Agaricus pakistanicus H. Bashir, A.N. Khalid, L.A. Parra & Callac, 2018

Bashir, Hira, Hussain, Shah, Khalid, Abdul Nasir, Niazi, Abdul Rehman Khan, Parra, Luis A. & Callac, Philippe, 2018, First report of Agaricus sect. Brunneopicti from Pakistan with descriptions of two new species, Phytotaxa 357 (3), pp. 167-178 : 172-174

Agaricus pakistanicus H. Bashir, A.N. Khalid, L.A. Parra & Callac sp. nov. FIGURES 2A–E , 3A–E

MycoBank no: MB 824094

Species-specific ITS markers:—aatcttCttcccg@200, attccTTggagca@420-421

Diagnosis:—This species is well distinguished from others in having a pileus with dark brown squamules, pileus and stipe surface immediately turning dark brown when touched, and a strong red to blackish brown discoloration upon bruising.

Etymology:—‘ pakistanicus’ in Latin means from Pakistan, the country where the species was recorded.

Description:— Pileus 4.5–5 cm in diam. and up to 16 mm thick at disc but very variable in different collections from very thin to thick, plano-convex, finally applanate, surface uneven, completely covered with appressed, conspicuous brown (2.7RP 5.4/3.3) large and triangular squamules becoming blackish brown (4.7RP 3.9/0.8) denser at the center on creamy white background (5.2P 9.5/2.7), not disseminated over the cap, surface dull and dry, immediately turning dark brown when touched. Margin entire, slightly wavy and vaguely ruptured at maturity. Lamellae 2–3 mm broad, dark brown (6.3RP 4.7/3.5), free with intercalated lamellulae, crowded, with eroded, unequal edges. Stipe 4–5.5 × 0.6–1 cm, fistulose, cylindrical or bulbous (1.5 cm) at the base in TTS19 collection, provided with an annulus in its upper part, smooth and white to dirty white above the annulus, below the annulus smooth, slightly fibrillose or with concolorous longitudinal striations, white with brown (8.5YR 3.1/1) tinge, becoming immediately dark brown (7.1RP 1.4/3.3) when touched in all collections, turning red (4.4R 4.3/7.3) or blackish brown (4YR 1.8/3.3) when bruised. Annulus superous, membranous, with thick double-edged margin, slightly fibrillose creamy white on the lower surface, light brown on the upper surface; edges immediately turning dark brown, sometimes rufescent, when rubbed. Context blood red when cut becoming dark brown with time. Odor fruity. Chemical tests KOH and Schäffer’s reactions negative on fresh sporocarps.

Basidiospores (5.3–) 5.5–8.6 (–8.9) × (3.9–) 4.2–6.3 (–6.8) μm, [avX = 6.6 ± 1.08 × 5.1 ± 0.79 μm, Q m = 1.29, n = 20 × 4], subglobose to broadly ellipsoid, rarely ellipsoid, light to dark brown in KOH, apiculus prominent, smooth, with granular content. Basidia 19–35.5 × 7.3–10.9 μm, 2 to 4-spored, clavate, thin-walled, hyaline in KOH, smooth, with olivaceous granular content. Cheilocystidia simple or septate at the base, smooth, thin-walled, hyaline in KOH, with olivaceous granular content; terminal elements 10.7–20 × 6.1–11.7 μm, broadly clavate to sphaeropedunculate; ante-terminal elements somewhat irregular in shape and size but frequently shortly cylindrical. Pleurocystidia absent. Pileipellis a cutis composed of hyphae 4–10 μm in diam., smooth, thin-walled, sometimes branched, frequently septate, hyaline to light brown in KOH, not constricted at septa, terminal elements with rounded ends. Stipitipellis composed of hyphae 3–8.5 μm in diam., parallel, hyaline in KOH, smooth, terminal elements with rounded tips, not constricted at septa.

Habit and habitat:—Solitary on roadside under Dalbergia sissoo .

Holotype:— PAKISTAN. Punjab, Lahore, University of the Punjab, on rich loamy soil in the grounds of the University under Dalbergia sissoo , 217 m a.s.l., 4 July 2017, Hira Bashir, PU 450, (LAH35299). GenBank no.: MG669256.

Additional material examined:— PAKISTAN. Punjab, Lahore, University of the Punjab, solitary on rich loamy soil on the grounds of the University near Dalbergia sissoo , 217 m a.s.l., 1 September 2016, Hira Bashir, PU 309 (LAH35298). GenBank no.: MG669258; Punjab, Lahore, Jallo Park, solitary on loamy soil in grassy ground, 217 m a.s.l., 2 September 2016, Hira Bashir, J 32 (LAH35300). GenBank no.: MG669257; Punjab, Toba Tek Singh, solitary in grassy ground near Dalbergia sissoo , 183 m a.s.l., 31 August 2016, Hira Bashir, TTS 19 (LAH35301). GenBank no.: MG669259.

Taxonomic comments:—In the phylogenetic analyses, A. pakistanicus is sister to Agaricus sp. 1 (CA800). CA800 is briefly described in Chen et al. (2015) and its basidiome is illustrated here in Fig. 4 . Agaricus pakistanicus is distinguished from its sister species in having a pileus with dark brown squamules, a basidiome immediately turning dark brown when touched, a context with a strong discoloration from red to blackish brown when cut (while the discoloration reported in A. sp. 1/CA800 was only red), cheilocystidia broadly clavate to sphaeropedunculate with granular content and very long basidia reaching up to 35 μm in length.

We note that the species-specific marker characterizing A. pakistanicus is located in the 5.8S region, which is highly conserved and not phylogenetically informative in A. sect. Brunneopicti . Indeed, the only other polymorphism found in this region is a heteromorphism (A/G), which is also present in a single species ( A. duplocingulatus /CA903).

The distribution range of this species likely extends to tropical regions of India. Indeed, we found that three ITS sequences deposited in GenBank as A. sp. were 100% identical to our sequence of PU309. In addition, two of them, KR155102 (BAB 5057) and KT186164 (BAB 5266) from the region of Ahmedabad ( India), are also deposited in BOLD (Barcode of Life Data Systems, Ratnasingham et al. 2007) as A. diminutivus Peck (1873: 53) (MGEN682- 15) and A. subrutilescens ( Kauffman 1925: 141) Hotson & Stunz (1938: 219) (MGEN731-15), respectively. The remaining sequence, KU366694 (HATFD14-3) from the region of Chennai ( India), which is incomplete, is listed as A. fuscofibrillosus ( Møller 1950: 28) Pilát (1951: 7) by Priyamvada et al. (2017) in their supplementary material. Without formal taxonomic study, these samples have been misidentified as three different non-tropical species that do not belong to A. sect. Brunneopicti .

Agaricus sparsisquamosus H. Bashir, S. Hussain, A.N. Khalid & H. Ahmed , sp. nov. FIGURES 5A–F , 6A–E MycoBank no: MB 824095

Species-specific ITS marker:—tgtttAtacat@255

Diagnosis:—This new species is characterized by its pileus covered by dark brown squamules denser at the center, globose to subglobose basidiospores, clavate to sphaeropedunculate cheilocystidia and squamulose stipe below the annulus.

Etymology:—From the Latin sparsus (sparse or scattered) and squamosus (scaly) by its dispersed squamules on the pileus surface.

Description:— Pileus 3–8 cm in diam., initially convex, then applanate to plane-concave, thin and fleshy when young, covered by grey to brown (9.4R 6.1/0.9) sometimes rufescent, irregular shaped, dispersed squamules, denser at disc, scattered towards the margin on a white creamy (3.3B 9.9/0.8) or brown (3.8YR 4.5/1.1) background in the collection LAH35297. Surface dull, fissured when mature, slightly uplifted with age. Margin straight, slightly exceeding the lamellae, sometimes appendiculate (LAH35294). Lamellae 3–5 mm broad, free, regular, crowded with intercalated lamellulae, pink to light brown when young and dark brown at maturity. Stipe 3.5–4.5 × 0.3–1 cm, cylindrical with slightly bulbous base, provided with an annulus in its upper half, smooth, white or light brown above the annulus, below the annulus white with light brown tinge, discoloring dark brown (5.7YR 1.7/3.1) when touched, smooth or hardly squamulose, sometimes with rhizomorphs at the base. Annulus superous, membranous, with thicker double-edged margin, white with brown tinge on the lower surface, white on the upper surface, turning brown when rubbed. Context changing to light brown (0.3Y 8/0.8) when cut. Odor fruity, strong. Chemical tests KOH and Schäffer’s reactions negative on fresh sporocarps.

Basidiospores (3.6–) 3.8–6.4 (–7.2) × (3.5–) 3.6–4.9 (–5.1) μm, [avX = 5.9 ± 0.81 × 4.4 ± 0.43 μm, Q m = 1.33, n = 20 × 6], subglobose to broadly ellipsoid, rarely globose or ellipsoid, light yellow to dark brown with prominent apiculus, smooth. Basidia 16.1–23 × 6.5–7.7 μm, clavate to narrowly clavate (PU256), thin-walled, hyaline in KOH, smooth and 2 to 4-spored. Cheilocystidia simple or septate at the base, numerous, in groups, hyaline in KOH, smooth, but absent in three of the six collections ( Table 1). Terminal elements 18.1–39.4 × 7.5–21.2 μm, pyriform, subglobose, some broadly clavate to sphaeropedunculate, mostly pedunculate, ante-terminal elements usually shortly cylindrical. Pleurocystidia absent. Pileipellis a cutis composed of hyphae 4.5–8 μm in diam., hyaline or with internal light brown pigment, frequently septate, slightly constricted at septa. Stipitipellis composed of hyphae 4.7–8.9 μm in diam., parallel, hyaline to light brown, smooth, terminal elements with rounded tips.

Habit and habitat:—Gregarious on grassy places covered with leaf litter.

Holotype:— PAKISTAN. Khyber Pakhtunkhwa, Qaldara Dargai, 3 km east of Swat-Malakand Highway , 490 m a.s.l., gregarious on loamy soil, 14 August 2014, Shah Hussain, SH160 ( LAH35201 ). GenBank no.: KY741892 .

Additional material examined:— PAKISTAN. Khyber Pakhtunkhwa, Qaldara Dargai, 3 km east of Swat-Malakand Highway , on loamy soil, 490 m a.s.l., 20 August 2014, Shah Hussain, Ag 11 ( LAH35211 ). GenBank no.: KY741893 ; Punjab, Lahore, University of the Punjab, solitary on rich loamy soil on the grounds of the University , at 217 m a.s.l., 1 August 2016, Hira Bashir, PU 251 ( LAH35294 ). GenBank no.: MG669252 ; Punjab, Lahore, University of the Punjab, solitary on rich loamy soil on the grounds of the University , at 217 m a.s.l., 1 August 2016, Hira Bashir, PU 254 ( LAH35295 ). GenBank no.: MG669253 ; Punjab, Lahore, University of the Punjab, solitary on rich loamy soil on the grounds of the University , at 217 m a.s.l., 1 August 2016, Hira Bashir, PU 256 ( LAH35296 ). GenBank no.: MG669254 ; Punjab, Lahore, University of the Punjab, solitary on rich loamy soil on the grounds of the University , at 217 m a.s.l., 1 September 2016, Hira Bashir, PU 320 ( LAH35297 ). GenBank no.: MG669255 .

Taxonomic comments:—According to our analyses, A. sparsisquamosus , A. pakistanicus , and CA800 form a subclade within A. sect. Brunneopicti . Agaricus sp. 1 (CA800) is characterized by having a pileus covered by triangular, predominantly dark grey to light brown squamules, strong red discoloration of the context ( Fig. 4 ), basidiospores 4.8−5.5 × 3.0−4.0 μm and absence of cheilocystidia, while A. sparsisquamosus has a dark brown pileus with rufescent tinge when rubbed, relatively longer basidiospores than CA800, 3.6–7.2 × 3.5–5.1 μm and absence or presence of large cheilocystidia, which are mainly pyriform, sphaeropedunculate to broadly ellipsoid. In A. pakistanicus the pileal squamules are dark to blackish brown, large, abundant, covering the entire surface of the pileus, and the context discolors strong red to very dark brown when cut, while in A. sparsisquamosus the pileal squamules are grey-brown with rufescent tint, small, dispersed and the context discolors light brown when cut. Despite the differences mentioned above, sometimes the distinction between A. sparsisquamosus and A. sp. (CA800) or A. pakistanicus might be difficult, and molecular data may be required to unequivocally identify some collections.

Polymorphisms in ITS sequences, which are reported in Table 1, reveal numerous heteromorphisms in sample PU251 and an allele (nucleotide A; position 255) that is present in only two collections (SH160 and Ag11) that, moreover, have relatively smaller spores and do not possess large cheilocystidia. However, they form a small clade that does not appear to constitute a reliable taxon since samples bearing allele T at position 255 may also lack cheilocystidia (PU320) or have relatively small spores (PU256). A single difference (without heteromorphism) among ITS sequences of the same species is not rare in Agaricus . Moreover, in the present small sample this difference is not correlated to any morphological trait. Therefore, we consider that the six samples belong to A. sparsisquamosus , which is a molecularly (ITS) and morphologically (spore size) variable species.

Figures

FIGURE 2. Macromorphological characters of A. pakistanicus. A, B = PU450 (Holotype); C = J32; D = PU309; E = TTS19. Bars = 10 mm. Photos by Hira Bashir.

FIGURE 3.Micromorphological charactersof A.pakistanicus.A–E (PU450,Holotype):A =Basidia,B =Cheilocystidia,C =Basidiospores, D = Pileipellis hyphae, E = Stipitipellis hyphae. Bars = 10 μm. Drawings by Hira Bashir.

FIGURE 4. Agaricus sp. 1 (A, B = CA800): Bars = 10 mm. Photo by Jacques Guinberteau (25 July, 2010), University park of Chiang Mai, Thailand.

FIGURE 5. Macromorphological characters of A. sparsisquamosus. A = SH160 (Holotype); B = Ag11; C = PU320; D, E = PU254; F = PU256. Bars = 10 mm. Photos by Hira Bashir.

FIGURE 6.Micromorphological characters of A. sparsisquamosus. A, C–E = (SH160, Holotype); B = (PU254): A = Basidia, B = Cheilocystidia, C = Basidiospores, D = Pileipellis hyphae, E = Stipitipellis hyphae. Bars = 10 μm. Drawings by Hira Bashir.