Austroboletus appendiculatus Semwal, D. Chakr., K. Das, Indoliya, D. Chakrabarty, S. Adhikari & Karunarathna

MycoBank number: MB 817672 Facesoffungi number: FoF 2952

Etymology: referring to stipes (of basidiomata) with appendages.

Holotype: CAL 1304

Diagnosis: is distinct from the closest species A. subvirens by larger and differently coloured pileus, longer and wider stipe, presence of pleurocystidia and caulocystidia and putative association with Shorea sp.

Pileus 75–90 mm. diam.; convex when young, to plano-convex, sometimes uplifted with age; surface areolate-squamose, then disrupted into several patches or segments with the expansion exposing yellowish-orange to greyish-yellow (4B7–4A7, 4B6) subpellis that becoming paler towards margin as pastel yellow to butter yellow, light yellow (3A4–4A5, 4A4); segments of suprapellis light brown to brown, cottony, slightly viscid when wet; margin plane, regular. Pore surface pinkish when young, gradually on bruising or maturity greyish-orange to brownish-orange (5B3–6C3), reddish-grey (12B2), or darker to brown, but paler toward the stipe juncture as light orange grey (6B2), depressed at junction to stipe, unchanging on bruising, pores often up to 2 mm broad at maturity, angular to rectangular, attached. Tubes 12–16 mm long, concolorous to slightly paler then pore surface. Stipe 80–120 × 20–30 mm, central, terete or more or less fusoid towards down, surface distinctly covered with pale orange to light orange (4–5A2–4) or sometimes yolk yellow (4B8) anastomosing broad ridges or appendages forming elevated or flaring to wing-like network (on yellowish-white background) which gradually becoming thin (less elevated) reticulum towards apex, but, never with typical lacerate surface, base with white mycelia elements; ridges cottony, darker with age as golden yellow to brownish-yellow (5B5–6, 5C6). Context white, unchanging, fluffy, partly hollow in stipe. Odour mushroomoid. Taste indistinct. Spore print rust brown to reddish-brown (6E8–8E8). Basidiospores 14.2–14.8–16.5 × 7.3–8–9.1 µm (n = 20, Q = 1.68–1.83–1.97), broadly fusoid to amygdaliform, depressed suprahilar part, surface appears warted by disruption of the outer wall, deeply pitted to form punctuate-warted; under SEM, pits minute at both the fusoid ends, gradually with intricate furrows towards mid region, at mid region with more broader and deeper pits forming warty surface with truncate warts, with disrupted adaxial surface. Basidia 22–32 × 12–16 µm, 4–spored, clavate. Pleurocystidia rare. Tube trama boletoid, gelatinous. Pileipellis trichoderm, composed of erect, septate hyphae; terminal cells cylindrical with rounded apex, width 6–9 µm. Stipitipellis fertile, composed of caulocystidia and caulobasidia. Caulocystidia 40–70 × 11–20 µm, clavate to subclavate, subventricose to appendiculate. Caulobasidia 30–45 × 9–12 µm, 2- to 4-spored, clavate.

Habitat and distribution: Under Shorea robusta Gaertn. in subtropical broadleaf forest.

Material examined: INDIA, Uttarakhand, Dehradun District, Tapovan forest, 650 m, 17 July 2010, K.C. Semwal, KCS 1401 (CAL 1304, holotype; isotype: BSHC). GenBank numbers ITS:KX530028.

Notes: Austroboletus appendiculatus is typically featured by combination of characters: brownish areolate-squamose pileus with exposed yellowish subpellis, distinctively reticulate-appendicute and non-lacerate stipe surface, pinkish pore surface that becomes brownish to greyish on bruising or after maturity, partly hollow stipe, almond-shaped deeply pitted basidiospores with distinct suprahilar depression and prominently disrupted adaxial surface and occurrence with putative association of Shorea in subtropical broadleaf forest.

In the field, Austroboletus subvirens (Hongo) Wolfe [≡ Porphyrellus subvirens Hongo], A. lacunosus (Kuntze) T.W. May & A.E. Wood [≡ A. cookie (Sacc. & Syd.) Wolfe] and A. novae-zelandiae (McNabb) Wolfe [≡ Porphyrellus novaezelandiae McNabb] are quite similar (as far as the stipe surface is concerned) to the present taxon. But, A. subvirens (probably the closest species being reported earlier from Japan, Papua New Guinea, China, Americas and Australia and labelled the Australian Material here with GenBank numbers KP242207, KP242214, KP242212 and KP242210) has distinctively smaller (25–60 mm) and differently coloured pileus, shorter and thinner stipe (50–90 × 4–10 mm), and lacks pleuro-, cheilo- and caulocystidia (Wolfe 1979; Jian-Zhe 1985). Moreover, A. subvirens are found to be associated with the trees of Fagaceae in Australasia and Americas and Acacia, Allocasuarina and Eucalyptus in Australia (Wolfe 1979; Horak 1980a, c; Halling et al. 2006) whereas, A. appendiculatus (present species) grows under Shorea robusta (Dipterocarpaceae). Austroboletus novae-zelandiae (reported from New Zealand and New Caledonia and labelled with GenBank numbers accession number HM060327) is separated from the present species by distinctively shorter and slender stipe (70 × 8 mm), bigger (17–19.5 × 8–10.4 µm) basidiospores (Wolfe 1979). Austroboletus lacunosus (reported from Australia and labelled with GenBank accession number KC552014) can also be separated from the present taxon by smaller pileus (15–25 mm), and stipe (20–50 × 5–6 mm) and smaller (32.5–56 × 10–15.5 µm) caulocystidia (Wolfe 1979).

Only two other species, hitherto known from India: A. olivaceoglutinosus K. Das & Dentinger (labelled with GenBank numbers KM597478 and KM597479) and A. gracilis (Peck) Wolfe can easily be differentiated in the field from the present novel species. A. olivaceoglutinosus which is found in subalpine Himalayan region under Tsuga and Picea has distinguishingly long and slender basidiomata (Pileus 30–55 mm diam., stipe 100–180 × 13–17 mm), highly glutinous olive coloured pileus, lacerate (never with ridges) stipe surface and narrower (12.7–19 × 5.9–7.7 μm) basidiospores showing different ornamentation pattern (Das and Dentinger 2015) whereas, A. gracilis has different pilear surface (subtomentose, velvety or fibrillose), smaller caulocystidia: “14–42 (–57) × 4–14 μm” and different pattern (“rugulose punctate”) of spore ornamentation, different host associations (Wolfe 1979; Lakhanpal 1996).

Considering the spore ornamentation, A. fusisporus (reported from Japan and China and labelled with GenBank numbers AB509830 and JX889719) also appears close to A. appendiculatus but the former has altogether different and very small (pileus 5–20 mm, stipe 20–45 × 2–4 mm) basidiomata (Wolfe 1979).

Our ITS phylogeny were obtained by the neighbour-joining analysis criterion considering the all available ITS sequences available in GenBank and/or appeared in BLAST search with the sequence of our Indian material. The new sequence forming a separate clade is clustered (80% bootstrap) with the sequences of A. subvirens derived from Australian Material. These two species in turn is clustered (80% bootstrap) with A. festivus (sequence derived from specimen collected from Guyana).

Unique combination of macro- and micromorphological features coupled with ITS phylogeny clearly support the novelty of Austroboletus appendiculatus.

Phylogenetic relationships inferred from neighbour-joining analysis of ITS-rDNA sequences of 22 taxa. The optimal tree with the sum of branch length = 0.82684195 is shown. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The novel Indian species: Austroboletus appendiculatus having GenBank numbers accession number KX530028 (ITS-rDNA) is shown in bold and blue. The topology is rooted with Boletus edulis. Evolutionary analysis was conducted in MEGA 6.0 (Tamura 2013).

Austroboletus appendiculatus (CAL 1304, holotype). a-c Fresh basidiomata in the field and base camp. d Hymenophoral trama. e Terminal cells of hyphae of pileipellis. f Cross section through stipitipellis showing anastomosing ridges. g, h Caulocystidia. i Basidiospores. j, k SEM micrograph of basidiospores. Scale bars: a = 100 mm, d = 50 µm, e, g-j = 10 µm, f = 100 µm, k = 5 µm.

Austroboletus appendiculatus (CAL 1304, holotype). a Basidia. b Caulocystidia. c Caulobasidia. d Radial section through pileipellis. Scale bars: a-d = 10 µm.