Laccaria populina Dovana, sp. nov.

Index Fungorum number: IF 557832; MycoBank number: MB 557832; Facesoffungi number: FoF 09202; Fig. 1

Etymology – The specific epithet populina is in reference to the genus Populus, because it was collected under poplar trees.

Holotype – GDOR411

Pileus 15–35 mm diam., convex to plane-convex centrally depressed to subumbilicate; surface glabrous when moist, on drying slightly squamulose at center, slightly translucentstriate, hygrophan, orange, brown–red at disk transitioning to pale orange toward margin, often with wine red areas; paler with age; margin striate and crenate. Lamellae up to 5 mm broad, moderately distant, emarginate, sinuate to adnate with decurrent tooth, with 2–4(–5) lamellulae between two lamellae of different size, sometimes anastomosing or forked, from flesh-pink to dark orange, when old often with wine red tinge. Stipe 10–60×2–6 mm, cylindrical, centrally inserted, solid to fistulose with age, generally darker than the pileus, orange to red-brown, when moist from glabrous to fibrillose, on drying generally squamulose to squarrose overall, squamules white to ochraceous. Context concolorous with surface, sometimes with red-vinaceous tinge, generally vinaceous in the base of stipe. Taste mild and fungoid. Basal mycelium white. Basidiospores (6.6–)7.2–8.8(–9.6)×(6.4–)7.1–8.8(–9.6) µm, Q=(0.98–)0.99–1.03(–1.06) µm, globose, hyaline, moderately echinulate, 1–2 µm long, 0.8–1.5 µm broad at the base. Basidia 40–50×11–13 µm, 4-spored, clavate, containing a yellow–brown pigmentation in 3% KOH. Pleurocystidia 40–50×3–7 µm, flexuous to narrowly-cylindrical. Cheilocystidia rare, similar to pleurocystidia. Hymenophoral trama, subregular, consisting of 15–56×3–6 µm subparallel hyphae. Pileipellis of interwoven hyphae with scattered fascicles of ± perpendicular hyphae; terminal elements variable in shape, from cylindrical to clavate, sometimes with median constriction, rarely with lobed to coralloid terminal elements. Stipitipellis a cutis consisting of thin, parallel, 18–40×4–9 µm hyphae. Clamp-connections frequent everywhere.

Habitat – Associated with Populus alba, Populus nigra, Populus spp. and Salix spp.

Material examined – ITALY, Province of Alessandria, Mombello, locality Casalino, 220 m a.s.l., in a grassland with scattered Poplars (Populus alba, Populus nigra, Populus spp.) and Willows (Salix spp.) nearby, 11 November 2019, F. Dovana (GDOR 411, holotype); ibidem, 3 September 2010, F. Dovana (GDOR 408).

GenBank numbers – GDOR 411: ITS = MN871895, LSU = MN873017; GDOR 408: ITS = MN871894, LSU=MN873018.

Notes – Macroscopically, Laccaria populina is characterized by its medium-sized basidiomata, with brown–red or orange pileus, crenulate at the margin, context concolour with pileus, with irregular red-wine spots especially near the base of stem and copious white basal mycelium (Fig. 1a, b). Laccaria populina observed appeared to be associated with Salicaceae. Microscopically, it can be recognized by the four-spored basidia, basidiospores echinulate with spines up to 2 µm long, and pileipellis with terminal elements variable in shape and size (Fig. 1c–h). A megablast search of GenBank nucleotide database at NCBI (24 Dec. 2019) using LSU sequence of L. populina (holotype) showed that the best hits were L. tortilis (GMM7635), L. proximella (F1081079), L. proxima (GMM7631) and L. bicolor (GMM2692), all with 98.71% similarity and 6 gaps. As for megablast analysis (24 Dec. 2019), ITS sequence of L. populina showed 99.86% (708/709) similarity and no gaps with two german environmental sequences (GenBank GU990359 and GU990354) that come from Salix viminalis and Populus maximowiczii x Populus nigra root respectively that probably are the same species and 98.71% similarity, and 1 gap with two Laccaria amethystina samples (GMM7592 and GMM7621). Laccaria proximella and L. proxima differ from L. populina mainly by their larger ellipsoidal basidiospores (Mueller 1992). Laccaria bicolor differs mainly in its remarkable violet basal mycelium and presence of subglobose to broadly ellipsoid basidiospores with smaller echinulae (1–1.8 in length and≤1 µm wide at base) (Mueller 1992). Compared to L. populina, L. tortilis has a smaller basidiocarp, 2-sterigmate basidia and larger globose basidiospores (9.2–)10–14.5(–16)×(8.3–)10–14.5(–16) µm (Mueller 1992). Laccaria amethystina is distinguished from L. populina by the bright grayish purple basidiomata and abundant filamentous, clavate or ventricose-rostrate conspicuous cheilocystidia (Mueller 1992). In the combined phylogenetic analysis of LSU and ITS sequences, the two collections of L. populina (holotype included) clustered in a supported clade (75% MLBS, 0.93 BYPP; Fig. 2) with two environmental sequences (GenBank GU990359 and GU990354) sister to the L. cf. laccata clade but lacking in statistical support. Laccaria laccata differs from L. populina mainly by their subglobose to ellipsoid basidiospores (Mueller 1992); Laccaria laccata var. pallidifolia is very similar to L. populina from which it can be distinguished by its slightly larger globose to subglobose spores (6.4–)7.4–10(–13)×(6–)7–10(–11.5) µm, with smaller echinulae (≤1 µm wide at base), context concolorous with pileus without a wine-red spot (Mueller 1992).

Figure 1 – Laccaria populina (GDOR 411, holotype). a Basidiomata in the field. b Section of stem. c Stipitipellis in KOH 3%. d Section of gills in Phloxin B. e Pileipellis in Phloxin B. f Basidiospore in KOH 3%. g Basidia in Phloxin B. h terminal elements of pileipellis in KOH 3%. Scale bars: c, e, g, h=50 µm, d=100 µm, f=10 µm

Figure 2 – Phylogram generated from maximum likelihood analysis based on LSU and ITS sequence data representing Laccaria of northern hemisphere and tropical areas. Related sequences aretaken from previous studies (Wilson et al. 2013; Popa et al. 2014; Luo et al. 2016; Ramos et al. 2017; Vincenot et al. 2017; Cho et al. 2018; Wang et al. 2019b). One hundred fifty-six strains are included in the combined analyses which comprise 1624 characters. Mythicomyces corneipes (AFTOL ID972) is used as the outgroup taxa. Single gene analyses were also performed to compare the topology and clade stability with combined gene analyses. Tree topology of the maximum likelihood analysis is similar to the Bayesian analysis. The best RaxML tree with a final likelihood values of − 9283.670199 is presented. Estimated base frequencies for the two partitions were as follows: the ITS partition A=0.248432, C=0.203290, G=0.210151, T=0.338126; substitution rates AC=1.729861, AG=6.506286,
AT=2.326472, CG=0.685905, CT=5.294619, GT=1.000000, gamma distribution shape parameter α=0.357610; the LSU partition A=0.269370, C=0.185989, G=0.290996, T=0.253645; substitution rates AC=1.151721, AG=25.007835, AT=2.282933, CG=0.751032, CT=11.631350, GT=1.000000, gamma distribution shape parameter α=0.144016. Bootstrap values for maximum likelihood (MLBS) equal to or greater than 70% and clade credibility values greater than 0.95 (the rounding of values to 2 decimal proportions) from Bayesian-inference analysis labeled on the nodes. Ex-type strains are in bold and newly generated sequences are in blue

Figure 2 – (continued)