropical to temperate areas and are infrequent in deserts and arctic-alpine habitats (Vellinga 2004) with more than 500 species worldwide (Singer 1986;Kirk et al. 2008; Razaq et al. 2012; Nawaz et al. 2013; Qasim et al. 2015; Sysouphanthong et. al 2016; Bashir et al. 2020; Niazi et al. 2021), including more than 30 species reported from Pakistan (Ahmad et al. 1997; Kirk et al. 2008; Kumar & Manimohan 2009; Liang & Yang 2011; Nawaz et al. 2013; Razaq et al. 2012; 2013; 2014; Qasim et al. 2015; Qasim et al. 2016; Bashir et al. 2020; Niazi et al. 2021). The genus Lepiota is characterized by its white to pale, mostly dextrinoid basidiospores that have different shapes i.e. ellipsoid to spurred, fusiform or penguin-shaped, various pileus coverings, ranging from hymeniderm, cutis, trichoderm, epithelium, a regular hymenophoral trama and usually the presence of clamp connections (Candusso & Lanzoni 1990; Vellinga 2003).

Pileus covering and basidiospore shape are the key characteristics that divide the genus Lepiota into several sections (Vellinga 2001a). Species within the genus Lepiota show quite close affinities on morphological analysis and have further been divided into several sections based on molecular data. Lepiota species have been supported by molecular studies indicating that the genus is polyphyletic, and thorough work focused on multigene phylogenetic analyses has placed the members of the genus in the relevant sections. (Sysouphanthong et al. 2011). However, there are still a lot of species that need to be identified and described.

Presently, there are six sections within the genus Lepiota and each section has a unique structure of pileus covering and basidiospore shape, viz., (1) species with fusiform-amygdaliform spores and trichodermal pileus covering (sect. Lepiota), (2) species with subglobose to ellipsoid or spurred spores and hymenidermal pileus covering (sect. Lilaceae M. Bon), (3) species with ovoid to ellipsoid spores and cylindrical pileus covering elements without clamp-connections (sect. Fuscovinaceae (J. Lange) Kühner), (4) species with ovoid to ellipsoid spores and trichodermal pileus covering (sect. Ovisporae (J. Lange) Kühner), (5) species with spurred spores and trichodermal or cutis pileus covering (sect. Stenosporae (J. Lange) Kühner) and (6) species with ovoid to ellipsoid or cylindrical spores with a spurred base and epithelium pileus covering (elements agglutinated in chains) (sect. Echinatae Fay.) (Candusso & Lanzoni 1990; Bon 1993; Vellinga 2001c).

Lepiota sect. Lilaceae is distinguished by a hymenidermal pileus covering which is made up of firmly packed clavate to narrowly clavate elements, and by subglobose to ellipsoid or spurred basidiospores (Bon 1981; Vellinga 2001a). Section Lilaceae is dissimilar from the other five sections of Lepiota based on morphological and molecular evidence (Vellinga 2001a; Vellinga 2003). However, some species, despite producing spurred spores like members of the sect. Liliaceae, were placed in some other sections of the genus in having close phylogenetic relationships with those sections (Candusso & Lanzoni 1990; Bon 1981; Horak 1981).

Morphological differences between Lepiota species are often quite subtle (Vellinga & Huijser 1998). Fortunately, the separation of different species has become more precise and exact based on nrITS phylogenetic analyses (Vellinga 2003).

Lepiotaceous fungi are highly diverse group, especially in tropical and subtropical areas, and a huge amount of work has been done on species from different sections of the genus, both based on morphology and phylogeny (Vizzini et al. 2014; Justo et al. 2015; Hosen et al. 2016; Sysouphanthong et al. 2020). They are found in many localities of Punjab Pakistan. Here, a new species of the genus Lepiota sect. Lilaceae, collected in three districts (Bahawalnagar, Sheikhupura, and Muzaffarghar) of Punjab, Pakistan, is presented with a description, illustrations, and phylogenetic analyses of its nrITS and LSU sequences.

Materials and methods

Locality description

The type specimen was collected from district Bahawalnagar, Punjab, Pakistan during the rainy season of 2019. Geographically, District Bahawalnagar is situated in eastern Punjab (Coordinates: 30°33′02″ N, 73°23′26″ E, 163 m) on the bank of Satluj River. The temperature of the district varies from a minimum of 11 ^oC to a maximum of 50 ^oC and the average annual precipitation is 119 mm (Ahmed et al. 2014a; 2014b). The second collection was collected from district Sheikhupura, in central Punjab (Coordinates: 31°42'47" N, 73°58' 41" E). The climatic conditions of the district remain moist sub-humid with annual rainfall from 250 to 500 mm which reaches a maximum of 635 mm (Nawaz et al. 2017). Another collection was made from District Muzaffargarh, in south-western Punjab, (Coordinates: 30°4′10″ N, 71°11′39″ E, 65 m), which lies in a sub-humid region with 127-150 mm average annual rainfall (Nickson et al. 2005; Akram et al. 2014; Zamir & Kazmi 2014; Mahmood et al. 2019).

Morphological Protocols

Basidiomata were collected, photographed, and the main fleeting features and superficial characters along with habitat and geographical data were noted in the field. Basidiomata were described using the terminology of Vellinga (2001b). Munsell’s soil color chart (Munsell 1975) was used to describe the colors. The collected samples were brought to the laboratory and dried at 40-50 °C using a fan heater. The dried specimens (Holotype) were deposited in LAH Herbarium, Institute of Botany, University of the Punjab, Lahore, Pakistan, and holotype (=isotype) was deposited in Islamabad Herbarium under herbarium voucher no. ISL-F02.

For anatomical analysis, dried tissues were rehydrated in 5 % KOH, stained with 1 % Congo Red; Melzer’s reagent was used for reactions of the spore walls. The microscopic features such as shape and size of basidiospores, cheilocystidia, basidia, structure, and elements of pileus covering and stipe covering were observed using a light microscope (CXRII, Labomed Labo America Inc., Fremont, CA, USA) with an HDCE-X5 microscopic camera under 40X and oil immersion 100X magnification. From each collection, a minimum of 25 measurements were taken for each character. Abbreviation: Dimension a-b × c-d = minimum and maximum values of length × width, avw = average width, L_m × W_m = mean spore length × mean spore width, spore quotient Q = length/width, Qm = mean quotient of all basidiospores. The notation ‘n/b/p’ indicates ‘n’ basidiospores measured from ‘b’ basidiomata from ‘p’ collections (Bas 1969; Yu et al. 2020).

Molecular Protocols

DNA extraction followed the 2 % CTAB protocol (Bruns 1995) using dried specimens. DNA was amplified using a combination of primers (i.e. ITS1F/ITS4) for ITS and LR0R/LR5 for 28S (Vilgalys & Hester 1990; Gardes & Bruns 1993; White et al. 1990). PCR was done in 25 µl reaction volume following Gardes & Bruns (1993). The PCR products were sequenced by a commercial laboratory and newly generated sequences in this study were deposited in GenBank under accession numbers nrITS OL331703 - OL331705 and LSU OL331514 - OL331516.

Phylogenetic analyses

The three obtained sequences from both ITS and LSU regions were prepared using BioEdit (Hall 1999). The final dataset of ITS sequences was 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) it consisted of 65 ingroup taxa and Macrolepiota dolichaulaPegler & R.W. Rayner (1969: 365) (JQ928939) as an outgroup. The LSU final dataset retrieved from GenBank comprised 26 sequences including Macrolepiota dolichaula (KY418836) as an outgroup taxon. We used the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990) against the GenBank database to find taxa with closely allied sequences, which were then retrieved for addition in the phylogenetic analyses dataset. All the sequences were aligned using the Multiple Sequence Alignment search tool (https://www.ebi.ac.uk/Tools/msa/muscle/) (Edgar 2004) and then adjusted manually in BioEdit (Hall 1999). Final alignment was deposited in TreeBASE (http://www.treebase.org/) under the accession numbers http://purl.org/phylo/treebase/phylows/study/TB2:S28978 and http://purl.org/phylo/treebase/phylows/study/TB2:S28980. Maximum Likelihood (ML) analysis was done using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) as applied on the CIPRES portal v 3.1 (Miller et al. 2010) and with 1000 rapid bootstrap iterations for both genes.

Results

Phylogenetic analyses of ITS dataset

The fragment size of the target region was 660 bp long. In BLAST results, the ITS sequences of Lepiota brunneogranulosa showed 86 % maximum similarity with Lepiota sp. (AF391052) from the USA. In this analysis, L. brunneogranulosa gets clustered within the L. sect. Lilaceae close to L. lilacea (1892: 3) (GQ203822 & AY176379) reported from California, USA and L. bengalensis (2016: 188) (KU563149 & KU563148) reported from Bangladesh (Fig. 1).

Figure 1
Molecular phylogenetic placement of Lepiota brunneogranulosa based on maximum likelihood (ML) method of ITS sequences.

Phylogenetic analyses of LSU dataset

The fragment size of the large subunit target region was 926 bp long. The ITS sequences of Lepiota brunneogranulosa showed 97 % maximum similarity with Lepiota sp. (OL652983), an unpublished species from Australia. In this analysis, L. scaberulaVellinga (2001: 290) (MK278271) reported from the USA is closely related to our new species (Fig. 2).

Figure 2
Molecular phylogenetic placement of Lepiota brunneogranulosa based on maximum likelihood (ML) method of LSU sequences.

Taxonomy

Lepiota brunneogranulosa M. Asif, A. Izhar, Haqnawaz, Niazi & Khalid sp. nov.

Figs. 3A-D, 4A-E

MycoBank No. MB841888

Etymology:brunneogranulosa (Latin), referring to the brown-colored granules on the pileus surface.

Diagnosis: This species is characterized by brown granules on pileus, stipe turning yellowish on handling, narrowly clavate cheilocystidia and hymeniderm pileus covering with obovoid to utriform terminal cells.

Type: Pakistan. Punjab: Haroonabaad, District Bahawalnagar, (Coordinates: 30°33′02″ N, 73°23′26″ E; 163 m above sea level (a.s.l.)), Scattered or in groups, on nutrient-rich loamy soil, July 31, 2019, Muhammad Asif BWN 23 (Holotype-LAH36803). GenBank: ITS = OL331703; 28S = OL331514. (=Isotype ISL-F02).

Description:Pileus 1.7-2.6 cm, plano-convex at younger stage, later become plane at maturity, yellowish-brown (10YR5/6) central disc with brown (7.5YR4/3) granules on very pale brown (10YR8/4) surface, denser around the central disc and becoming sparse towards margins, showing the pale brown surface; dull and dry; margin undulating and dentate. Lamellae free, sub-distant, broad, light brown (6YR7/1), wavy with eroded edges, a single tier of lamellulae, concolorous. Stipe 2.8-3.5 × 0.2-0.5 cm light brown (6YR7/1), becoming yellowish upon handling, central, equal, with shiny surface, concolorous fibrils at the apex of stipe. Annulus sub-peronate, white (2.5YR5/12), single-edged, upturned, attached at the upper part of the stipe, disappearing at maturity. Volva absent. Smell acrid. Taste mild

Basidiospores (100/4/4) (4.5-) 4.6-5.2 × 3.2-3.8 (-3.9) µm, avl × avw = 4.9 × 3.5 µm, Q = 1.4 - 1.7, Qm = 1.5, thin-walled, pale yellow in KOH, slightly brownish in Melzer’s reagent, but not so strong, ellipsoid, oblong in side view, rounded triangular in frontal view, non-guttulate, smooth with a prominent apiculus. Basidia 18-22 × 6.8-9 µm, avl × avw = 20.19 × 7.69 µm, Q = 2.62, thick-walled, mostly clavate, rarely narrowly clavate, frequently bisporic, rarely tri and tetra sporic, hyaline in KOH, non-guttulate. Lamella edge sterile, cheilocystidia crowded. Cheilocystidia 17-23 × 6-7.5 µm, avl × avw = 19.6 × 7.02 µm, Q = 2.8, thick-walled, narrowly clavate, hyaline in KOH, non-guttulate. Pleurocystidia absent. Pileus covering hymeniderm, thick-walled, hyaline in KOH, septate, irregularly arranged with obovoid, clavate to broadly clavate or utriform with median constriction terminal cells 25-47 × 17-27 µm, hyphae 8.7-6.3 µm, avl × avw = 19.3 × 11.5 µm. Stipitipellis a cutis, hyphae 6.6 - 13.2 µm, avw = 8.4 µm, thick-walled, hyaline in KOH, septate, parallel arrangement, long and narrow. Caulocystidia absent. Clamp connections are present in all tissues.

Habitat and Ecology: Scattered or in groups, on nutrient-rich loamy soil, under Eucalyptus camaldulensis Dehnh., Aug to Sep.

Geographical distribution range: Known only from Punjab, Pakistan.

Additional specimens examined: PAKISTAN. Punjab: Haroonabad, District Bahawalnagar, at 163 m a.s.l, September 07, 2020, Muhammad Asif BWN 142 (LAH36804). GenBank: ITS = OL331704; 28S = OL331516. Pakistan-Punjab: District Sheikhupura, (Coordinates: 31° 71′67″N, 73°98 50″E; 236 m a.s.l) scattered on loamy soil, September 20, 2019, Aiman Izhar SKP 1211 (LAH36974). Pakistan-Punjab: Easan Wala Forest, District Muzaffargarh, (Coordinates: 30°4′10″N, 71°11′39″E; 123 m a.s.l) August 29, 2020, Muhammad Haqnawaz EF 02, (LAH36954). GenBank: ITS = OL331705; 28S = OL331515.

Figure 3
Basidiomata of Lepiota brunneogranulosa (LAH36803, Holotype) Photos by Muhammad Asif & Aiman Izhar.

Figure 4
Microscopic characters of Lepiota brunneogranulosa (LAH36803, Holotype) A. Basidia, B. Basidiospores, C. Cheilocystidia, D. Pileus covering, E. Stipitipellis, Bars: A-E = 10 µm. Drawings by Aiman Izhar

Discussion

Lepiota brunneogranulosa is characterized by pileus with brown granules on a very pale brown background, presence of annulus on stipe, becoming yellowish on handling, narrowly clavate cheilocystidia, ellipsoid to broadly basidiospores, and obvoid, clavate to broadly clavate or utriform terminal elements in pileus covering (Tab. 1).

Table 1
Comparison of the diagnostic characteristics of L. brunneogranulosa with closely related species (Vellinga 2001b; 2001d; Hosen et al. 2016; Orton 1960).

Characters	L. brunneogranulosa	L. lilaceae	L. bengalensis	L. scaberula	L. ochraceofulva
Pileus	Plane, very pale brown	Violet, plano-convex to applanate,	Pastel red to brownish red, campanulate to conicoconvex, expanding to hemispherical to applanate with age	Paler around centre and whistish at margins, hemispherical to plano-convex	Orange brown to leather brown
Central disc	Yellowish-brown	Dark violet	Dark ochraceous or reddish brown	Cream to yellow-brownish	Reddish brown
Squamules	Brown granules	Violet, radially arranged appressed scales	Brownish red to reddish orange, appressed or radially arranged squamules	Irregularly fibrillose v-shaped squamules	Pileus broken into squamules
Lamellae	Sub-distant, light brown	Crowded, white	Distant to sub-crowded, pale yellow to yellowish white or light ochraceous-cream	Moderately crowded to moderately distant	Crowded
Basidiospores	Ellipsoid to broadly ellipsoid or oblong, av = 4.9 × 3.5 µm,	Ellipsoid to oblong, av = 5.2 × 3.1 µm	Ellipsoid to cylindrical, av = 5.6 × 3 µm	Ellipsoid to oblong, av = 5.7 × 3.6 µm	Broadly ellipsoid, av = 5.5 × 3.7 µm
Cheilocystidia	Narrowly clavate	Narrowly cylindrical, narrowly utriform	Fasciculate, clavate to narrowly clavate	Cylindrical and slightly widened at apex	Clavate
Pileus covering	Hymeniderm, irregularly arranged with obvoid, clavate to broadly clavate or utriform terminal cells	Hymeniderm made up of clavate to broadly clavate cells	Hymeniform, composed of clavate to broadly clavate or narrowly clavate cells	Cylindrical to slightly inflated hyphae	Hymeniform
Habitat	Scattered or in groups, on nutrient-rich loamy soil under Eucalyptus camaldulensis	Gregarious in small groups, on irrigated soil, on rich nitrophilous shrubs in woods under Sequoia sempervirens	Solitary, caespitose or gregarious in small groups on debris of Albizia and Eucalyptus	Gregarious, saprotrophic on soil in Sequoia sempervirens forests	On loamy soil, in gardens and parks

Lepiota lilacea (GQ203822 & AY176379) and L. bengalensis (KU563149 & KU563148) are found to be close relatives of our newly described taxon in ITS based phylogenetic analysis (Fig. 1). But both species can be differentiated from our new species morpho-anatomically.

Lepiota lilacea is widely reported around temperate areas. It was originally described and widely reported in Europe and is also present in North America. It differs from L. brunneogranulosa macroscopically in having plano-convex to applanate pileus with dark violet central disc that has radially arranged scales, white lamellae with free margins, and microscopically in having ellipsoid to oblong basidiospores with 5.2 × 3.1 µm average size (Tab. 1) (Vellinga 2001ab; Vellinga et al. 2011).

Lepiota bengalensis reported from Bangladesh differs from L. brunneogranulosa in having pastel red to 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 squamules on the pileus and no color change on touching (Hosen et al. 2016). Clamp connections are only present in pileus covering (Tab. 1) (Hosen et al. 2016).

In LSU based phylogenetic analysis (Fig. 2), Lepiota scaberula, a species originally reported from the California, USA, is the closest relative to L. brunneogranulosa; it differs from latter by its scurfy pileus surface with fibrillose v-shaped squammules, moderately crowded and pinkish lamellae, whitish-wooly stipe and non-dextrinoid basidiospores (Tab. 1) (Vellinga 2001d).

Lepiota ochraceofulva, which is originally reported from Europe, differs from L. brunneogranulosa by its crowded lamellae, long and broad stipe 52-70 × 6-9 mm with an almost clavate bulbous base, tinged ochraceous-cream color at stipe apex, and slightly wider basidiospores 5.5-7 × 3.5-4.5 µm (Tab. 1) (Orton 1960).

Previously, 32 species of Lepiota have been reported from Pakistan (Ahmad et al. 1997; Razaq et al. 2012; Nawaz et al. 2013; Qasim et al. 2015; 2016; Bashir et al. 2020; Niazi et al. 2021; Haqnawaz et al. 2022). Our current study, based on morphological and molecular data of the species collected from Punjab, Pakistan, indicated that this is a genuine member of the genus Lepiota and also a new taxon to science. So, future mycological expeditions are appreciated to explore more hidden species of the genus, their variabilities, and ecology from Pakistan.

Acknowledgments

We are sincerely thankful to Dr. Else C. Vellinga (University of California, Berkeley, USA) for her critical review and valuable comments and suggestions on an earlier version of this manuscript which helped us to improve the article. We thank all the anonymous reviewers for their corrections and suggestions to improve this paper.

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Publication Dates

• Publication in this collection
  21 Nov 2022
• Date of issue
  2022

History

• Received
  24 Dec 2021
• Accepted
  29 Aug 2022