Research Article |
Corresponding author: Arooj Naseer ( arooj.hons@pu.edu.pk ) Academic editor: María P. Martín
© 2023 Shazia Ashraf, Arooj Naseer, Muhammad Usman, 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:
Ashraf S, Naseer A, Usman M, Khalid AN (2023) Two new species of genus Leucoagaricus (Agaricaceae, Agaricales) from Pakistan. MycoKeys 96: 159-171. https://doi.org/10.3897/mycokeys.96.101745
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The genus of basidiomycetous fungi, Leucoagaricus, occurs worldwide, from subtropical to boreal latitudes. Several collections of Leucoagaricus were made during mycological field trips conducted in different forests of Margalla, Pakistan. An integrative framework combining morphological and phylogenetic data was employed for their study. As a result, the two species La. margallensis and La. glareicolor are here described as new to science. Detailed macro- and micro-morphological descriptions, and a molecular phylogenetic reconstruction based on nrITS and LSU sequence data are provided and used to discriminate the new species from morphologically and phylogenetically close taxa. Whereas, our phylogenetic tree inference gave unequivocal support for the inclusion of these two species within the section Leucoagaricus.
Bayesian analysis, Islamabad, Margalla, systematics
The genus Leucoagaricus Locq. ex Singer, is a relatively well known mushroom-forming genus of basidiomycetous fungi, characterized by the small to medium-sized, thin or fleshy basidiomata; pileus surfaces ranges from radially fibrillose, floccose, squamulose to fibrillose-scaly or rarely granulose; entire or very short striated margins; and central, equal to bulbous stipe that have membranous, sometimes movable annuli; thin-walled and smooth basidiospores generally lack well-defined germ pores; and the pileipellis is either a trichoderm or a cutis of repent and radially arranged hyphae lacking sphaerocysts (Singer 1986;
Taxonomic studies on Leucoagaricus throughout the whole of Pakistan are, in fact, scant. Only 12 species of Leucoagaricus have been reported from Pakistan so far (
Basidiomata were collected following
Genomic DNA was extracted from portions of lamellae following a modified CTAB extraction method (
The online tool BLAST and the databases GenBank (http://www.ncbi.nlm.nih.gov/) was used to check for possible PCR-product contamination and to identify and retrieve available, highly similar Leucoagaricus nrITS and LSU sequences to the newly produced sequences. A comprehensive representation of currently available sequences, in NCBI database with similarity up to 92% identity and 95% query cover for ITS Phylogenetic tree and 95% identity and 98% query cover for LSU Phylogenetic tree, were used for the phylogenetic analyses and all the sequence of section Leucoagaricus from recent publications were also included. The final dataset consists of 54 sequences as ingroup and one sequence of Cystolepiota seminuda (Lasch) Bon (AY176350 for ITS and AY176351 for LSU) from the Netherlands was used as outgroup. The dataset for the phylogenetic tree was made by MUSCLE alignment in SEA VIEW software version 5.0.5 (
For the tree reconstruction based on Bayesian inference, the program MrBayes 3.2.7 (
Sequences of the nr DNA ITS region basidiomata MH63 (LAH37453), MH111 (LAH37454), MH65 (LAH37575) and MH169 (LAH37456) were obtained with both primers and the final sequences consisted of 780, 770, 753, and 656 base pairs, respectively and 1090, 936, 977 and 953 base pairs, respectively for LSU. These four samples belong to two different taxa described here under the name of Leucoagaricus margallensis and Leucoagaricus glareicolor. For ITS, the aligned final dataset comprised 719 characters including gaps; out of these, 422 characters were conserved, 286 were variable, 199 were parsimony informative and 87 were singletons. For LSU, the aligned final dataset comprised 947 characters including gaps; out of these, 763 characters were conserved, 178 were variable, 131 were parsimony informative and 47 were singletons. Our taxa are separating from their closest species with strong bootstrap value of 100 for ML and 1 PP for BI and there was no conflict in both analyses in the position of our taxa.
The specific epithet margallensis (Latin) refers to type locality Margalla.
Leucoagaricus margallensis can be distinguished by small, umbonate pileus with minute, fragile annulus, bulbous stipe, smaller basidiospores (6.27 × 4.67 µm) and cheilocystidia without crystals on its apex.
Basidiomata medium-sized, shiny, smooth, moderately fleshy, solitary. Pileus 1.5–2.5 cm in diameter, hemispherical to parabolic when young, expanding convex to plano convex, plane at maturity, umbonate, radially fibrillose to rugulose, whole pileus orange (0.5YR 3.2/8.7) when immature, then yellowish orange (9.4YR 6.1/7.2) on maturity, margin incurved in mature and striated, context white, smooth, thick at center. Lamellae free to thin, milky white (2.2GY 6.8/0.9), spacing fine and close, 1–1.5 mm wide, edges entire, lamellae regular, 3-4 tiers. Lamellulae irregular, alternating with lamella, in 2-3 tiers. Stipe 4.5–7 × 0.5–1 cm, cylindrical, central, thin, white (2.2GY 6.8/0.9), smooth, shiny, even, clavate. Annulus simple, white, located in upper half of stipe, membranous upturned. Flavor and odor not distinctive.
Basidiospores [90/5/3] (6–) 6.2 – 8.0 (–8.2) × (3.43–) 4.11 – 4.7(–4.9) µm, 6.1–8.1 × 3.7–4.8 µm, av. Q = 1.58–1.42, Qav = 1.49, hyaline to light yellowish in 5% KOH, oval to ellipsoid at face view, lacrymoid to amygdaliform in side-view, guttulate, smooth, dextrinoid, thin-walled and apiculate. Basidia (12.06–) 13.08 – 23.5 (–24.03) × (6.86–) 7.7 – 8.72 (–9.72) µm, 12.9–18.5 × 5.8–7.7 µm, av. L =19.29 µm, av. W = 7.54 µm, narrowly clavate, hyaline in 5% KOH, smooth, with 2–4 prominent sterigmata, oil droplets present,no clamp at base. Cheilocystidia (15.12–) 16.12 – 24.9 (–25.98) × (6.88–) 7.8 – 8.4 (–9.43) µm, 15–26 × 6.8–9.4 µm, hyaline in 5% KOH, thick-walled, broadly clavate and smooth, without internal content and clamp. Pleurocystidia absent. Pileipellis an intricate trichoderm, made up of 5.6–7.7 µm, av w=5.99 µm, wide, septate, interwoven, thin-walled hyphae, hyaline in 5% KOH, clamp connections absent.
Stipitipellis as compactly arranged cutis, made up of septate, cylindrical hyphae, 3.4–4.9 µm in diam., thin-walled, light yellowish in 2% KOH, clamp connections absent.
Leucoagaricus margallensis has a combination of striking features like small, umbonate pileus with minute, fragile annulus and broader bulbous stipe. Anatomically, it has smaller basidiospores (6.27 × 4.67 µm), smaller cheliocystidia and absence of crystals at apex of cheliocystidia.
Leucoagaricus margallensis resembles La. rubrotinctus including similar pileus size and shape, fibrillose surface, stipe color and shape. But Leucoagaricus margallensis differs from La. rubrotinctus by minute fragile annulus while La. rubrotinctus has prominent white annulus. Anatomically, Leucoagaricus margallensis has ellipsoid, smaller (6.27 × 4.67 µm) basidispores as compared to La. rubrotinctus larger and amygdaliform basidiospores. Furthermore, Leucoagaricus margallensis has smaller cheliocystidia (15–26 × 6.8–9.4 µm) as compared to La. rubrotinctus (30–50 × 5–10 µm), narrow pilleipellis hyphae (5–10 µm) in La. rubrotinctus. The other closely related taxa in phylogenetic tree is L. rubroconfusus Migl. & Coccia, characterized by centrally depressed, larger (up to 7.5 cm) pileus with orange fibrillose squamules as compared to umbonate, smaller (2.5 cm) yellowish orange fibrils on white pileus of Pakistani taxon. Anatomically, both taxa lack crystals at apex of cheilocystidia. Our taxon Leucoagaricus margallensis is differentiated from L. rubroconfusus due to smaller (6.27 × 4.67 µm) basidiospores as compared to basidiospores of L. rubroconfusus (5.5–8(9.5)) µm.
Leucoagaricus subpurpureolilacinus Z.W. Ge & Zhu L. Yang, from southwestern China differs in its broad, brown to dark ruby umbo, larger basidiospores and clavate cheilocystidia with gelatinized covering intermixed with crystals (
The specific epithet glareicolor (Latin) refer to the brownish black color of umbo and fibrils on pileus.
Is distinguished due to brown to blackish brown, broadly umbonate, white pileus with blackish brown fibrillose to rugulose, smooth sub-bulbous stipe with annulus in upper 1/3 of stipe, smaller (5.65–7.70 × 3.44–5.63 µm) basidiospores and cheilocystidia without crystals at apex.
Basidiomata medium to large-sized, soft shiny, fragile solitary. Pileus 4-4.2 cm, plano-convex to plane at maturity, radially fibrillose to rugulose towards margins, broadly umbonate, umbo brown when young, becoming blackish brown with age, disc creamy white (1.7GY 6.4/1.6) with dark brown (3.8Y 3.5/3.2) to blackish brown (4.8Y 5.5/8.2) rugulose towards margins, shiny and sericeous, on maturity broadly striated and overextended blackish brown fibrils darker in center and lighter towards margin, margin incurved and appendiculate. Lamellae free to approximate, adnexed, distant, fragile, edges entire, yellowish creamy (1.7GY 6.4/1.6) to grayish brown (4.8Y 5.5/8.2). Lamellulae irregular, alternating with lamella, in 2-3 tiers. Stipe 6.2–6.7 cm in length, tapering upwards, 0.5 cm at apex, 1.7 cm at base, sub bulbous, yellowish creamy (1.7GY 6.4/1.6) central, cylindrical, smooth, shiny. Annulus present, superior, thin, made up of cottony scales, non-persistent in nature. Flavor and odor not distinctive. Basidiospores [60/3/2] (5.65–) 5.66 – 7.73 (–7.76) × (3.43–) 4.11 – 4.6 (–5.63) µm, 5.65–7.70 × 3.44–5.63 µm, Q = 1.23–1.70, Qav = 1.48, hyaline in 5% KOH, oval at face view, lacrymoid to amygdaliform in side-view, guttulate, smooth, dextrinoid, thin-walled and apiculate. Basidia (12.06–) 13.08 – 23.5 (–24.03) × (6.86–) 7.7 – 8.72 (–9.72) µm, 12.9–18.5 × 5.8–7.7 µm, narrowly clavate, hyaline in 5% KOH, smooth, with 2–4 prominent sterigmata, oil droplets present, no clamp at base. Cheilocystidia (15.12–) 16.12 – 24.9 (–25.98) × (6.88–) 7.8 – 8.4 (–9.43) µm, 15–26 × 6.8–9.4 µm, hyaline in 5% KOH, thin-walled, clavate to narrowly clavate, smooth, without internal content and clamp. Pleurocystidia absent. Many layers of isodiametric irregular epithelial cells av = 7.87 µm in diameter, at the base of basidia and cystidia. Pileipellis trichoderm, made up of septate, cylindrical hyphae 5.6–7.7µm in diam., thin-walled, light brown to reddish pigment in the center, light orange to yellowish pigment towards margin in 2% KOH, clamp connections absent. Stipitipellis cutis compactly arranged, made up of septate, cylindrical hyphae 3.4–7.1µm in diam., av. w = 5.86 µm thin-walled, hyaline in 2% KOH, clamp connections absent.
Leucoagaricus glareicolor is characterized by broad umbo that is brown to blackish brown, fibrillose to rugulose white pileus. The stipe is smooth, sub-bulbous with annulus that is present in upper 1/3 of stipe. Anatomically, Leucoagaricus glareicolor has smaller (5.65–7.70 × 3.44–5.63 µm) basidiospores and cheilocystidia without crystals at apex.
Leucoagaricus glareicolor shows close relationship with L. subvolvatus and L. menieri. Leucoagaricus subvolvatus and L. glareicolor share many similarities like same pileus color, size and shape. However, L. subvolvatus has broad yellow umbo with yellow, fine fibrils while L. glareicolor has very prominent, blackish brown umbo with blackish brown fibrils. Leucoagaricus subvolvatus is characterized by stipe base with a marginal bulb. Leucoagaricus glareicolor has slightly bulbous stipe. Furthermore, Leucoagaricus subvolvatus has annulus at lower part of stipe while Leucoagaricus glareicolor has prominent annulus in upper 1/3 half of stipe. Furthermore, Leucoagaricus subvolvatus has cheilocystidia with crystals at apex whereas Leucoagaricus glareicolor cheilocystidia lacks crystals at apex (
Compared to Leucoagaricus menieri, characterized by light yellow, slightly umbonate pileus which is milky white while Leucoagaricus glareicolor has brownish black umbo, creamy white pileus with blackish brown fibrils. The stipe in the former is more bulbous (4–5 mm) as compared to subbulbous (0.5–1.7 cm) stipe in Leucoagaricus glareicolor. Anatomically Leucoagaricus menieri is differentiated from Leucoagaricus glareicolor by longer basidiospores {(6.9–)7.4(–8.7)} and the presence of crystals at apex of cheilocystidia. Another closely related taxon is Leucoagaricus sardous that is differentiated by our taxa by broader {5–6.8 (7.3)} basidiospores and larger (upto 75 µm) cheilocystidia. The next closely related taxon is L. volvatus that is characterized by the gelatinized, white pileus with olivaceous tinges and presence of crystals on cheilocystidia.
Molecular phylogenetic analyses based on ITS and LSU sequences also support La. glareicolor as a distinct species with strong bootstrap support.
In this paper, two new species of Leucoagaricus were studied morphologically and sequences of two DNA regions were analyzed for each species. With the new data provided in the present study, the number of Leucoagaricus species for all of Pakistan increases to fourteen. All in all, these data suggest that our knowledge of the diversity of Leucoagaricus in high mountain areas in Asia and the Margala forests of Pakistan is still in its infancy. These two new species provide evidence that further research is needed to collect and identify the fungal diversity of Asia.
We are thankful to reviewers for their valuable suggestions to improve the quality of manuscript. The corresponding author is thankful to University of the Punjab, Lahore for providing financial support (Research Grant 2022-2023) to Arooj Naseer for this Research work.