Agaricus agharkarii P.N. Singh, S.K. Singh, S. Rana & A.C Lagashetti, sp. nov.
Index Fungorum number: IF 559727; Mycobank number: MB 559727; Facesoffungi number: FoF11794; Figs. 1, 2, 3
Etymology – species named ‘agharkarii’ in the surname of Professor Shankar Puroshattam Agharkar, founder Director of Agharkar Research Institute.
Holotype– AMH 10341.
Colour code follows – Methuen Handbook of Colour (Kornerup and Wanscher 1978).
Basidiomes solitary or in groups of 2–4, variable in size, agaricoid, stipitate. Pileus 80–110 mm in diam, broadly umbonate, broadly ovate when young, 8 mm thick at the disk, fleshy, convex to plano-convex, when mature the pileus becomes upwards, surface dry, squamulose, color rufescent to greyish brown (8F3) when young, paraboloid when young, surface dry with greyish brown spots (8F3) when mature, margin entire, nonappendiculate. Lamellae free, regular, become dark brown with age (6F8); lamellulae present in 1–3 tiers, 8–10 mm broad, wavy, greyish brown (8F3). Stipe 70–96 × 20–22 mm, compact, attenuated towards length, base slightly bulbus with white (1A1) mycelial threads, fragile, greyish brown (8F3), surface dry, after cutting or touching with finger becomes dark brown (7F5). Volva absent. Annulus present, drooping or skirt like, membranous covering around the stipe, with double edged margin, white (1A1) to light brown (5D8). Volva absent. Spore print dark brown (7F5). Odour mushroomy. Taste not recorded. Context thick and fleshy, spongy, whitish (1A1) in pileus and pithy in stipe. Macrochemical reactions; negative in Schäffer’s reaction, no reaction with Ammonia and Potassium hydroxide.
Basidia 21.80–36.10×3.5–9.55 µm, clavate to narrowly cylindrical, subhyaline to light olivaceous, pigmented, smooth walled, variable in size, frequently tetrasporic. Sterigmata straight to curved sometimes resembles like an incisor canine, hyaline smooth walled, up to 3.52×1.90 µm. Basidiospores 5.15–9.19 × 3.92–5.67 µm ( x = 6.55 × 4.70 μm, n=30), Qm=1.43, Q=1.11–1.94, oval, ellipsoid to oblong, sometimes slightly tapered towards base with one or two guttulate, dark olivaceous brown, smooth walled, wall thickened and darkened (up to 0.85 µm thick). Hilum protuberant. Lamellar surface fertile, made-up of basidia, cystidia, and marginal cell. Cheilocystidia pedicillate, ampulliform to narrowly clavate, apex rounded, non-fertile, base narrow, subhyaline to light olivaceous, smooth walled, wall thickened and darkened, 19.75–42.47×7–8.12 µm. Pileocystidia hyphoid, fusiod, unbranched to branched, smooth walled, hyaline, up to 45.5–230 × 13–35 µm. Subhymenium 22.5–25 µm thick, composed of globose to oval cells, 6.9–18.65 × 6.55–15.25 µm, light olivaceous, Hymenial trama hyphoid, upto 113.35 μm wide, composed of parallel to interwoven thick and thin hyphae, subhyaline to light olivaceous, with distant septation in hyphae, branched, variable in dimensions. Pileipellis (pileus trama) dark brown, interwoven, hyphae septate, up to 34.80 μm in diam, branched, pigmented, light olivaceous. Clamp connections absent. Stipitipellis hyphal, arranged in parallel bundles of hyphae, irregularly septate, subhyaline to light olivaceous, cells irregular, variable in dimensions, 20.38–26.75 µm wide.
Materials examined – INDIA, Maharashtra, Pune District, on garden soil, 9 July 2021, P.N. Singh, AMH10341 (holotype).
Host and habitat – Solitary or in groups, free living in botanical garden under Albizia tree.
GenBank numbers – ITS=MZ198899, LSU=MZ198900.
Notes – In the present taxon, the basidiomes are medium to large sized, squmulose with off-white linings with greyish brown pileus that have negative reaction with Schäffer’s test. There is no reaction with KOH and Ammonia on the flesh of fruiting body. The stipe base is white with swollen base and mild mushroomy odour. The pileipellis hyphae are hyphoid and variable in shape and size (10.5–34.80 μm in diam). The presence of cheilocystidia in the present taxon is a key feature of the A. sect. Flocculenti under the A. subg. Pseudochitonia (Zhao et al. 2011; He et al. 2018). Morphologically, new taxon, Agaricus agharkarii is different from its related taxa in having a larger convex pileus with broader lamellae as compared to A. erectosquamosus and A. pallidobrunneus (Zhao et al. 2016b). The length of cheilocystidia in A. agharkarii is comparatively larger than in A. erectosquamosus and A. pallidobrunneus. In addition to this, the dimension of basidiomes and basidiospores is apparently larger compared to A. erectosquamosus and A. pallidobrunneus (Table 1).
ITS and LSU sequence comparison has revealed that the sequences of the new species Agaricus agharkarii differ at 33 positions in ITS and 10 positions in LSU. Beside this, one deletion and five insertions (four of 1 bp and one of 3 bp) were observed in ITS sequence of the present taxon. The position of the present taxon within Agaricus sect. Flocculenti was further confirmed by molecular phylogeny based on combined ITS and LSU rDNA data. Phylogenetic analysis based on combined ITS and LSU sequence data indicates that Agaricus agharkarii (AMH 10341) is a new species of A. sec. Flocculenti, which is different from the other known species of A. sec. Flocculenti. The present taxon forms a distinct clade from other species of the A. sec. Flocculenti (A. erectosquamosus and A. pallidobrunneus) with a moderately supported ultrafast bootstrap value of 85% (Fig. 4).
On megablast analysis, ITS sequence of Agaricus agharkarii showed 95% (654/691) identity and 8 gaps (1%) with Agaricus erectosquamosus SDBR-NK0080 and Agaricus erectosquamosus SDBR-CJ0032, 95% (653/690) identity and 8 gaps (1%) with Agaricus pallidobrunneus SDBRNK0368, 94% (642/684) identity and 6 gaps (0%) with Agaricus sparsisquamosus PU320, 94% (641/684) identity and 6 gaps (0%) with Agaricus sparsisquamosus PU256, 93% (640/685) identity and 8 gaps (1%) with Agaricus sparsisquamosus PU320, and 94% (641/684) identity and 9 gaps (1%) with Agaricus sparsisquamosus PU320.
The distinct morphology compared to other species of A. sec. Flocculenti (A. erectosquamosus and A. pallidobrunneus), as well as the phylogenetic analysis, clearly establishes Agaricus agharkarii as a novel species.
Figure 1 – Agaricus agharkarii (AMH10341, holotype). a Basidiomes habitat. b, c Surface view of growing basidiomes. d–f Gill view with attached stipe and disintegrated annulus. f A complete gill view. h Spore print
Figure 2 – Agaricus agharkarii (AMH10341, holotype). a A basidium with attached basidiospores on sterigmata. b Basidia with basidiospores and cheilocystidia on lateral side. c A basidium with sterigmata. d A clavate cheilocystidium. e Numerous basidiospores arranged in tetrad manner (photographs taken from surface of gill). f, g Numerous basidiospores (arrow showing slightly tapered basal end). h An elongated basidiospore with thickened wall (showing arrow). i–k Pileocystidia (pileal tissues). l–m Stipitipellis (stipe tissues arranged in parallel manner). Scale bars: a–m=10 µm
Figure 3 – SEM images of Agaricus agharkarii (AMH10341, holotype). a tetroid basidiospores (showing arrow). b Basidiospores in higher magnifications (arrow showing protruburent hilum). Scale bars: a=10 µm, b=1 µm
Figure 4 – Phylogenetic tree of Agaricus agharkarii (AMH 10341) by Maximum-Likelihood method based on combined sequence data of ITS and LSU. Agaricus biberi LAPAG687 was used as an outgroup. The analysis involved 17 nucleotide sequences. Evolutionary analyses were conducted in IQ–TREE multicore version 1.6.11 (Nguyen et al. 2015) by the Maximum–Likelihood method using the best suitable model (TPM3u+F+R2 model). One–thousand bootstrap replicates were analysed to get ultrafast bootstrap values, and the values above 50% were represented on nodes in the tree