Research Article |
Corresponding author: Kanad Das ( daskanadbsi@gmail.com ) Academic editor: Kentaro Hosaka
© 2018 Dyutiparna Chakraborty, Alfredo Vizzini, Kanad Das.
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Citation:
Chakraborty D, Vizzini A, Das K (2018) Two new species and one new record of the genus Tylopilus (Boletaceae) from Indian Himalaya with morphological details and phylogenetic estimations. MycoKeys 33: 103-124. https://doi.org/10.3897/mycokeys.33.23703
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Tylopilus himalayanus and T. pseudoballoui are described as new species from two Himalayan states (Sikkim and Uttarakhand) in India. Tylopilus himalayanus is characterised by a unique combination of features: reddish- or brownish-grey to purplish-grey then brown to reddish-brown or darker pileus, absence of olive or violet tinges on stipe surface, angular pores, stipe without reticulum or rarely with a faint reticulum restricted to the very apex, bitter taste of the context and positive macrochemical colour reaction of the stipe context with KOH (dark orange) and FeSO4 (dark green), medium sized (10.9–14.4 × 3.9–4.9 µm) basidiospores and occurrence under coniferous trees; T. pseudoballoui is distinguished by orange-yellow to brown-yellow sticky pileus, pale yellow pore surface with pinkish hues that turns pale to greyish-orange on bruising; angular pores, stipe concolorous to pileus with pruinose but never reticulate surface, ixocutis pattern of pileipellis and occurrence under broadleaf trees. Another species, T. neofelleus, which was reported earlier from China and Japan, was also collected from Sikkim and reported for the first time from India. All three species are described with morphological details and two-locus based (nrLSU and nrITS) phylogenetic data.
Agaricomycetes , Basidiomycota , Boletales , macrofungi, phylogeny, Sikkim, taxonomy
The genus Tylopilus P. Karst., one of the less attractive to eye-catching ectomycorrhizal taxa (associated mainly with Fagales and Pinaceae) in the family Boletaceae, is featured by its dry, glabrous to subvelvety pileus, white to greyish pore surface usually becoming flesh pink to purple-brown at maturity, immutable to slightly brownish or becoming blue-green context on bruising, solid stipe with pruina or reticulation over the surface, absence of annulus or veil, flesh-pink to dull flesh-ochre spore print, smooth pink-coloured basidiospores, presence of pleurocystidia and absence of clamp-connections (
The entire Indian Himalayan region comes under one (“Himalaya”) of the globally acclaimed biodiversity hotspots and thus has immensely diverse mycobiota (including macrofungi) apart from its myriad flora and fauna. A wide range of phytogeographic variations with the presence of large numbers of ectomycorrhizal host plants, cold to warm monsoon, favourable humidity and plenty of rainfall, supports the growth of ectomycorrhizal mushrooms of Boletaceae. However, due to the lack of mushroom-explorers or mushroom-taxonomists, most of the areas of Indian Himalaya remain unexplored in terms of Boletaceae (only 80 species belonging to 23 genera, while more than 1050 species from 66 genera are reported from the world) (
Macromorphological characters and habitat details were noted from fresh, young to mature basidiomata in the field and in base-camp. After recording the macromorphological characters, basidiomata were dried with a field drier. Photographs of these fresh and dry basidiomata and microphotographs were taken with the aid of Canon Power Shot SX 50HS, Canon SX 220 HS and Nikon-DS-Ri1 (dedicated to Nikon Eclipse Ni compound microscope) cameras. Colour codes and terms are mostly from Methuen Handbook of Colour (Kornerup and Wanscher, 1978). Micromorphological characters were observed with compound microscopes (Nikon Eclipse Ni-U and Olympus CX 41). Sections from dry specimens were mounted in a mixture of 5% KOH, 1% Phloxine and 1% Congo red or in distilled water. Micromorphological drawings were prepared with a drawing tube (attached to the Nikon Eclipse Ni microscope) at 1000×. The basidium length excludes that of the sterigmata. Basidiospore measurements were recorded in profile view from 30 basidiospores. Spore measurements and length/width ratios (Q) are recorded here as: minimum–mean–maximum. Herbarium codes follow Thiers (continuously updated).
Genomic DNA (for all the species) was extracted from 100 mg of dry basidiomata using the InstaGeneTM Matrix Genomic DNA isolation kit (Biorad, USA) following the manufacturer’s instructions. PCR amplification primers were ITS1 and ITS4 (nrITS region) and LR0R and LR7 (nrLSU region) (
The nrLSU and nrITS datasets were assembled according to recent previous studies on this genus (
The nrLSU- and nrITS-based phylogenetic analyses (Figs
nrLSU based phylogram generated from Maximum likelihood (ML) analysis under GTRGAMMA substitution model depicting the placement of Tylopilus neofelleus, T. pseudoballoui and T. himalayanus within Tylopilus s.s. Two species of Xanthoconium (X. sinense and X. purpureum) were used as outgroup taxa. ML Bootstrap percentage (MLB) derived from this analysis (MLB >50%) are shown above or beneath the branches. Two novel species and a new record for Indian mycobiota are highlighted in bold and red font. GenBank accession no. and country name (when available) for each species are shown after the species name.
nrITS based phylogram generated from Maximum Likelihood (ML) analysis under GTRGAMMA substitution model depicting the placement of Tylopilus neofelleus, T. pseudoballoui and T. himalayanus within Tylopilus s.s. Two sequences of Gyroporus castaneus were used as outgroup. ML Bootstrap percentage (MLB) derived from this analysis (MLB >50%) are shown above or beneath the branches. Two novel species and a new record for Indian mycobiota are highlighted in bold and red font. GenBank accession no. and country name (when available) for each species are shown after the species name.
Similarly, in our nrITS-based ML and BI analyses (Figs
India. Sikkim: East District, Upper Chandmari, 1977 m alt., N27°23'13.7", E88°46'42.9", 26 Aug 2017, D. Chakraborty & K. Das, DC 17-25 (CAL 1649).
Distinct from all allied taxa by a combination of sequence data (nrITS and nrLSU), reddish- or brownish-grey to purplish-grey, then brown to reddish-brown pileus in basidiomata, absence of olive or violet tinges on stipe surface, presence of angular pores, stipe without reticulum or rarely with a faint reticulum restricted to the very apex, bitter taste of the context, positive reaction of the stipe context with KOH (dark orange) and FeSO4 (dark green) and medium sized (10.9–14.4 × 3.9–4.9 µm) basidiospores.
Referring to Indian Himalaya, the type locality.
Pileus 71–120 mm diam., initially convex then plano-convex to applanate, surface dry, matte to somewhat subvelvety, faintly areolate, brownish-grey, dull red, reddish-grey to purplish-grey or greyish-magenta (11–13B–C2–3) when young, gradually brown to reddish-brown (7E4–9D4) or darker, paler greyish-yellow (4C4) towards margin, pale yellow (2A3) at margin; margin entire, decurved to plane with a narrow flap of tissue, blond (4C4). Pore surface greyish-yellow (3C4) when young, pinkish-white (8A2) with age, turning greyish-brown (6D3) on bruising; pores angular, 2/mm. Tubes adnexed to subdecurrent, 5–6 mm long, whitish-brown to brownish, light brown to brown (26B2–3) on bruising. Stipe 95–155 × 20–32 mm, mostly subclavate, hollow, pale yellow (1–2A3) at apex, brownish towards base but never violaceous; surface usually without any reticulum, but sometimes faintly reticulate at apex (1/8th from the juncture), the rest longitudinally striate. Context up to 16 mm thick in pileus, milk white (1A2), unchanging when exposed. Stipe context turning dark green with FeSO4, dark orange with 5% KOH, orange with 10% NH4OH. Taste bitter. Spore print not obtained.
Basidiospores 10.9–12.5–14.4 × 3.9–4.5–4.9 µm, (n = 30; Q = 2.51–2.75–3.25), elongated to fusiform, inequilateral, thin-walled, smooth under light microscope. Basidia 30–40 × 9–10 µm, four-spored, clavate. Pleurocystidia 27–54 × 8–10.5 µm, emergent up to 30 µm, fusoid to ventricose, appendiculate. Tube edge sterile; cheilocystidia 32–48 × 5.2–8 µm, common, clavate to cylindrical, subfusoid to ventricose. Hymenophoral trama divergent, hyphae septate, gelatinous, up to 6 µm wide. Pileipellis a trichoderm, up to 150 µm thick, composed of erect hyphae, somewhat interwoven, encrusted, brown pigmented; terminal elements 20–50 × 5–10 µm, cylindrical to subcylindrical, sometimes subfusoid, content brown pigmented. Stipitipellis a cutis, made up of sub-parallel repent hyphae; caulocystidia not observed; caulobasidia similar to that of hymenial basidia.
Tylopilus himalayanus (DC 17-25, holotype). a, b Fresh basidiomata in the field and in basecamp c Pore surface after bruising d Surface of stipe apex with reticulation e Pleurocystidia f Pileipellis g Tube edge h Basidiospores. Scale bars: 50 μm (f); 10 μm (e, g, h); 5 mm (c, d); 5 cm (a).
India. Sikkim: Upper Chandmari, 1977 m alt., N27°23'13.7", E88°46'42.9", under Pinus sp. in temperate mixed forest, 26 Aug 2017, D. Chakraborty & K. Das, DC 17-31 (CAL); Uttarakhand: Champawat district, Abbot mount, 1933 m alt., N29°25.465', E80°06.422', under Cedrus deodara in temperate coniferous forest, 18 Jul 2017, A. Ghosh, KD B-03 (CAL).
INDIA. Sikkim, South District, Maenam WLS (Maenum 3), 2136 m alt., N27°15'34.7" E88°21'25.7", 23 Aug 2017, Quercus spp., D. Chakraborty & K. Das, DC 17-30 (CAL 1651)
Distinct from all allied taxa by sequence data (nrITS and nrLSU) and morphologically by its sticky orange-yellow pileus surface, pale yellow pore surface which turns to pale orange or greyish-orange when bruised and absence of reticulation on stipe surface.
referring to its being a look-alike of T. balloui, a North American species.
Pileus 60–150 mm diam., initially convex then plano-convex, surface sticky, orange-yellow to brownish-yellow (5B–C8), paler at margin; margin entire, plain, without any sterile flap of tissue. Pore surface pale yellow (3A3), turning pale orange to greyish-orange (6A–B3) on bruising; pores angular, 5–8/mm. Tubes subdecurrent, 6–10 mm long, yellowish-white, brownish on bruising. Stipe 55–110 × 20–40 mm, mostly subclavate, solid, concolorous with pileus; surface pruinose, never reticulate; basal mycelium white. Context 20 mm thick in pileus, chalky white (1A1), unchanging on exposure but turning turquoise grey (24D2–D1) with FeSO4 (chalk), pale yellow (4A3) with 5% KOH, yellowish-grey (4B3) in Guaiacol. Pileus surface brownish-red (8C8–7) on bruising, turning dark green to greenish-grey (25E–F3–2) in FeSO4, vivid yellow (3A8) in KOH, unchanging in NH4OH. Stipe 55–110 × 20–40 mm, mostly subclavate, solid, concolorous with pileus; surface pruinose, never reticulate; basal mycelium white. Odour pleasant. Taste slightly pungent. Spore print not obtained.
Basidiospores 6.4–7.4–9.9 × 3.8–4.5–5.7 µm (n = 30, Q = 1.5–1.73–2.04), ellipsoidal, thin-walled, smooth under light microscope. Basidia 22– 30 × 8–10 µm, four-spored, clavate. Pleurocystidia 40–54 × 7–10 µm, emergent up to 30 µm, fusoid to ventricose, appendiculate, yellow pigmented or hyaline, mostly with dense globular to oily content. Tube edge fertile; cheilocystidia 33–55 × 7–10 µm, common, clavate to cylindrical, subfusoid to ventricose. Hymenophoral trama divergent, hyphae septate, gelatinous, up to 5 µm wide. Pileipellis an ixocutis up to 150–280 µm thick, composed of subparallel to suberect, somewhat interwoven hyphae; terminal elements 20–70 × 6–11 µm, cylindrical to subcylindrical, sometimes subfusoid, content orange-brown pigmented. Stipitipellis up to 150 µm thick, fertile, composed of basidia and cystidia in several clusters; caulobasidia similar to that of hymenial basidia; caulocystidia 40–76 × 10–12 µm, broadly clavate to subclavate or appendiculate.
India. Sikkim: South District, Maenam WLS (Maenam 3), 2136 m alt., N27°15'34.7", E88°21'25.7", 23 Aug 2017, Quercus sp., D. Chakraborty & K. Das, DC 17-35 (CAL); Uttarakhand: Champawat district, Abbot mount, 1885 m alt., N29°25.466', E80°06.085', 18 July 2017, A. Ghosh, KD B-02 (CAL). Uttarakhand: Pauri district, 1971 m alt., N30°02.874', E79°08.221', 4 Aug 2017, K. Das, KD 17-24 (CAL).
Pileus 70–120 mm broad, convex to broadly convex; surface distinctly tomentose to subvelvety, dry, subviscid when wet; reddish-brown (8–9F4) when young, rosewood (9C5) to vinaceous-brown (16C5) with maturity, fawn (7E4) towards margin. Pores yellowish-white or cream with a pinkish tinge, orange-grey (6B2) with age; pore 2–3/mm, rounded. Tubes 10–15 mm, adnate-sinuate, white in colour, yellow-brown or orange white with maturity. Stipe 60–100 × 18–22 mm, cylindrical, solid, surface dry, glabrous to subvelutinous, typically distinctly reticulate at apex, reticulation greyish-ruby (12C–D4) to dark ruby (12F5); surface pinkish brown to vinaceous or violaceous, dark brown to reddish-brown with maturity. Context chalky white, but pinkish-brown when exposed. Spore print not obtained.
Basidiospores 10–11.5–13.5 × 4–4.6–5.2 µm (n = 30, Q = 2.05–2.48–2.76), ellipsoid to narrowly subfusoid, inequilateral, smooth, thin-walled. Basidia 30–36 × 10–11 µm, 4-spored, clavate, thin-walled, hyaline or pale grey in KOH. Pleurocystidia 35–66 × 14–24 µm, scattering and numerous, fusoid-ventricose or subclavate, with orange brown contents. Cheilocystidia 33–38 × 9–12 µm, ventricose to fusoid, shorter and smaller than pleurocystidia thin-walled, with orange brown contents. Pileipellis 100–150 µm thick, an ixotrichoderm of suberect, branched, septate hyphae; terminal elements ventricose to fusoid, vaculolated, 28–50 × 12–14 µm, with granular yellowish to brown orange contents in KOH; subterminal elements mostly with incrustations. Stipitipellis 35–65 µm, fertile, composed of basidia and cystidia. Caulocystidia 52–63 × 8–13 µm, fusoid to subfusoid, ventricose to ventricose-rostrate or narrowly cylindrical, content granular. Clamp connection absent in all tissues.
Under Castanopsis sp. in temperate broadleaf forest.
Japan (
India. Sikkim: East district, Fambonglo WLS, 2021 m alt., N27°21'47.5" E88°34'13.2", 26 Aug 2016, D. Chakraborty & K. Das, DC 16-63 (CAL); ibid., D. Chakraborty & K. Das, DC 16-64 (CAL).
Our first novel species in Tylopilus s.s. (
Tylopilus balloui, as currently circumscribed (pileus orange-yellow and short elliptical, pale-coloured basidiospores), represents a species complex rather than a single species, based on morphological data (
The combination of morphological features in Indian collections of T. neofelleus and two-fold phylogeny (MG777529, MG777525 in Figs
Two (DC & KD) of us are grateful to the Director, Botanical Survey of India (BSI), Kolkata and Scientist-in-Charge, BSI, Gangtok for providing facilities and to Aniket Ghosh (H.N.B. Garhwal University, Uttarakhand) for sending the collections of Tylopilus from Uttarakhand and helping us in many ways. Literature provided by A.C. Magnago (Brazil) was highly appreciated. Field assistance to KD rendered by M.E. Hembrom (Central National Herbarium, BSI, Howrah, India) is duly acknowledged.
Figure S1
Figure S2