https://orcid.org/0000-0002-0917-5224
Figures
Abstract
The genus Agaricus includes more than 500 species mostly containing the edible and cultivated species worldwide. As part of the ongoing studies on the biodiversity of genus Agaricus in Pakistan, our objective was to focus on A. sect. Minores which is the largest section of the genus. In the first phylogenetic analyses based on the ITS region of the nuclear ribosomal DNA, our sample included specimens of 97 named species, 27 unnamed species, and 31 specimens (29 newly generated sequences in this study) from subtropical climate zones of Pakistan that likely belong to this section based on their morphology. The 31 specimens grouped into five distinct, well-supported clades corresponding to five species: A. glabriusculus already known from Pakistan and India, A. robustulus first recorded from Pakistan and briefly described here but already known from Bénin, Malaysia, China, and Thailand, and three possibly endemic new species described in detail A. badiosquamulosus sp. nov., A. dunensis sp. nov., and A. violaceopunctatus sp. nov. The sixth species currently known in Pakistan, including A. latiumbonatus also found in Thailand, were included in a multigene tree based on ITS, LSU, and Tef-1α sequence data. They all belong to a large pantropical paraphyletic group while most temperate species belong to a distinct clade, which includes about half of the species of the section. The current study aims to propose three novel species of genus Agaricus based on comprehensive morphological as well as molecular phylogenetic evidences from Pakistan.
Citation: Bashir H, Asif M, Ghafoor A, Niazi AR, Khalid AN, Parveen G, et al. (2024) Multigene phylogeny and morphological descriptions of five species of Agaricus sect. Minores from subtropical climate zones of Pakistan. PLoS ONE 19(7): e0302222. https://doi.org/10.1371/journal.pone.0302222
Editor: Dharmendra Kumar Meena, CIFRI: Central Inland Fisheries Research Institute, INDIA
Received: October 6, 2023; Accepted: March 30, 2024; Published: July 11, 2024
Copyright: © 2024 Bashir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: https://www.treebase.org/treebase-web/home.html;jsessionid=1F4ADD78BB9C492CEC9BE51AE74A7115 https://www.mycobank.org/ https://www.ncbi.nlm.nih.gov/nucleotide/ All the accession numbers have already been mentioned in the manuscript.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The genus Agaricus (Basidiomycota) is the type genus of Agaricaceae containing saprophytic species. The species are adapted to live in various climatic regions as subtropical, tropical, temperate, Caribbean islands and deserts. Several species are consumed and cultivated because of their nutritional and medicinal importance such as A. bisporus (J.E. Lange) Imbach, A. arvensis Schaeff. and A. subrufescens Peck [1, 2–4].
The classification of the genus is mainly proposed by Zhao et al. [5] which was revised using molecular clock methods [6]. Five monophyletic clades A, B, C, D, and E were proposed having stem ages ranging from 30–33 My ago. Each clade is considered as subgenus. Amongst these five subgenera, Clade A is considered as a subgenus Minores [6]. It includes mainly the section Minores reported previously by Zhao et al. [5] that diverged 30 My ago and is sub-divided into Clade A1/ sect. Minores, Clade A2/sect. unknown or sect. Laeticolores and sect. Laeticolores. Agaricus sect. Laeticolores was represented by only one species A. rufoaurantiacus (LAPAM15) [6] which was reported as subgen. Minoriopsis by Chen et al. [7]. In the same study by Chen et al. [7] another new section Leucocarpi has been introduced and Clade A2/sect. 1 still remains unnamed. The classification of subgen. Minores is recently revised where subgen. Minoriopsis is splitted into A. sect. Kerrigania and Minoriopsis. Agaricus sect. Minores is sub-divided into three sections viz. A. sect. Pantropicales L.A. Parra, Angelini, B. Ortiz, Linda J. Chen & Callac, A. sect. Minores (Fr.) Henn., and A. sect. Leucocarpi Linda J. Chen & Callac [6–8]. Later, He et al. [9] has introduced a new section of Agaricus subg. Pseudochitonia and now the number of sections are increased to 24. According to Index Fungorum data, there are about 6000 species of Agricus recorded from all over the world so far (accessed in April 2022).
Generally the species of A. sect. Minores are distributed world wide in tropical and temperate areas [5, 10] and some are found in extreme climates like secotioid species, adapted well to desert conditions [11, 12]. However, the newly reported species in this study were collected mainly from subtropical areas of Pakistan and the desert but none of them is sectioned. Previously 35 species of the genus Agaricus have been reported from Pakistan [7, 13–20] amongst these only two species belong to Agaricus sect. Minores [17]. Recently, two new species viz. A. midnapurensis and A. purpureosquamulosus and a new record of A. glabriusculus have been documented using ITS region from India [21] and Agaricus parviumbrus is documented as a new species in Fungal Planet description sheets [22].
The data of all the species in this study is a significant contribution to the knowledge of genus Agaricus from Pakistan. A few studies have been conducted based on multigene and morphological data so far. The heteromorphic sites of ITS region within and between the closely related taxa are analyzed in this study.
Materials & methods
Specimen collection and sampling sites description
A total of 29 specimens of genus Agaricus were collected from different semi-arid and arid localities of Punjab province, Pakistan. Based on morphological observations and fresh basidiomata field notes, the specimens likely belonged to Agaricus sect. Minores. As part of the current study on the genus, surveys were conducted in different subtropical areas of Punjab province i.e., Lahore; Toba Tek Singh; Haroonabad, district Bahawalnagar and Lal Suhanra National Park, Cholistan desert, district Bahawalpur, Pakistan.
Lal Suhanra National Park lies in southeastern Punjab, Pakistan. This national park is part of the Cholistan desert having fungal flora that has not yet been studied so far. It is a subtropical region characterized by very low annual rainfall (90–200 mm) in monsoon season, low relative humidity (60%) and high temperature (50°C) in summer [23]. It is located 32 km away from district Bahawalpur.
District Bahawalpur lies in southern side of Punjab, Pakistan. This subtropical region receives average annual rainfall fluctuating from 90 to 200 mm only [24]. The average annual temperature is more or less similar to Lal Suhanra National Park ranging from 6 to 10°C in winters and 49 to 52°C in summers [25].
The other collection site lies in Haroonabad, District Bahawalnagar, Punjab, Pakistan adjacent to Cholistan desert which is characterized by hot and dry climate with an extreme temperature of 50°C and minimum temperature of 11°C. The average annual rainfall is just 99 mm [26, 27]. The basidiomata (BWN 34, BWN 67, BWN 85) were collected along the bank of three R (3R) irrigation canal under Acacia nilotica from the loamy soil.
Toba Tek Singh is a district of Punjab with a subtropical climate. The maximum temperature is recorded at 40.7°C and a minimum of 6°C with an average annual rainfall of 254–381 mm [28].
Changa Manga Forest lies in the southeast of the district Lahore. This is a subtropical area with minimum to maximum temperature ranges from 5.9 to 39.6°C, respectively, and the annual rainfall is about 650 mm [29].
Morphological characterization
The fresh specimens were photographed during field surveys at collection sites. Collected basidiomata were carefully tagged and dried in sunlight or by using a fan heater. All the dried specimens were packed in zipper bags and brought to the laboratory for further processing. Morphological observations such as color, size, and shape of pileus, stipe, and lamellae were noted [30]. Other fresh sporocarp characters were also observed and noted during collection survey including discoloration upon bruising/handling, odor, and different chemical reactions such as KOH and Schaeffer’s reaction [31]. Color codes were given from Munsell’s Soil Color Charts [32].
Tissues from different parts of fruiting bodies were rehydrated in 5% KOH (w/v) solution followed by staining in 1% aqueous Congo red (w/v). Anatomical characeterization included size and shape of basidia, basidiospores, cheilocystidia, pileipellis and stipitipellis and their measurements were taken using a light microscope at 100X (MX4300H, Meiji Techo Co., Ltd., Japan). All the microscopic features were represented by at least 20 measurements each. The dimensions of basidiospores are provided as (a) b–c (d) × (e) f–g (h), [avX, Qm, n = i × j] where b–c and, f–g includes the spore length and width, respectively, between the 5th percentile and the 95th percentile, (a) and (d) the minimum or maximum values of spore lengths recorded, similarly (e) and (h) the minimum or maximum values of basidiospores width were recorded, avX the mean of length by width ± SD (standard deviation), Qm the mean of Q coefficient (length/width ratio), n represents the total number (i) of basidiospores measurements of each sample/collection and (j) indicates number of total samples/collections measured. Measurements of other anatomical structures as basidia, cheilocystidia, hyphal structures including pileipellis, and stipitipellis were available in dried samples included the range between the minimum or maximum values calculated in length and width. The measurement of basidia was considered excluding sterigmata. Short descriptions of all the new species have been deposited in MycoBank.
Phylogenetic observations.
For ITS phylogenetic analyses, a dataset of 158 specimens belonging to 95 nammed and 27 unammed species within A. sect. Minores was used. The remaining taxa belong to A. sect. Leucocarpi (01), A. sect. Pantropicales (03) and A. subg. Minoriopsis (02). The sequences for phylogenetic analyses were selected from initial BLAST results and in previously referenced studies on A. sect. Minores [17, 31].
For multigene phylogenetic analyses, dataset of 100 specimens with characters was included belonging to 72 named and 26 unnamed species including A. sect. Leucocarpi (01), A. sect. Pantropicales (03), Incertae sedis (02), and 02 from A. sect. Agaricus as outgroup taxa.
DNA extraction, PCR amplification and DNA sequencing.
DNA was extracted from the dried specimens using the 2% CTAB method with some modifications as described by Zhao et al. 2011 [5]. PCR amplification was performed of three nrDNA regions viz. ITS (Internal Transcribed Spacer region) using ITS1F/ITS4 primers [33, 34] LSU (large subunit fragment) using LROR/LR5 set of primers [35] and EF1 (translation elongation factor) utilizing EF1-983F/EF1-1567R combination of primers [36]. In PCR cycle, denaturation was done at 95°C followed by annealing of 35 cycles at 94°C and final extension at 72°C. The PCR products were sequenced from BGI (Beijing Genomic Institute) and Genewiz, UK. All the newly generated sequences during this study have been submitted to GenBank.
DNA sequence alignments.
Sequence alignment for ITS and multigene datasets was performed independently, using T-cofee version 8.99 [37] then final adjustment was done manually in Bioedit version 7.2.0. The CIPRES PORTAL v. 3.1 [38] was used for maximum likelihood (ML) analyses. Both ITS and multigene phylogeentic analyses were conducted for each alignment separately using RAXML–HPC2 v 8.1 [39]. Topology of phylogram was retrieved at 1000 bootstrap replicates. Significant support was considered to be BS≥50. No significant inconsistency between the ITS and multigene sequence data was observed, therefore ITS, LSU, and TEF1 sequences were integrated in BioEdit for final phylogenetic analyses. The multigene data was segmented into ITS, LSU, TEF1 (intron and coding sites).
Data analyses.
The most suitable substitution model for each region was inferred with MrModeltest version 2.2 [40]: SYM+G for all partitioned DNA regions. Bayesian Inference (BI) [41] was completed with MrBayes v. 3.1.2. Six Markov chains were run for one million generations and sampled every 100th generation. Burn-in was indicated in TRACER v. 1.6 [41] from trace plots of likelihood and finally discarded. The resulting phylograms were opened in FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/sofware/fgtree/). The final alignments of both ITS and multigene dataset were deposited in TreeBASE (ID = 30723).
Species-specific ITS markers.
Taxa, clades, sub-sections, or sections characterizing markers were located in the final alignment of 153 total ITS sequences of A. sect. Minores. These markers are provided with capital letters and are shown along with flanking sequences. IUPAC codes, for example Y, do not indicate ambiguous nucleotide (C or T) but heteromorphic site (C and T) likely reflecting allelic polymorphic conditions in the heterokaryotic (n+n) basidiomata. Deletions or insertions are mentioned in square brackets. The locality/position of the markers are numbered with respect to their position either in the 5’-3’ ITS1–5.8S–ITS2 sequence data (starting from tygaatt) of the taxon to be characterized or in the final alignment deposited in TreeBASE for the clades or higher taxa characterization.
Results
Phylogenetic analyses
The phylogenetic analyses comprised of 42 sequences that were newly generated (30 ITS, 06 LSU, and 06 TEF1) from 30 specimens. These 30 specimens belonged to six species including five new species described in this study and a new record (A. robustulus) from Pakistan. ITS sequences of all the specimens were included for the first time in phylogenetic analyses. The alignment for ITS dataset consisted of total sequence of 158 specimens and 718 characters (Fig 1). Among these, seven samples were used as outgroup belonging to seven different species of: A. sect. Leucocarpi (A. leucocarpus), A. subg. Minoriopsis (A. rufoaurantiacus and A. sp. ZRLWXH3064), A. sect. Pantropicales (A. candidolutescens, A. sp. LAPAM14 and A. sp. ZRLWXH3161) and A. sect. Agaricus (A. campestris). All samples with their GenBank accession numbers are listed in Table 1 and references in Table 2.
Agaricus campestris is used as outgroup. Bootstrap support values are indicated (BS). New species and new record from Pakistan are in bold. T = type specimen.
The alignment for multi-gene analyses consisted of sequences (ITS/LSU/TEF1) of 105 sequences and 2052 characters (Fig 2). The five samples used as outgroup belonging to five different species of: A. sect. Leucocarpi (A. leucocarpus), A. sect. Pantropicales (A. candidolutescens, A. sp. LAPAM14 and A. sp. ZRLWXH3161) and A. sect. Agaricus (A. campestris). Agaricus huijsmanii, a European species, is separated from all the clades in multigene phylogenetic analyses as referenced in He et al. 2017 [32] also, however, this taxon got aligned within A. sect. Minores in ITS phylogenetic analyses. The remaining 99 samples belonged to 96 species or putative species of A. sect. Minores (Table 1).
Agaricus campestris is used as outgroup. Bootstrap support values are indicated (BS). New species from Pakistan are in bold red. T = type specimen.
For both multigene and ITS analyses, the topologies of the phylogenetic trees using the Maximum likelihood (ML) or the Bayesian inference (BI) methods were highly similar. The bootstrap support values (BS) and Bayesian posterior probabilities (PP) are reported in the ML phylogenetic trees of Fig 1 for ITS analyses, and Fig 2 for multigene analyses. The multigene data of all the newly reported species and those available of previously reported samples were completed in our multigene analyses, however, still there remain many species that only have ITS sequence data. For this reason, we presented first the phylogeny of the sections based on multigene-analyses. The ITS analyses remain useful mainly at the species level to delimit new taxa and identify their most closely related taxa.
Taxonomy
Agaricus badiosquamulosus H. Bashir & Khalid sp. nov. (Figs 3 and 4).
Bars = 1 cm. Photographed by Dr. Hira Bashir.
(A–H); (A-B) Basidia, (C) Basidiospores, (D-F) Cheilocystidia, (G) Pileipellis hyphae, (H) Stipitipellis hyphae. Bars = 10 μm. Drawings by Dr. Hira Bashir.
Species–specific ITS markers: aatcttCttcccg@200, attccTTggagca@420–421
Etymology: The specific epithet ‘badio’ means in Latin brown and ‘squamulosus’ means scales, ‘badiosquamulosus’ referred to the brown colored squamules on the pileus.
Holotype: PAKISTAN, Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M3) near Eucalyptus camadulensis, 217 m a.s.l., 01 September 2016, Hira Bashir, M28 (LAH35751), (GenBank # ON137221(nrITS).
Diagnosis: This species is characterized by having a dark brown pileus covering with purplish tinged squamules, immediately gives strong fulvescent discoloration when bruised. Annulus membranous, single edged with erroded edges in most of specimens and superous. Basidia frequently bisporic to tetrasporic and monosporic observed in only one collection, cheilocystidia abundant, multi-septate and broadly clavate.
Description: Pileus 3–5 cm in diam., parabolic at first then convex, slightly umbonate, dark brown (4.3R 2.3/2.4) at disc, dark brown with purple tinged (4.5RP 6.6/3.4) slightly appressed squamules when young (M28), disc completely covered with dark brown (0.5YR 4/2.8) squamules in younger sporocarp (F3), later scattered towards margin on creamy white (1.3PB 9.6/2.2) background outside disc or umbo. Surface fresh and fragile, light yellow surface observed in F3 and F4 after rain. Margins appendiculate, slightly wavy at maturity in some specimens (M28), vaguely exceeding the lamellae, ruptured at maturity, orange-yellow (10YR 8.3/2.5) discoloration when rubbed. Lamellae light pink (2.8R 8.1/3.6) to mouse-grey (9RP 6.3/0.8), then light (8.6R 6.5/3.2) to dark brown (3.3YR 3.5/1.8), free, crowded, with intercalated lamellulae in 2–3 tiers, sometimes erroded. Stipe 3.0–6 × 0.5–1 cm, cylindrical in some specimens, mostly curved at maturity, slightly bulbous at base, partially stuffed, provided with annulus in its upper part near lamellae, white (N10) with concolorous longitudinal striations above and below the annulus, immediately discoloring yellow-brown when rubbed, ends with rhizomorphs. Annulus superous, single-edged, membranous, fragile, non-persistent in some specimens (M22, M28, F53, CM09) only ring zone observed, or pendent (F4, F5, M4), white (N10), fibrillose, edges uneven. Context 2–4 mm wide, thick, white, immediately becomes strongly fulvescent (7.7YR 4.3/6.5) when bruised. Odor almond-like, strong. (Fig 3).
Macrochemical Reactions: KOH reaction positive, strong yellow. Schäffer’s reaction positive, strong reddish-orange.
Basidiospores (5.5–) 6–6.5 (–7) × (4.2–) 4.5–4.8 (–5.1) μm, [avX = 6.2 ± 0.67 × 4.6 ± 0.45 μm, Qm = 1.32, n = 20 × 10], subglobose to broadly ellipsoid, light to dark brown in KOH, smooth with a prominent apiculus, without apical pore and with granular content. Basidia 16–22 × 7–8.5 μm, narrowly clavate in most specimens, broadly clavate in F53 only, with abundant olivaceous granular content, frequently bisporic to tetrasporic, monosporic observed in F11. Cheilocystidia abundant, short and multiseptate and broadly clavate to rarely simple with terminal elements 11.5–20.5 × 6.5–11 μm, [avX = 15.9 ± 4.6 × 8.8 ± 2.3 μm], with ante-terminal elements mostly short, broader, cylindrical, 4–11 × 2.5–6 μm, olivaceous granular content observed but not abundant. Pleurocystidia absent. Pileipellis constituted by interwoven hyphae 4–10 μm in diam., frequently septate, branched, slightly constricted at septa, smooth, hyaline in KOH, terminal elements with rounded comparatively narrow ends. Stipitipellis constituted by hyphae 3–6.5 μm in diam., cylindrical, septate, unbranched, hyaline in KOH, no constriction at septa, terminal elements with narrow rounded tips. (Fig 4).
Habitat and distribution: Gregarious on grassy grounds, some under Eucalyptus camaldulensis Dehnh. trees along Pak. Motorway (M2 & M3) and TS57 found near Dalbergia sissoo trees along roadside in sandy soil. It is only known from Punjab province of Pakistan so far.
Additional material examined: PAKISTAN, Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M3) near Eucalyptus camadulensis, 217 m a.s.l., 14 August 2016, Hira Bashir, M4 (LAH35752), (GenBank # ON137222); Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M3) near Eucalyptus camadulensis, 217 m a.s.l., 14 August 2016, Hira Bashir, M6 (LAH35753), (GenBank # ON137223); Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M3) under Eucalyptus camadulensis, 217 m a.s.l., 1 September 2016, Hira Bashir, M22 (LAH35754), (GenBank # ON137224); Punjab, Lahore, solitary on rich loamy soil along motorway Eucalyptus camadulensis, 217 m a.s.l., 4 August 2015, Hira Bashir, F3 (LAH35755), (GenBank # ON137225); Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M2) under Eucalyptus camadulensis, 217 m a.s.l., 4 August 2015, Hira Bashir, F4 (LAH35756), (GenBank # ON137226); Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M2) near Eucalyptus camadulensis, 217 m a.s.l., 4 August 2015, Hira Bashir, F5 (LAH35757), (GenBank # ON137227); Punjab, Lahore, solitary on rich loamy soil along Pak. Motorway (M2) near Eucalyptus camadulensis, 217 m a.s.l., 4 August 2015, Hira Bashir, F11 (LAH35758), (GenBank # ON137228); Punjab, Lahore, gregarious on rich loamy soil along Pak. Motorway (M2) near under Eucalyptus camadulensis, 217 m a.s.l., 6 August 2015, Hira Bashir, F53 (LAH35759), (GenBank # ON137229); Punjab, Toba Tek Singh, at 183 m a.s.l., solitary in grassy ground near Dalbergia sissoo, 30 June 2017, Hira Bashir, TS57, (LAH35760), (GenBank # ON137230). Punjab, Kasur district, Changa Manga forest, scattered on decomposed matter under deciduous trees, 218 m a.s.l., 2 September 2021, Aneeqa Ghaffor and A.R. Niazi, CM09 (LAH02921), (GenBank # ON137231).
Notes:
Phylogenetic analyses revealed that A. badiosquamulosus and A. glabriusculus clustered close to A. glabriusculus (SH7, SH291L) reported from Pakistan also [17]. There are 12 nucleotide differences of A. glabriusculus from A. badiosquamulosus excluding heteromorphic sites. The other three taxa form sister subclade with A. purpureosquamulosus (CUH AM716), A. goossensiae (GF929) and an undescribed Agaricus sp. (MATA774) within A. sect. Minores. In the ITS sequences of A. badiosquamulosus and A. glabriusculus, numerous heteromorphisms were observed in the samples (Table 3) and a nucleotide G at position 701 is present only in two samples (M4 and TS57) instead of T. A single nucleotide difference is not considered very significant in Agaricus, especially in case of species having numerous polymorphic positions. Also, the morphological characters strongly constrained the phylogenetic results. So, these two specimens are considered to be the same as A. badiosquamulosus. Agaricus glabriusculus has five unique sites when compared with A. badiosquamulosus. As far as the A. sp. (MATA774) is concerned, there are 18 nucleotide differences from A. badiosquamulosus and 19 from A. glabriusculus excluding heteromorphic sites. The data indicate that our species A. badiosquamulosus is unique from all other taxa.
Agaricus dunensis H. Bashir & M. Asif (Figs 5–7)
Macromorphological characters of A. dunensis [BWN-34 (A-B), BWN-64 (C-D)] collected from Bahawalnagar. (A–D) basidiomata in the field. Bars = 1 cm. Photographed by Dr. Hira Bashir and Muhammad Asif.
Macromorphological characters of A. dunensis [L99 (A-B), LS4 (C)] collected from Cholistan desert. (A–C) basidiomata in the field. Bars = 1 cm. Photographed by Dr. Hira Bashir.
Micromorphological characters of A. dunensis (A–F); (A) Basidia, (B) Basidiospores, (C-D) Cheilocystidia, (E) Pileipellis hyphae, (F) Stipitipellis hyphae. Bars = 10 μm. Drawings by Dr. Hira Bashir.
Etymology: ‘dunensis’ in Latin referred to sand dune area from where the type specimen was recorded for the first time.
Holotype: Punjab, Lal Suhanra National Park, Cholistan desert, Bahawalpur, at 140 m a.s.l., scattered on sandy soil under Acacia nilotica trees, 23 June 2017, Hira Bashir, L99, (LAH 35747), GenBank # ON137217 (nrITS).
Diagnosis: This species is distinguished well having a pileus with bright yellow to rust squamules becoming brownish at maturity, pileus and stipe surface immediately turning dark brown when touched, and a strong red to blackish–brown discoloration upon bruising, monosporic basidia frequently observed in one sample (L2), frequently bisporic to tetrasporic in others, cheilocystidia polymorphous.
Description: Pileus 2.5–9 cm in diam., convex then plano-convex, slightly appressed and flattened at disc (LS4), sometimes disc depressed (BWN-64) at maturity, thoroughly covered by orange-yellow (5.8YR 5.9/6.2) squamules (predominant in L99 collection) in young stages, at maturity surface has dotted bright yellow (2.1Y 4.1/5.4) squamules becoming yellowish brown (10YR6/8) in some fruiting bodies, few scattered towards margin more dense at the center in mature sporocarp on a creamy (3.1G 8.5/0.7) background. Surface dull and dry. Margins slightly appendiculate, white, slightly incurved when young become straight at maturity, slightly exceeding lamellae. Lamellae light pink (7.2R 7.5/2) at young stage to dark brown (5.7R 2.2/1.4) at maturity, free, crowded, intercalated with lamellulae, entire edges sometimes slightly wavy. Stipe 1.5–6.5 × 0.5–3 cm, cylindrical to slightly bulbous at the base to ventricose with bulbous from center tapering towards base (BWN-64), stuffed, provided with an annulus in its upper part close to lamellae, covered with creamy white squamules from above and below annulus, creamy when young becomes brown from top at maturity, discoloring yellow or rusty at stipe base when rubbed, becoming light yellowish brown (1.3Y 6/2.4) when cut in L2, no significant discoloration observed in L99, brown (9.7YR 5.5/2.8) color appears mildly at stipe base in other specimens. Annulus superous, single edged, white (8.2PB 9.3/0.9), ruptured at young stage leaving behind a ring zone at maturity, fragile, smooth, membranous or thick and floccose on both sides (BWN-64). Context whitish (0.9PB 8.6/2.4), stuffed, no discoloration when cut. Odor strong almond-like. (Figs 5 and 6).
Macrochemical Reactions: KOH positive yellow. Schäffer’s reaction positive, yellow then strong reddish-orange on fresh sporocarp.
Basidiospores (5.5–) 6.3–6.9 (–7.5) × (4.2–) 5–5.3 (–5.6) μm, [avX = 6.5 ± 0.78 × 5.2 ± 0.46 μm, Qm = 1.3, n = 20 × 9], subglobose to ellipsoid, light to dark brown in KOH, smooth with prominent apiculus, without apical pore, with granular content. Basidia 21–29 × 6.5–10 μm, frequently bisporic to tetrasporic in all specimens, monosporic basidia observed frequently in L2 only and rarely trisporic found in BWN-34 and BWN-64, clavate or slightly truncate at apex, with abundant olivaceous granular content. Cheilocystidia abundant, polymorphous, short, broadly clavate, simple with terminal elements 9.5–18.5 × 5.5–10 μm, while fusiform abundantly observed in L2 and L99 with terminal elements 22.5–26 × 7.5–9.5 μm, some having round apices, ante-terminal elements subglobose to cylindrical measuring 5–9.5 × 4.2–7 μm, with abundant olivaceous granular content. Pleurocystidia absent. Pileipellis constituted by cylindrical hyphae 2.5–11.5 μm in diam., frequently septate, sometimes branched, no constriction at septa, some hyaline others with internal brown vacuolar pigmentation, terminal elements with rounded ends. Stipitipellis constituted by hyphae 2–10.5 μm in diam., mostly cylindrical few broader, no or slight constriction at septa, hyaline in KOH, terminal elements with rounded tips. (Fig 7)
Habit, habitat and distribution: Growing solitary and in groups on sandy soil under Acacia nilotica trees in the desert and its surrounding areas. The distribution of this species is known from arid regions of Pakistan until now.
Additional material examined: PAKISTAN, Punjab, Lal Suhanra National Park, Cholistan desert, Bahawalpur, at 140 m a.s.l., scattered on sandy soil near Acacia nilotica trees, 30 August 2016, Hira Bashir, L2, (LAH35748), (GenBank # ON137218); Punjab, Lal Suhanra National Park, Cholistan desert, Bahawalpur, at 140 m a.s.l., scattered on sandy soil near Acacia nilotica, 7 August 2013, Hira Bashir and Muhammad Usman, LS4, (LAH35749), (GenBank # ON137219); Punjab, Toba Tek Singh, at 183 m a.s.l., solitary in grassy ground near Dalbergia sissoo, 30 June 2017, Hira Bashir, TS56, (LAH35750), (GenBank # ON137220). Punjab, District Bahawalpur, at 163 m a.s.l, from loamy soil along the canal bank under Acacia nilotica, 5 September 2020, Aneeqa Ghafoor and A.R. Niazi, BWL 01 (LAH21719), (GenBank # ON158598). Punjab, District Bahawalnagar, at 163 m a.s.l, from loamy soil along the canal bank under Acacia nilotica, 31 July 2019, Muhammad Asif, BWN 34 (LAH36808), (GenBank # ON158597). Punjab, District Bahawalnagar, at 163 m a.s.l, from loamy soil along the canal bank under Acacia nilotica, 11 August 2019, Muhammad Asif, BWN 64 (LAH36807) (GenBank # ON158596). Punjab, District Bahawalnagar, at 163 m a.s.l, from nutrient rich loamy soil along the canal bank under Acacia nilotica, 11 August 2019, Muhammad Asif, BWN 67, (LAH36805), (GenBank # ON158600). Punjab, District Bahawalnagar, at 163 m a.s.l, from muddy soil along the canal bank under Acacia nilotica, 04 October 2019, Muhammad Asif, BWN 85, (LAH36806), (GenBank # ON158599).
Notes:
Phylogenetically, A. dunensis forms a small subclade with A. sp. (LAPAM35 and LAPAM64) and A. parvibicolor. These two species (LAPAM35 and LAPAM64) were introduced by Parra et al. 2018 [8] in the phylogenetic analyses and collected from Dominian Republic but not formally described yet. A few heteromorphic sites were observed in three samples (L2, L99 & TS56) of A. dunensis at differenet positions (Table 4). However, A. parvibicolor is characterized by its white and violaceous pileus with reddish or violet brown triangular fibrillose squamules while A. dunensis has pileus with bright yellow to orange yellow dotted squamules. Discoloration is yellow when stipe is cut and orange by handling in A. parvibicolor, in contrast, A. dunensis gave no significant discoloration when cut or very light yellow observed (L2) or faint yellowish brown at stipe base (BWN34 and BWN64). Agaricus parvibicolor has ellipsoid to elongate basidiospores having an average size of 6.3 × 3.9 μm [31] while A. dunensis has subglubose to ellipsoid basidiospres with an average size of 6.5 × 5.2 μm.
Agaricus robustulus Linda J. Chen, Callac, L.A. Parra, K.D. Hyde & De Kesel 2017 (Figs 8A, 8B and 9)
Macromorphological characters of A. robustulus (A-D) and A. glabriusculus (E-F). (A–F) basidiomata in the field. Bars = 1 cm. Photographed by Dr. Hira Bashir.
Bars = 10 μm. Drawings by Dr. Hira Bashir.
Material examined: PAKISTAN, Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary or in groups on rich loamy soil on the grassy grounds of the University Botanical Garden, 13 July 2016, Hira Bashir, PU217 (LAH35764).
Description: Pileus 5–7 cm in diam., broadly convex to plano-convex, brown (3.8RP 4.6/2.9) squamules with reddish tinge dense at disc, sometimes disc becomes depressed (PU212), covered with irregular shaped, few roughly triangular or dotted squamules, sparse towards margin on creamy (3.1P 7.6/2.5) background. Surface dry and dull. Margin irregular, ruptured at maturity, slightly appendiculate, slightly incurved at maturity, vaguely exceeding lamellae, immediately become strongly fulvescent on handling or rubbing. Lamellae whitish (N10) to brown (3YR 1.3/3.8), free and approximate, crowded, intercalated with lamellulae and with entire edges. Stipe 3–7 × 0.5–2 cm, cylindrical having bulbous base, stuffed, provided with annulus in its upper half close to lamellae, above and below annulus covered with concolorous fibrils, strong yellow discoloration by rubbing or bruising, provided with multiple rhizomoprphs at the base. Annulus superous, single edged, membranous, fragile, smooth, white, non-persistent leaving behind ring zone on stipe at maturity. Context white, light yellow discoloration when cut predominantly from upper half of stipe. Odor of almond, mild. (Fig 8A and 8B)
Macrochemical Reactions: KOH reaction positive, yellow. Schäffer’s reaction negative.
Basidiospores (5.5–) 5.9–6.3 (–6.6) × (3–) 3.5 (–4) μm, [avX = 5.8 ± 0.3 × 3.6 ± 0.2 μm, Qm = 1.5, n = 4 × 20], ellipsoid, rarely oblong, light to dark brown in KOH, smooth with a prominent apiculus. Basidia 14–22 × 6.5–9 μm, narrowly to broadly clavate, slightly truncate at apex, with abundant olivaceous granular content in KOH, frequently tetrasporic rarely bisporic. Cheilocystidia abundant, simple and septate at the base, with terminal elements 15–40 × 13.7–22.5 μm, polymorphous, broadly clavate, few pyriform, mostly hyaline in KOH, sometimes granular content present but not abundant, ante-terminal elements mostly cylindrical, broad, rarely subglobose, 3–10.5 × 2–6.9 μm. Pleurocystidia absent. Pileipellis constituted of interwoven hyphae 4–12.5 μm in diam., frequently septate, rarely branched, vaguely constricted at septa, with internal brown vacuolar pigmentation or hyaline in KOH, terminal elements with more or less rounded ends, few having attenuated apices. Stipitipellis constituted by interwoven hyphae 2.5–9 μm in diam., cylindrical, septate, rarely branched, hyaline in KOH, slightly constricted at septa, with terminal elements having rounded ends. (Fig 9).
Habit, habitat and distribution: In groups, on grassy grounds of Botanical Garden, University of the Punjab and near Eucalyptus trees. This species is known from China and now from Pakistan so far.
Additional material examined: PAKISTAN, Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary on rich loamy soil on the grounds, 13 July 2016, Hira Bashir, PU212 (LAH35765); Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary on rich loamy soil on the grounds of the University, 1 September 2016, Hira Bashir, PU302 (LAH35766).
Notes:
Phylogenetically, four of our specimens get clustered with a recently described Chinese taxon, A. robustulus. This species is characterized by its reddish brown squamules on pileus and strongly fulvescent discoloration in mature sporocarps when rubbed or bruised. Cheilocystidia are polymorphic, having simple, broadly clavate to pyriform shaped. All these morphological characters are similar to that reported by Chen et al. 2017 [7]. Other species of this section also resemble morphologically to A. brunneolus, A. goossensiae, and A. megalosporus. However, the spore size in A. goosensiae is bigger (6.3 × 4.4) and cheilocystidia are inconspicuous while the other two taxa can be distinguished having large sized pileus exceeding 7 cm in diameter. This species is first time recorded from Pakistan.
Agaricus glabriusculus S. Hussain (Figs 8D–8F and 10)
Bars = 10 μm. Drawings by Dr. Hira Bashir.
Diagnosis: This species is distinguished well by having a pileus with brown fibrillose squamules dense at disc, context with pale brown discoloration when cut, annulus not very conspicuous, fragile, non-persistent and left in the form of ring zone on upper half of stipe. Basidia frequently bisporic to tetrasporic, cheilocysytidia broadly clavate and some have globose elements at apices.
Description: Pileus 4.5–6 cm in diam., broadly convex finally applanate, brown (3.8RP 4.6/2.9) squamules dense at disc, disc slightly depressed at maturity, few roughly triangular shaped, sparse towards margin at maturity on creamy background (3.1P 7.6/2.5), later surface turned light pinkish-brown. Surface fleshy, fragile. Margin serrate, ruptured at maturity, slightly appendiculate, not exceeding lamellae, light brown when rubbed. Lamellae pink (4.8RP 5.9/4.5) to brown (3YR 1.3/3.8), free and approximate, crowded, intercalated with lamellulae and with entire edges. Stipe 3.5–5 × 0.7–1 cm, having slightly bulbous base, cylindrical, stuffed, provided with annulus in its upper half close to lamellae, above and below covered with white fibrils, light pink and fleshy from top, solid, no significant discoloration or faint brown when rubbed, with rhizomorphs at the base in some speciemns. Annulus superous, single edged, membranous, fragile, smooth, white, non-persistent leaving behind ring zone on stipe. Context white, solid, no immediate discoloration when cut, faint brown color appears after 5–10 minutes. Odor faint almond-like. (Fig 8D–8F)
Macrochemical Reactions: KOH reaction positive, yellow. Schäffer’s reaction negative.
Basidiospores (5.5–) 5.9–6.2 (–6.5) × (3.9–) 4.1 (–4.4) μm, [avX = 6.2 ± 0.44 × 4.2 ± 0.27 μm, Qm = 1.5, n = 20 × 3], ellipsoid, light to dark brown in KOH, smooth with a prominent apiculus. Basidia 20.5–24.2 × 6.8–8 μm, narrowly clavate, slightly truncate at apex, with abundant olivaceous granular content, frequently bisporic to tetrasporic. Cheilocystidia abundant, septate at the base, with terminal elements 11–16 × 6.5–9.5 μm, [avX = 13.3 ± 2.7 × 8 ± 1.4 μm, broadly clavate, sometimes having globose or rounded elements at apex, smooth, mostly hyaline in KOH, in some granular content observed but not abundantly, ante-terminal elements short cylindrical, broad, subglobose, 4–11 × 2.5–6 μm. Pleurocystidia absent. Pileipellis constituted by interwoven hyphae 4–12.5 μm in diam., frequently septate, frequently branched, constricted at septa, with internal brown vacuolar pigmentation or hyaline in KOH, terminal elements with rounded ends, clavate elements also observed. Stipitipellis constituted by interwoven hyphae 3–6 μm in diam., cylindrical, frequently septate, branched, hyaline in KOH, slightly constricted at septa, with terminal elements having rounded ends. (Fig 10)
Habit, habitat and distribution: In groups, on roadside in grassy soil and around wheat fields. It is known only from arid region of Pakistan so far.
Material examined: PAKISTAN, Punjab, Toba Tek Singh, at 183 m a.s.l., in groups on grassy ground along roadside, 30 June 2017, Hira Bashir, TS10, (LAH35761), (GenBank # ON158590). Punjab, Toba Tek Singh, at 183 m a.s.l., gregarious in grassy ground along roadside under bushes, 30 June 2017, Hira Bashir, TS2, (LAH35762), (GenBank # ON158591); Punjab, Toba Tek Singh, at 183 m a.s.l., in groups in a village about 29 km away from the city, on grassy ground near Dalbergia sissoo and Populus trees, 30 June 2017, Hira Bashir, TS11, (LAH35763), (GenBank # ON158592).
Notes:
Agaricus glabriusculus is an already reported species from Pakistan on the basis of ITS and LSU data [17] and India only on ITS data analysis [21]. In this study, Tef-1α of this species is also added. Agaricus sp. (MATA774), A. purpureosquamulosus and A. goossensiae are also close to A. glabriusculus. The polymorphic positions in different samples of A. badiosquamulosus and A. glabriusculus are given in Table 3. Agaricus badiosquamulosus is characterized by having dark brown with purplish tinged squamules, A. glabriusculus has fine pinkish fibrils on pileus surface at young stage later dark brown squamules covering the pileus thoroughly, dense at disc. In A. badiosquamulosus, strong yellowish-orange discoloration on pileus covering and stipe when rubbed/touched and context becomes orange-brown or rusty upon bruising or cutting, in A. glabriusculus no or faint brown discoloration appears upon cutting. Agaricus sp. (MATA774) has delicate, pinkish brown in young to dark brown pileus covering in mature sporocarps. Stipe white, light pink from the top above annulus with concolorous squamules and gives yellow discoloration when bruised.
Agaricus violaceopunctatus H. Bashir (Figs 11 and 12)
Bars = 1 cm. Photographed by Dr. Hira Bashir.
Bars = 10 μm. Drawings by Dr. Hira Bashir.
Etymology: ‘violaceo’ originate from Latin meaning of violet color and ‘punctatus’ means condensed at some point, which referred that our species has violet-colored squamules condesnsed at disc of pileus.
Holotype: PAKISTAN, Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary on rich loamy soil on the grassy grounds of the University, 11 July 2016, Hira Bashir, PU125 (LAH35767).
Diagnosis: This species is well characterized by its violet squamules on pileus covering dense at disc and scattering towards margin at maturity, delicate and fleshy sporocarp, annulus fragile, white, and context with yellow discoloration on stipe when rubbed or bruised.
Description: Pileus 3–4.5 cm in diam., parabolic first, then convex, finally broadly convex at maturity, disc subumbonate in mature sporocarp, squamules predominantly provided with bright violet (8.9RP 3.9/5.3) coloration covering the pileus thoroughly at young stage, then sparse towards margin on creamy white background. Surface fleshy, fragile, becomes light yellow (8.4YR 7.3/2.8) when rubbed. Margins entire in young sporocarp, radially ruptured at maturity, appendiculate, vaguely incurved, slightly exceeding lamellae. Lamellae whitish pink (0.9YR 7.1/2.9) at first, then light brown (6.6R 5.8/1), free and approximate, crowded, intercalated with lamellulae and with entire edges. Stipe 4–7.5 × 1–2 cm, cylindrical with slightly bulbous base, stuffed, provided with annulus in its upper half close to lamellae, above and below annulus covered with white appressed fibrils, white, pinkish abover annulus. Annulus superous, single edged, membranous, fragile, smooth, having white fibrils. Context white, discoloring light yellow on bruising. Odor of almonds, mild. (Fig 11)
Macrochemical Reactions: KOH reaction positive, yellow. Schäffer’s reaction: positive, reddish orange on dry sporocarp.
Basidiospores 5–5.7 × 3.5–4.3 μm, [avX = 5.2 ± 0.23 × 3.8 ± 0.21, Qm = 1.39 ± 0.1, n = 2 × 20], ellipsoid, light brown in KOH, smooth with a prominent apiculus. Basidia 14–25 × 6.2–7.5 μm, narrowly clavate, slightly truncate at apex, hyaline in KOH, frequently tetraspoic rarely bisporic. Cheilocystidia simple or septate at the base, with terminal elements 15–30 × 6–10 μm, long and clavate, broadly clavate, ante-terminal elements mostly cylindrical, sometimes subglobose, hyaline in KOH. Pleurocystidia absent. Underside of annuluu. Pileipellis constituted by interwoven hyphae, 6–11 μm in diam., septate, branched, slightly constricted at septa, smooth, cylindrical, mostly with internal brown pigmentation and some hyaline in KOH, terminal elements with rounded ends. Stipitipellis constituted by hyphae 4–12.5 μm in diam., some broader hyphal elements observed interspersed in narrowly cylindrical hyphae, slightly constricted at septa, with terminal elements having rounded ends. (Fig 12).
Habit, habitat and distribution: In groups, on grassy grounds of Botanical Garden, University of the Punjab, near Eucalyptus trees. This species is known only from Lahore, Pakistan until now.
Additional material examined: PAKISTAN, Punjab, University of the Punjab, Lahore, at 217 m a.s.l., solitary on rich loamy soil on the grounds of the University, 13 July 2016, Hira Bashir, PU234 (LAH35768). Punjab, Kasur disctrict, Changa Manga forest, scattered on ground, 218 m a.s.l., 21 July 2019, Aneeqa Ghaffor and A.R. Niazi, CM16 (LAH21719).
Notes:
In phylogenetic analysis A. violaceopunctatus formed a subclade with all the undescribed taxa except A. microviolaceous. One of the closest taxon is the Pakistani specimen (MCR59), which was misidentified as A. goossensiae and the sequence is also submitted in GenBank (LAH5972011) with wrong identification. Our species has three nucleotide differences from A. sp. (MCR59) at position 37, 43 and 690 in ITS sequence data. When the original description of MCR59 (taken from the dissertation of author) was compared with our taxon, morphologically, pileus in MCR59 was recorded obtuse to umblicate with dark reddish squamules against reddish grey to hazel background and uplifted margin while in our species the squamules are strongly violaceous with creamy background. Annulus was not observed in MCR59 while in our collections annulus is very prominent, membranous and white. Clavate cheilocystidia were observed in MCR59 which are absent in our specimens. On the otherhand, A. microviolaceus also has violet squamules, small sized sporocaps as observed in our newly described taxon but phylogenetically both species are separated having more than 15 nucleotide differences in their ITS sequence data. So, our new species does not similar to any of the already described taxa.
Discussions
From Pakistan, three new species viz., A. badiosquamulosus, A. dunensis, and A. violaceopunctatus have been recorded during this study that are grouped within A. sect. Minores. In addition, one new record, A. robustulus, and an already reported species, A. glabriusculus, have also been documented providing their ITS, LSU, and Tef-1α sequence data. Two of those, A. badiosquamulosus and A. glabriusculus, clustered close to each other and are sister to A. sp. (MATA774) and with A. goossensiae (GF929; type specimen). Agaricus sp. (MATA774) was collected from a dune in 2007 growing near Tulostoma sp. by Philippe Callac & Gérardo Mata 2 Km away from Montepio, San Andrés Tuxtle, Mexico. Agaricus cf. goossensiae (ADK2751) was initially used in the phylogenetic analysis by Zhao et al. 2011 [5] and later as A. sp. [7, 9] so the species status was unclear. Therefore, in this study, we have requested to get a sequence of the type specimen of ADK2751 to Belgium herbarium which was provided to us for analysis. After comparisons, it was concluded that the ITS sequence of A. goossensiae (GF929) is far from the sequence of ADK2751 and both of these sequences do not share any genetic character. Therefore, ADK2751 is considered to be another taxon, different from A. goossensiae. So, the later concept was taken into account and accepted. Similarly, another taxon from Pakistan was misidentified as A. goossensiae (MCR59; LAH5972011) retrieved from the Genbank that is revised as A. sp. in this study as the ITS sequence is quite different from the type of A. goossensiae. This species is, in fact, closely related to an undescribed species (ZRL2011039) from China. The same species from Pakistan A. sp. (MCR59) clustered close to A. violaceopunctatus, another new taxon reported in this study.
All of the four newly described species in this section were collected from subtropical semi-arid and arid regions of Pakistan. Agaricus dunensis was collected from Cholistan desert (L2, L99 and LS4) and forth collection (TS56) from Toba Tek Singh, from sandy soils having a dry arid climate. All the collections of A. glabriusculus were collected from Toba Tek Singh district having subtropical and arid climate. Agaricus badiosquamulosus, is a common species found abundantly in different areas of Lahore and one collection was made from Toba Tek Singh which are subtropical semi-arid and arid areas, respectively. Agaricus robustulus and A. violaceopunctatus, were collected from subtropical region of Lahore.
Mainly, the species of A. sect. Minores have been reported from China (Yunnan) and Thailand. From Thailand, the collections were made from tropical areas while the climate of Yunnan is subtropical. Previously, 27 species were reported from Thailand which are distributed in six clades and 14 species from Yunnan while only three species are shared by both of these regions [7]. After the detailed study on A. sect. Minores by Chen et al. 2017 [7], the section comprised of 93 species in total. He et al. 2017 [31] added sixteen new taxa in A. sect. Minores, from tropic origins, China. In this study, multigene and molecular dating analyses have been conducted and very interesting conclusions were made regarding the origin of A. sect. Minores. The study showed that this section has a tropical origin with four major routes: 1) ca. 9–13 Ma, species migrated to the Tibetan Plateau and Europe from South Asia; 2) ca. 22 Ma, species from outside South Asia dispersed to Europe; 3) around ca. 9 Ma, species spreaded to Alaska via North Asia, then West America from South Asia; and 4) species reached south and Oceania from South Asia by three invading occurrences around ca. 9, 12 and 16–18 Ma, respectively. He et al. 2018 [9] reported three new species of A. sect. Minores based on ITS and multigene phylogenetic data. Recently, Hussain and Sher added two new species in this section from Pakistan [17]. In the present study, 41 sequences are added of three newly described species including 29 ITS and six of LSU and Tef-1α regions. So, this section is very diverse having great number of species of tropic origin.
Conclusion
The ITS and multigene phylogenetic analyses interpreted three new species A. badiosquamulosus, A. dunensis, and A. violaceopunctatus, a new record A. robustulus belonging to the Agaricus section Minores from subtropical regions of Pakistan. An already reported taxon from Pakistan and India, A. glabriusculus [17, 21] Tef-1α sequences are also added in this study which were not provided in the previous studies. All the micro- and macro-morphological data supported the molecular evidence. The species status of Agaricus cf. goossensiae (ADK2751) and A. goossensiae (MCR 59) on GenBank is clarified by obtaining the ITS sequence data from the type specimen of A. goossensiae (GF929) on special request and Agaricus sp. is assigned to both of these species. In this regard, the current investigation is an important contribution to the previously reported data on this largest section Minores of the genus Agaricus.
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