Strobilomyces longistipitatus – Facesoffungi number: FoF 2954

Strobilomyces longistipitatus D. Chakr., K. Das & S. Adhikari                                        

MycoBank number: MB 817357 Facesoffungi number: FoF 2954

Etymology: referring to the very long stipe (of basidiomata)

Holotype: CAL 1336

Diagnosis: Distinct from the European species Strobilomyces strobilaceous by its exceptionally long stipe, significantly short tubes and frequent occurrence of tongue-like appendages on the wall of the ridges of basidiospores. Pileus 37–50 mm. diam.; convex when young, becoming pyramidal to convex with maturity; surface densely squamulose to floccose in numerous conical clusters of somewhat repent pattern, greyish-brown to violet brown (11E3–11F4) to blackish, reddish with KOH; margin wavy with sterile flap of tissue, brownish-black. Pore surface covered with thick cottony partial veil when young, white to dingy white; depressed near stipe, yellowish-white (3A2), turning rusty or darker after bruising; pore 2 mm, simple, angular. Tube 6 mm long, sinuate, chalky white or yellowish-white (3A2), turning reddish-orange after bruising. Stipe 110–200 × 17–28 mm, central, dark grey to (11F4) or black; surface cotton like or floccose with white basal mycelium. Context solid in pileus and stipe; context in pileus and stipe chalky white but immediately turning pastel red (8A4) and then greyish-red (8C5) or darker on exposure, turning orange (6A7) with KOH, greyish-green (25E5) with FeSO₄, greyish-green (25D4) with guiacol. Spore print blackish-brown. Odour like Boletus edulis. Basidiospores 10–11.1–12.7 × 8–9.1–9.9 µm (n = 20, Q = 1.16–1.22–1.33), broadly ellipsoid to ellipsoid, ornamentation composed of high ridges forming complete reticulum; under SEM with broad and confluent ridges (of irregularly wavy walls) forming complete reticulations and with frequent tongue like erect appendages on the wall of ridges, blackish-brown. Basidia 40–58 × 14–18 µm, 2 to 4 spored, clavate to subclavate. Pleurocystidia 34–75 × 8–17 µm, pigmented, emergent 15–30 µm, common, subfusiform to fusiform, ventricose to ventricose with somewhat moniliform or capitate head; content dense, slightly fibrillose. Tube edge fertile, composed of basidia, cystidia. Cheilocystidia 39–65 × 11–13 µm, subclavate to subventricose, some are appendiculate, mostly hyaline. Tube trama divergent, tramal hyphae 5–13.5 µm wide, gelatinous, encrusted, septate, branched. Pileipellis 1000–1200 µm thick, trichododerm to palisadoderm, composed of erect elements, sometimes septate; terminal cells up to 20 µm wide, slightly thick walled (up to 0.8 µm), mostly with rounded to fusoid apices, brown pigmented, sometimes faintly encrusted; finely warted when observed under SEM. Stipitipellis trichoderm to palisadoderm, hyphae brown pigmented, wall up to 1 µm thick, often minutely encrusted; fertile, with basidia and cystidia. Caulobasidia 4-spored, similar to tube basidia. Caulocystidia 23–65 × 14–20 µm, clavate to subclavate, or fusoid to ventricose. Clamp connections not found.

Habitat and distribution: Under Abies densa in subalpine mixed (broadleaf and coniferous) forest.

Material examined: INDIA, Sikkim, East District, Memainchu, 3692 m, N 2721’25.0” E 8836’24.9”, 4 July 2015, D. Chakraborty, DC 15-010 (CAL 1336, holotype). GenBank numbers ITS:KX364694.

Notes: Characteristic features of S. longistipitatus are presence of short pileus with almost blackish dense squamules on surface in numerous conical clusters arranged repently (never erect), exceptionally long stipe (3 to 4 times or more of pileus diameter) without any striation or reticulation on the surface, distinguishingly short white sinuate tubes which becomes reddish-orange on bruising, basidiospores with complete reticulum with the confluent ridges and frequent occurrence of tongue-like appendages on the ridge-walls and occurrence in association with the coniferous trees.

Our ITS based phylogeny clearly supports the existence of two distinct and strongly supported clades (Clade A and Clade B) in Strobilomyces which are also reported repeatedly by the earlier workers (Gelardi et al. 2012; Antonin et al. 2015) however, another clade represented by the only species S. annulatus Corner from Malaysia (sequence of ITS region is not yet available in GenBank numbers) is also known when phylogeny is conducted based on partial sequences of largest subunit of RNA polymerase II (RPB1) as shown by Gelardi et al. (2012) and Antonín et al. (2015). Here, Clade A includes S. confusus Singer, S. seminudus Hongo, S. verruculosus Hirot. Sato where basidiospores are never reticulated as also stated by Gelardi et al. (2012) and Antonín et al. (2015). Distinguishingly, Clade B represents S. strobilaceous (Scop.: Fr) Berk., S. appendiculatus sp. nov., S. echinocephalus Gelardi & Vizzini., S. pteroreticulosporus Antonín & Vizzini, S. mirandus Corner where basidiospores are typically reticulate (Gelardi et al. 2012, Antonín et al. 2015). Basidiospore-ornamentation pattern is known to be the reliable parameter in the delimitation of major groups of Strobilomyces. Recently, on the basis of morphology and ITS data Petersen et al. (2012) showed the existence of only a single species of Strobilomyces in Europe, as S. strobilaceus. Therefore, we considered only the sequences derived from European collections as S. strobilaceus sensu stricto in our phylogeny. Our LSU based phylogeny which was conducted considering all the Strobilomyces LSU sequences appeared on BLAST (or available in GenBank numbers) search with our specimen (GenBank numbers KF112459, KF112460, KF112463, KF030345, DQ534626) and some other species of Boletaceae also clearly supports the position of the present specimen amongst Strobilomyces and confirms the novelty. Here three sequences labelled as Strobilomyces sp. (Wu et al. 2014) are placed in the weakly supported sister clade of present species (94–95% identity with S. longistipitatus for 91% query coverage using BLAST).

Morphologically, this Indian specimen is quite similar to S. strobilaceous (an European species represented here by GenBank numbers JQ319001, JQ318998, JQ318984, JQ318975, JQ318977 and JQ318988 and S. echinocephalus (reported from China) but, the former (probably the closest relatives of present species) differs from S. longistipitatus by its considerably shorter stipe [60–140 mm and 80–150 mm as mentioned by Breitenbach and Kränzlin (1991) and Knudsen and Vesterholt (2012) respectively] and longer tubes (10–15 mm) whereas, the latter is distinct from the present Indian specimen by its much shorter stipe (74–105 mm), presence of striation on stipe apex and longer tube (9 mm). Phylogenetically, both the species are well distinct. Three more species: S. alpinus, S. pteroreticulosporus, S. mirandus are also partly close to S. longistipitatus. Strobilomyces alpinus can be separated in field by the presence of longitudinal striations on stipe and unchanging exposed context and larger spores (12.5 to 15 µm) (Gelardi et al. 2012). Strobilomyces pteroreticulosporus is distant by its pileus surface which is covered with pyramidal fibrillose dirty whitish scales, longer (30 mm) tubes and shorter (100–140 mm) stipe (Antonin et al. 2015) whereas, S. mirandus has yellow or golden orange to brownish-orange pileus, longer (up to 10 mm) tubes and distinctly smaller (40–70 mm) stipe (Sato et al. 2005).

            Strobilomyces polypyramis Hook. f., another species recently reported from the state Sikkim can easily be separated by presence of erect, small conical to pyramidal, pointed or rarely spinoid black scales, longer tube (up to 13 mm), shorter stipe (80 mm), absence of complete reticulum in basidiospores (Horak 1980a, c; Das et al. 2014). Strobilomyces dryophilus Cibula & N.S. Weber (96% identity with S. longistipitatus for 95% query coverage using BLAST) and S. floccopus were also appeared close (though well separated) in the LSU phylogeny along with our Indian material. But, S. dryophilus is morphologically distinct from S. longistipitatus by its whitish ground coloured pileus with coarse, woolly appressed or erect, greyish-pink or pinkish-brown scales, 10–17 mm long tubes, and much shorter (40–80 mm) stipe (Bessette et al. 2010). Whereas, S. floccopus (sequence derived from European material) is considered as the synonym (Peterson et al. 2012; Gelardi et al. 2012) of S. strobilaceous (morphological comparison given in the earlier paragraph).

According to unique combination of morphological data and phylogenetic analyses of ITS and LSU sequences, our specimen from India should be treated as an independent species, proposed as S. longistipitatus which is placed in Clade B along with other species featured with reticulate basidiospores.