Tephrocybella constrictospora Clericuzio, Dovana & Vizzini

Index Fungorum number: IF553797 Facesoffungi number: FoF 03640

Etymology: the epithet, from the Latin words constrictus and spora refers to the slight central constriction often present in the spores.

Holotype: TO HG3329

Habit collybioid. Saprobic in grasslands, graminicolous. Pileus 15–30 mm broad, conico-convex, then convex, with a constant central umbo, which varies from small, acute to larger and more rounded; surface dry, slightly hygrophanous, ochraceous brown, reddish-brown, ochraceous date, with faint or no greyish hues, at first entirely covered by a white pruina which is partially visible also in mature specimens (towards the pileus margin); margin undulate, not striate. Lamellae ascendant, adnate or slightly emarginated, rather crowded, narrow, with 1–3(4) lamellulae between two lamellae, initially pale yellow, ceraceous, then more reddish or ochraceous reddish, sometimes also spotted of a darker colour. Stipe 20–40 × 3–6 mm, cylindrical, slightly enlarged towards the apex, attenuated at the base, but not rooting, somehow darker than the pileus, reddish ochraceous, darker towards the base, covered, mainly in lower half, by the same white pruina present on the pileus. Flesh thin, ochraceous, in the pileus; darker, reddish, in the stipe; not staining or blackening in any part. Smell and taste strongly farinaceous. Spores (3.8–) 4.1–4.9 (–5.2) × (2–) 2.2–2.9 (–3.1) µm (n = 40), on average 4.5 × 2.6 µm, Q = (1.34–) 1.49–2.08 (–2.23), Qm = 1.79, elliptic to sub-cylindrical, often constricted in the middle in side view, hyaline, smooth, cyanophilic, inamyloid and non-dextrinoid, often pluriguttulate. Basidia 13.7–16.7 × 3.9–5.3 µm, 4-spored, clavate, with inner siderophilous granulation. Lamellae edge mainly fertile, with ripen basidia mixed with basidiola and rare sterile elements. Cheilocystidia 23.3–29 × 3.9–6.2 µm, scattered, fusoid to sub-lageniform, lanceolate, with acute tip, thin-walled, some seems to originate from the hymenophoral trama. Pleurocystidia similar to cheilocystidia. Hymenophoral trama subregular, consisting of 15–56 × 4–12 µm subparallel hyphae. Pileipellis a dry cutis of radially arranged, 5–7 µm wide cylindrical hyphae, with some ascendant, slightly enlarged (clavate) terminal elements; pigment both encrusting, with large plaques covering the outer walls, and in addition, diffused, vacuolar, yellow-brown. Stipitipellis a cutis consisting of thin, parallel, 18–70 × 3–9 µm hyphae, with densely encrusting pigment. Clamp-connections frequent everywhere.

Material examined: Italy, Tuscany, Grosseto province, Scansano, Monte Auto, 480 m a.s.l., in a grassland with scattered deciduous oaks (Quercus pubescens) nearby; on neutral to slightly calcareous soil, 11 October 2014, M. Clericuzio (TO HG3329, holotype). GenBank numbers ITS:MF614962; LSU:MF614963.

Notes: Phylogenetically, the new species clusters with T. griseonigrescens in a strongly supported clade (0.99 BY, 94%ML) sister to Lyophyllum cf. maas-geesterani PAM06082615 (0.94 BY, 62%ML). Tephrocybella constrictospora shares collybioid habit, shape and dimensions of the spores, presence of thin-walled cheilocystidia and pileipellis structure with T. griseonigresces (Crous et al. 2015c) However, T. constrictospora clearly differs in having immutable basidiomes, which do not turn black on handling, an umbonate pileus, rare but well-differentiated pleurocystidia, and constricted spores; in addition the pigment was recorded as intracellular only in T. griseonigrescens (Crous et al. 2015c), while it is both extra-cellular (encrusting) and intracellular in T. constrictospora. Lyophyllum maas-geesterani Clémençon & Winterh. is distinguished by yellowish lamellae, a blackening flesh, more elongate, ellipsoid-elongate to subcylindrical, 4.7–5.7 (–7) × 2.6–3.3 (–3.8) µm size spores, irregularly fusiform, and slender cheilocystidia (24–30 × 3–4 µm) (Clémençon and Winteroff 1992). Thus, based on both morphological and molecular characters, T. constrictospora is introduced as a new species. With the inclusion of this species, the definition of Tephrocybella has to be emended and extended to encompass also species with immutable lamellae (not changing colour when bruised).

Morphological comparison between the two Tephrocybella species.

Characteristics T. constrictospora T. griseonigrescens
Habit and pileus size Collybioid with umbo 15–30 mm Collybioid without umbo 15–42 mm
Basidiome colour Ochraceous brown, reddish-brown Light brown, grey-brown
Flesh Unchanging, smell strongly mealy Blackening, smell fruity + a mealy component
Spores Narrowly elliptical to sub-cylindrical, (3.8–) 4–5 (–5.2) × (2–) 2.2–2.9 (–3.1) µm, Q = (1.3–) 1.5–2.1 (–2.2) Elliptical to narrowly elliptical, (3.2–)3.5–4.5(–5) × (1.5–)2–2.5(–3)µm, Q = 1.6–1.9
Cheilocystidia Present, scattered, lanceolate Present, scattered, sub-lageniform
Pleurocystidia Present, scattered, lanceolate Absent
Pileipellis A cutis with ascendant, clavate terminal cells A cutis with some ascendant terminal cells
Pigment Both encrusting and intracellular Exclusively intracellular

Tephrocybella constrictospora (TO HG3329, holotype). Basidiomes in field. Scale bars 20 mm

Tephrocybella constrictospora (TO HG3329, holotype). a–c Spores (a in Cotton blue, b in ammoniacal Congo red, c in 3% KOH). d Basidia and basidioles (in 3% KOH). e Cheilocystidia (in ammoniacal Congo red). f Pileipellis (in water). g Stipitipellis (elements with encrusting pigment, in water). Scale bars a, b = 5 µm, c–g = 10 µm

Phylogram generated from a Bayesian analysis based on combined ITS and LSU sequence data for species of Lyophyllaceae. Based on the BLASTn algorithm. Based on the BLASTn results and outcomes of sound phylogenetic studies focused on Lyophyllaceae (Hofstetter et al. 2002, 2014; Bellanger et al. 2015; Crous et al. 2015c; Vizzini et al. 2015), sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/). Entoloma sericeonitidum (EF421108, AF261315) and Entoloma sericeum (DQ367430, DQ367423) (Entolomataceae) are used as outgroup taxa. Tree topology of the Bayesian analysis is similar to the maximum-likelihood analysis. Bootstrap support (BPP) values (in bold) ≥ 0.7 and maximum-likelihood bootstrap (MLB) values ≥ 50% are given above or below the nodes. Newly generated sequences are in blue and in bold