Bangladesh J. Plant Taxon. 32(1): 83-92, 2025 (June) DOI: https://doi.org/10.3329/bjpt.v32i1.82396 © 2025 Bangladesh Association of Plant Taxonomists AGARICUS BRUNNEOSPORUS, A NEW SPECIES FROM PUNJAB, PAKISTAN MUHAMMAD AHMED RAZA, MUHAMMAD BILAL SHARIF, HIRA IJAZ, MEMOONA AZEEM, MEHBOOBULLAH KHAN AND SANA JABEEN * Department of Botany, Division of Science and Technology, University of Education, Township, Lahore, 54770, Punjab, Pakistan Keywords: Hot desert; Saprobic; New species; Taxonomy. Abstract Agaricus species are ecologically and economically significant, yet their diversity in underexplored regions like Pakistan remains poorly documented. This study investigated Agaricus species from Bahawalnagar district, Punjab, Pakistan. Specimens were collected from different locations in Bahawalnagar, followed by morphological examination based on macroscopic and microscopic features. For molecular identification, DNA was extracted, and the ITS region of the nrDNA was sequenced. Phylogenetic analysis was conducted using Maximum Likelihood methods to determine the taxonomic placement of the species under investigation. The newly discovered species is morphologically similar to A. dunensis, with a brownish-orange central disk and light brown squamules becoming sparser at the margins. Basidiospores were subglobose to broadly ellipsoid, measuring 3.9–4.8 × 3.1–4.1 μm, and the species displayed clavate to narrowly clavate cheilocystidia. Phylogenetic analysis of the ITS region confirmed the distinctiveness of this species within Agaricus sect. Minores. The findings highlight the diversity of macrofungi in this region, with this species representing a previously undocumented taxon. This discovery enhances knowledge of Pakistan's fungal diversity and emphasizes the importance of exploring understudied regions. Introduction Agaricus L. (Agaricales, Agaricaceae) is a saprobic genus with a wide ecological distribution, ranging from arctic tundra to tropical rainforests. Species within this genus are found in diverse habitats, including alpine meadows, grassy dunes, salt-tolerant seaside grasslands, coniferous and deciduous forests, and various soil types. However, they generally avoid highly acidic and waterlogged soils (Bas, 1991). The genus is characterized by species that have white, yellow, or brown pileus, free gills lamellae with a regular trama when young, which later becomes irregular. The spore print appears in dark brown, featuring spores of a dark to purple-brown coloration, devoid of any discernible germ pore basidiospores smooth with a compound wall not visibly pseudo amyloid. The genus commonly displays a partial veil that develops into a ring on the stipe (Mitchel and Bresinsky, 1999; Karunarathna et al., 2016; Kuo, 2018; Saini et al., 2018; Ismail et al., 2023). Agaricus is represented by 500 species divided into seven subgenera and twenty-eight sections (Heinemann, 1974; Stoichev and Lacheva, 2002; Lacheva and Stoichev, 2004; Zhao et al., 2016; Chen et al., 2017; Callac and Chen, 2018; He et al., 2018; Parra et al., 2018; Bashir et al., 2018, 2021, 2023, 2024; Jaichaliaw et al., 2021; Wang and Bau 2024). From Pakistan, more than 40 species in this genus are known. Among these, 16 species have been reported based on *Corresponding author. E-mail: sanajabeen@ue.edu.pk https://doi.org/10.3329/bjpt.v32i1.82396 84 RAZA et al. morphological characteristics; however, two species are given in Ahmad et al. (1997); A. callipelus Berk. & Br. and A. lateritiocolor Heinm. lack records in Index Fungorum. The remaining species have been reported on molecular basis (Thongklang et al., 2014; Chen et al., 2016; Bashir et al., 2018, 2021, 2023, 2024; Hussain and Sher, 2019; Niazi et al., 2022; Nawaz et al., 2024; Ullah et al., 2024). Agaricus subgen. Minores comprises three sections: A. sect. Leucocarpi Linda J. Chen & Callac, A. sect. Minores (Fr.) Henn., and A. sect. Pantropicales L.A. Parra, Angelini, B. Ortiz, Linda J. Chen & Callac (Zhao et al., 2016; Chen et al., 2017; Parra et al., 2018). The characteristic feature of the A. sect. Minores is that the lower surface of the annulus is neither floccose nor squamose. The universal veil is poorly developed or absent (Zhao et al., 2016). Agaricus sect. Minores stands out as the most diverse section within the genus comprising 80 known species (He et al., 2017, 2018). It is anticipated that this section encompasses around 200 species (Chen et al., 2017). Among these, approximately 40 species in this section are documented in Asia and 21 are reported from Europe (Chen et al., 2017; Hussain and Sher, 2019). Data of the A. sect. Minores in Pakistan is extremely limited, with only two known species; A. glabriusculus S. Hussain and A. latiumbonatus S. Hussain (Hussain and Sher, 2019). A field survey of Haroonabad, Bahawalnagar district, Punjab Pakistan was conducted during the rainy season of 2023 to collect the fungal specimens for a research project. During the survey, various basidiomata were collected. Among these, two specimens from two collections were found similar and closely resembled the species in Agaricus. The specimens were observed morphologically and phylogenetically, which represent a new species in A. sect. Minores. Materials and Methods Sample collection, isolation, and specimen examination The specimens were collected from Haroonabad, located in the Bahawalnagar district of Punjab, Pakistan. Based on the Köppen climate classification, this area experiences a hot desert climate (BWh), characterized by extremely high temperatures and low precipitation. The temperature in the region varies between 11°C and 50°C, with an average annual rainfall of approximately 99 mm (Asif et al., 2023). The vegetation in this area predominantly includes species such as Azadirachta indica A. Juss., Albizia lebbeck (L.) Benth., Dalbergia sissoo Roxb., Eucalyptus camaldulensis Dehnh. and Vachellia nilotica (L.) P.J.H. Hurter & Mabb. (Ahmed et al., 2014). The specimens were photographed at the collection site, morphological features were noted, and the collections were dried using a fan heater. Key characteristics, such as the pileus' size, shape, and color, lamellae attachment and color, and the presence and type of annulus on the stipe, were noted. For color specification, Munsell color chart mobile application was used (https://munsell.com/color-blog/tag/color-app/). An EU 2230436 microscope with a 100X objective lens was used to observe anatomical features. Various chemicals, including 5% KOH for rehydration and Congo red for staining hyaline tissues, were used as mounting media. Measurements were recorded using the Calibrated Motic Images Plus 2.0 software. Microscopic characteristics were based on a minimum of 20 measurements for each structure. The symbol '(e) f–g (h)' was used to indicate the basidiospore size, where 'f–g' represented 90% of measured values, and 'e' and 'h' indicated extreme values. 'Q' denoted the length-to-width ratio of a single basidiospore from the side view, while 'avQ' represented the average 'Q' value for all specimens. Other microstructural measurements included the range between extreme length and width measurements. The specimens were deposited in the Herbarium at the Department of Botany, University of Education, Lahore, Pakistan (UEH). AGARICUS BRUNNEOSPORUS, A NEW SPECIES FROM PUNJAB DNA extraction, PCR amplification, and sequencing The DNA extraction was done by Bruns (1995) CTAB method. PCR and sequencing used the ITS1F and ITS4 primers (White et al., 1990). Sequences were analyzed in BioEdit version 7.2.5 (Hall, 1999). Phylogenetic reconstruction involved selecting close matches from GenBank, excluding sequences with inadequate query coverage. The phylogenetic tree included published sequences of species' closest relatives with corresponding GenBank accessions. To root the tree, sequences from A. sect. Agaricus were chosen as the out group (Bashir et al., 2021, 2024; Hussain and Sher, 2019). Multiple sequence alignment was performed with the online MUSCLE tool at EMBL-EBI (https://www.ebi.ac.uk/jdispatcher/msa/muscle). Phylogenetic analysis was performed to investigate the evolutionary relationships of the sequences. The best-fit nucleotide substitution model was selected based on model selection criteria, using the MEGA version 6 software (Tamura et al., 2013). Kimura 2-parameter model (Kimura, 1980) was found to be the most suitable model and applied to account for the nucleotide substitution patterns. Phylogeny was inferred using Maximum Likelihood (ML) for evolutionary distance estimation. The support for the branches was evaluated by performing 1000 bootstrap replicates to assess the robustness and statistical significance of the tree topology. Phylogenetic analyses The ITS sequence of 677 base pairs from our Pakistani collection UEH-F0020 was BLAST searched at NCBI. It showed 100 % similarity with a sequence from Pakistan and 99.12–99.38% similarity with Agaricus sequences from Pakistan and India. These sequences along with closely related sequences of Agaricus spp. from A. sect. Minores were retrieved from GenBank. The final aligned dataset contained a total of 50 nucleotide sequences, including two sequences: A. campestris L. (KM657927) and A. langei (F.H. Møller) F.H. Møller (JF797181) as out group (Table 1). The final dataset contained a total of 706 positions in the final dataset. Out of which, 493 sites were conserved, 201 variable, 125 parsimony informative and 74 singletons. The sequences from our collections appeared in a clade of A. sect. Minores separating from other Agaricus spp. in the same section. The sequences generated from the current collections clustered in a clade that includes recently published taxon, A. dunenesis H. Bashir & M. Asif sequences from India and Pakistan along with A. parvibicolor Linda J. Chen, R.L. Zhao & K.D. Hyde (NR_151751) from Thailand with 86% boot strap value. The clade representing A. dunensis is split into two lineages. The sequences generated during this investigation are separated from the clade that includes the type sequence of A. dunensis (ON137217) and two additional sequences labeled as A. dunensis (ON158599 & ON158600), suggesting that these sequences represent a separate taxon distinct from A. dunensis (Fig. 1). Results and Discussion Taxonomy Agaricus brunneosporus Raza, Bilal & Jabeen sp. nov. (Figs 2, 3) MycoBank: MB 852517 Etymology: The specific epithet “brunneosporus” (Latin) refers to the brown basidiospores. Diagnosis: Agaricus brunneosporus differs from A. dunensis by its brownish orange central disk and light brown squamules which become sparse towards margins, subglobose to broadly ellipsoid smaller (3.9–4.8 × 3.1–4.1 μm) basidiospores and clavate to narrowly clavate cheilocystidia. Holotype: PAKISTAN. Punjab, Bahawalpur division, Bahawalnagar district, Haroonabad, 160 m a. s. l., on soil, 26 July 2023, Muhammad Bilal Sharif P325 (UEH-F0020; GenBank for ITS: PP262619). 86 RAZA et al. Table 1. Taxa used for constructing the phylogenetic tree, along with their voucher numbers, geographical localities and GenBank accession numbers. Species Voucher Country ITS References Agaricus armandomyces ZRL2015992 China KX684860 He et al. (2017) A. bonussquamulosus ZRL2010106 China KX657047 He et al. (2017) A. brunneolus LAPAG938 Spain KU975082 Chen et al. (2017) A. brunneosporous as A. dunensis LAH36806 Pakistan ON158599 Bashir et al. (2024) A. brunneosporus UEH-F0020 Pakistan PP262619 This study A. brunneosporus UEH-F0021 Pakistan PP262620 This study A. brunneosporus UEH-F40027 Pakistan PV796086 This study A. brunneosporus as A. dunensis BWN-67 (LAH36805) Pakistan ON158600 Bashir et al. (2024) A. campestris LAPAG370 China KM657927 Ling et al. (2021) A. coccyginus 275412 China KU245981 Unpublished A. comtulus 2692 Canada KM248904 Unpublished A. diminutivus WC912 USA AY484681 Geml et al. (2004) A. dulcidulus PRM-909627 Czech Republic KF447894 Parra (2013) A. dunensis LAH36806 Pakistan ON158599 Bashir et al. (2024) A. dunensis LAH35748 Pakistan ON137218 Bashir et al. (2024) A. dunensis LAH 35747 Pakistan ON137217 Bashir et al. (2024) A. dunensis CUHAM737 India OM654930 Bashir et al. (2024) A. dunensis LAH36808 Pakistan ON158597 Bashir et al. (2024) A. dunensis LAH21719 Pakistan ON158598 Bashir et al. (2024) A. dunensis LAH36807 Pakistan ON158596 Bashir et al. (2024) A. dunensis LAH35749 Pakistan ON137219 Bashir et al. (2024) A. edmondoi LAPAG80 Spain KF447902 Parra (2013) A. elongatestipes ZRL2013271 China KX657002 He et al. (2017) A. friesianus LAPAG592 France KT951316 Parra (2013) A. glabriusculus SH7 Pakistan MK751852 Hussain and Sher (2019) A. heinemannianus LAPAG302 Spain KF447906 Parra (2013) A. indicus TBGT16128 India OR661746 Arya and Pradeep (2024) A. jacobi AH-44505 Spain NR_158300 Parra (2013) A. jingningensis ZRL20151562 China KX684877 He et al. (2017) A. kerriganii AH-44509 Spain KF447893 Parra (2013) A. lamelliperditus MDBF61/96 Australia JX984559 Lebel (2013) A. langei LAPAG141 Spain JF797181 Zhao et al. (2011) A. latiumbonatus SH166 Pakistan MK751861 Hussain and Sher (2019) A. luteomaculatus CA331 France KF447901 Parra (2013) A. marisae LAPAG138 Spain KU975083 Chen et al. (2017) A. matrum AH-44506 Spain KF447896 Parra (2013) A. megalosporus MFLU:100774 Belgium NR_119951 Unpublished A. midnapurensis CUH AM718 India OL467539 Tarafder et al. (2022) A. neimengguensis HMAS:254648 China NR_189789 He et al. (2017) A. pallens LAPAG441 Spain KF447898 Parra (2013) A. palodensis TBGT17483 India OR661748 Arya and Pradeep (2024) A. parvibicolor MFLU:12-0953 Thailand NR_151751 Liu et al. (2015) A. parvibrunneus HMAS0278356 China MG137001 He et al. (2018) A. pseudopallens ZRL20151552 China KX684874 He et al. (2017) AGARICUS BRUNNEOSPORUS, A NEW SPECIES FROM PUNJAB A. purpurellus TRgmb01309 Italy KF447903 Parra (2013) A. purpurellus - Canada MN620489 Unpublished A. purpureosquameus MFLU:17-1306 Thailand NR_157484 Unpublished A. purpureosquamulosus CUH AM716 India OL467541 Tarafder et al. (2022) A. robustulus CA847 (MFLU16- 0973) Thailand KU975086 Chen et al. (2017) A. yanzhiensis HMAS0281083 China MG137003 He et al. (2018) Fig. 1. Molecular phylogenetic analysis of Agaricus brunneosporus and related species based on ITS sequences. GenBank accession numbers are provided for each taxon. Sequences generated during this study are marked with bullets. Bold face represents sequence from type species. 88 RAZA et al. Fig. 2. Agaricus brunneosporus. a, b. UEH-F20020 (Holotype), c, d. UEH-F0021. Scale bars: 1 cm. Description Pileus 3–7 cm diam., parabolic to hemispherical; surface squamulose, dense at center, sparse towards margins; margins entire to eroded; central disk brownish orange (7.5YR 7/8), light brown (10YR 8/6), white fibrillose context. Lamellae free, alternate with lamellulae; brownish pink (5RP 7/4); lamellulae frequent, variable in length. Stipe 4.2–5.0 × 0.6–0.8 cm, central, cylindrical, bulbous base; surface smooth to slightly fibrillose, brown (7.5YR 7/3) from base to annulus, white above annulus. Annulus superior, white. Context white, firm, no color change on exposure to air. Basidiospores [40/2/2] (3.5)3.9–4.8(4.9) × (3)3.1–4.1(4.2) µm, Q = 1–1.3, avQ = 1.15, subglobose to broadly ellipsoid, smooth, thick walled, apiculus prominent, monoguttulate, brown in 5 % KOH. Basidia (11)13–14(15.5) × (5.1)5.5–5.6(5.7) µm, clavate, basal cell present, 2–4 sterigmata. Cheilocystidia (10)11.5–12(12.7) × (4.1)4.7–5.1(5.3) µm, clavate to narrowly clavate, septa present at the base. Pileipellis hyphae (4.5)5–5.8(6.7) µm wide, septate, cylindrical, AGARICUS BRUNNEOSPORUS, A NEW SPECIES FROM PUNJAB branched. Stipitipellis hyphae (4.1)5.1–6(6.9) µm wide, septate, branched. Clamp connections absent in all tissues. All tissues hyaline in 5 % KOH. Fig. 3. Microscopic characters of Agaricus brunneosporus UEH-F0020 (holotype). a. Basidiospores; b. Basidia; c. Cheilocystidia; d. Stipe hyphae; e. Pileipellis hyphae. Scale bars: a–c = 5 μm; d & e = 10 μm. Habitat and distribution: Found solitary in a hot desert climate with dominant vegetation of Azadirachta indica, Albizia lebbeck, D. sissoo, E. camaldulensis, and V. nilotica. Additional material examined: PAKISTAN. Punjab, Bahawalpur division, Bahawalnagar district, Haroonabad, 160 m a. s. l., on the soil, 26 July 2023, Muhammad Bilal Sharif P220 (UEH-F0021; GenBank for ITS: PP262620); 9 August 2024, Muhammad Bilal Sharif P654 (UEH- F40027; GenBank for ITS: PV796086). Agaricus brunneosporus is characterized by its hemispherical pileus covered with brownish orange squamules on white fibrillose surface, having pink and later brownish lamellae and slightly fibrillose stipe bearing a white superior annulus. The basidiospores are brown, subglobose to broadly ellipsoid (3.9–4.8 × 3.1–4.1 μm) without a germ pore. Agaricus dunensis, a recently reported taxon from Punjab, Pakistan differs from A. brunneosporous by its pileus thoroughly covered by orange-yellow squamules, comparatively small and thicker stipe, subglobose to ellipsoid larger (6.3–6.9 × 5–5.3 μm) basidiospores and polymorphous cheilocystidia (Bashir et al., 2024). Agaricus parvibicolor, a closely related taxon, differs from A. brunneosporus by its hemispherical to convex pileus surface having violet-brown 90 RAZA et al. fibrils and a crenulate annulus on the stipe. Its basidiospores are ellipsoid to oblong and larger (4.7–5.5 × 3–3.5 μm) (He et al., 2018). Agaricus glabriusculus S. Hussain, a Pakistani taxon, differs from A. brunneosporus by its fibrillose pileus surface having pinkish fibrils, pendant annulus and relatively larger (6–6.5 × 4–4.5 μm), cylindrical to broadly ellipsoid basidiospores (Liu et al., 2015). Agaricus latiumbonatus S. Hussain another Pakistani species differs from A. brunneosporus by a white pileus having grayish-red to dark-red squamules, arranged in rings and by a broadly umbonate disc, a stipe bearing pendant ascending annulus and by ellipsoid to broadly ellipsoid or amygdaliform, and relatively small (4.8–5.5 × 3.2–3.8 μm) basidiospores (Hussain and Sher 2019). Agaricus megalosporus J. Chen, R.L. Zhao, Karun. & K.D. Hyde from Thailand is distinct from A. brunneosporus by its hemispherical, purplish-brown to brown pileus with an eroded surface, and by a membranous pendant annulus. Its basidiospores are relatively larger (5.5– 6.5 × 7–7.5 μm) and ellipsoid to oblong, rarely cylindrical, smooth, reddish-brown, and thick- walled (Chen et al., 2012). Agaricus parvibrunneus M.Q. He, K.D. Hyde & R.L. Zhao another Asian species from China is different from A. brunneosporus by its brown fibrillose pileus surface, pinkish brown lamellae and a white pendent annulus. It further has smooth, thick-walled, and larger (5.0–5.8 × 3.7–4.1 μm) basidiospores (He et al., 2018). Agaricus velutinosus T. Bau & S.E. Wang from Northeast China differs from A. brunneosporus by fibrillose minutely floccose pileus and stipe surfaces and by initially white lamellae, and a stipe with superior white annulus. The basidiospores are relatively smaller (2.9–3.0 × 3.7–3.8 μm) ellipsoid to elongate-ellipsoid, thick-walled, and guttulate (Wang and Bau, 2024). Besides these morpho-anatomical differences, molecular phylogenetic analysis based on sequences from ITS region of nrDNA showed that this species formed its own lineage separated from already known taxa in A. sect. Minores with a strong bootstrap support. Acknowledgments Students are grateful to their parents for financial support. References Ahmad, S., Iqbal, S. and Khalid, A.N. 1997. Fungi of Pakistan. Sultan Ahmad Mycol. Soc. of Pakistan, Department of Botany, University of the Punjab, pp. 1–248. 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