Tricholoma olivaceonigrum, a new species of the section Tricholoma (Agaricales) from Japan

Abstract

A novel species of Tricholoma section Tricholoma, namely, T. olivaceonigrum, is described and illustrated based on samples found in an oak woods dominated by Quercus myrsinifolia, an evergreen oak, in Tottori Prefecture, western Japan. It is characterized by a conic-umbonate, dark-greenish olivaceous pileus with blackish innate fibrils; a whitish silky-fibrillose stipe, often faintly tinted pale yellow and with a narrowed subpointed base; subglobose to broadly elliptic spores; and fruiting in early winter. Phylogenetic analysis targeting the internal transcribed spacer region of the ribosomal RNA gene revealed that T. olivaceonigrum forms a well-supported clade sister to T. portentosum. Other morphologically and phylogenetically closely related species of the section Tricholoma are discussed.

Since 2000, for nearly 20 y, the first and the second authors (SU and EN) have repeatedly collected a Tricholoma fungus in oak woods dominated by evergreen Quercus myrsinifolia Blume occasionally intermixed with deciduous oaks (Q. acutissima Carruth. and Q. serrata Murray) in Tottori City near the Tottori Mycological Institute. The fungus was considered to be a member of Tricholoma section Tricholoma (Singer, 1986; Christensen & Heilmann-Clausen, 2013), having morphological and ecological characteristics very similar to those of T. portentosum (Fr.) Quél. and T. sejunctum (Sowerby) Quél. of that section. In this study, we aimed to clarify its taxonomic status and its phylogenetic relationship within the section.

Specimens examined (Table 1) in this study were deposited in the herbarium of the Tottori Mycological Institute (TMI), Tottori, Japan, and the mycological herbarium of Tottori University (TUMH), Tottori, Japan. Color names as alphanumeric terms in double quotation marks were from the Online Auction Color Chart, e.g., “oac858” (Anonymous, 2004), and Methuen Handbook of Colour, e.g., “4E7” (Kornerup & Wanscher, 1967). In the description, the following abbreviations are used: Q = spore length/spore width, Q_m = mean Q, L_m = mean spore length, W_m = mean spore width. For microscopic observations, fresh and dry specimens were hand-sectioned using steel razor blades and then mounted in Melzer’s reagent, 1% (w/v) Congo-Red solution, Lacto-Fuchsin, 2.5% (w/v) KOH solution and distilled water. All microscopic observations were made using a Nikon Ni-U microscope (Nikon, Tokyo, Japan) outfitted with differential interference contrast optics, a drawing tube, and a digital camera unit. For comparison, five specimens of T. portentosum were examined in this study (Table 1). DNA extraction from dry and fresh specimens was carried out using the Kaneka Easy DNA extraction kit (KN-T 110005; Hyogo, Japan), following the manufacturer’s protocol. Polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region was performed on a KOD FX (Toyobo, Osaka, Japan) using the primers ITS1-F and ITS4-B (Gardes & Bruns, 1993). PCR products were purified by using Exo SAP-IT (Affymetrix USB, Cleveland, USA) following the manufacturer’s protocols. DNA sequencing and assembling of obtained PCR products, using the same forward and reverse primer pair, were outsourced to FASMAC sequencing services (Kanagawa, Japan, http://fasmac.co.jp/dnaseq). The sequences obtained in this study were deposited in the DNA Data Bank of Japan (DDBJ; accession nos. LC260648, LC335836–LC335842, and LC375759–LC375763). The obtained sequence data were aligned using MUSCLE (Edgar, 2004) in MEGA7 software (Kumar, Stecher, & Tamura, 2016) and manually refined with MEGA7. The refined alignment was submitted to TreeBase (http://www.treebase.org; accession no. S26030). A phylogenetic tree was constructed with the Maximum-likelihood (ML) analysis using MEGA7 software with bootstrapping 1,000 replicates. To determine the posterior probabilities for individual branches, the Bayesian inference (BI) analysis was performed using MrBayes v.3.2.1 software (Ronquist et al., 2012). Two runs with four chains of the Markov chain Monte Carlo iteration were performed for 1,000,000 generations when the average standard deviations of split frequencies were below 0.01 (the first 25% of generations were treated as burn-in). Trees were kept for every 100 generations, and the latter 75% of the trees were used to calculate the 50% majority-rule consensus topology. The best substitution model, Hasegawa-Kishino-Yano (HKY) + Gamma Distributed with Invariant Sites (G+I) was applied for both ML and BI analyses. Tricholoma saponaceum (MF034221) was selected as the outgroup.

Table 1-Tricholoma specimens examined in this study.

Scientific name	Specimen number	Locality in Japan	Forest canopy vegetationa	Accession nos. of ITS sequence
T. olivaceonigrum	TMI 26351	Kokoge, Tottori, Tottori	Qm	–
T. olivaceonigrum	TMI 26352	Kokoge, Tottori, Tottori	Qm	–
T. olivaceonigrum	TMI 26353	Kokoge, Tottori, Tottori	Qm	LC335836
T. olivaceonigrum	TMI 26354	Kokoge, Tottori, Tottori	Qm	LC335837
T. olivaceonigrum	TMI 26355	Kokoge, Tottori, Tottori	Qm	LC335838
T. olivaceonigrum	TMI 26356	Kokoge, Tottori, Tottori	Qm	LC335839
T. olivaceonigrum	TMI 26357	Kokoge, Tottori, Tottori	Qm	LC335840
T. olivaceonigrum	TMI 26358	Kokoge, Tottori, Tottori	Qm	LC335841
T. olivaceonigrum	TMI 26359	Kokoge, Tottori, Tottori	Qm	LC335842
T. olivaceonigrum	TMI 26360 (holotype)	Kokoge, Tottori, Tottori	Qm	LC260648
T. olivaceonigrum	TMI 26367	Kokoge, Tottori, Tottori	Qm	–
T. olivaceonigrum	TMI 37389	Kokoge, Tottori, Tottori	Qm	–
T. portentosum	TMI 37390	Matsumoto, Nagano	Qc, Td	LC375759
T. portentosum	TMI 37391	Shinano, Nagano	Qs, Qc, Fc	LC375760
T. portentosum	TMI 37392	Nakagawa, Nagano	Qs	LC375761
T. portentosum	TMI 37393	Minami-minowa, Nagano	Pd	LC375762
T. portentosum	TMI 37394	Kushiro, Hokkaido	Pp	LC375763

^a Qm: Quercus myrsinifolia, Qc: Q. crispula, Qs: Q. serrata, Fc: Fagus crenata, Pd: Pinus densiflora, Pp: P. pumila, Td: Tsuga diversifolia.

Taxonomy

Tricholoma olivaceonigrum Ushijima, Nagas. & N. Endo, sp. nov. Figs. 1, 2.

MycoBank no.: MB 823556.

Fig. 1-Tricholoma olivaceonigrum. A: Fresh basidiomata (TMI 26359). B: Differential interference contrast (DIC) micrograph of the pileipellis element in water (TMI 26360, holotype). C: DIC micrograph of the basidiospores in Lacto-Fuchsin (TMI 26360, holotype). D: DIC micrograph of the basidia in Congo-Red (TMI 26360, holotype). Bars: A 10 mm; B, D 20 µm; C 10 µm.

Fig. 2– Micro-morphological features of Tricholoma olivaceonigrum (TMI 26360, holotype).

A: Basidiospores.B: Basidia. Bar: 10 µm.

Diagnosis: Tricholoma olivaceonigrum is characterized by a medium-sized to large basidioma with a slightly viscid, conic-umbonate, dark-greenish olivaceous pileus streaked with innately radiating blackish fibrils, white lamellae often with a pale yellow-flush, a white silky-fibrillose stipe often tinted pale yellow and tapering to a subpointed base, and microscopically, subglobose to broadly ellipsoid basidiospores (6–10 × 5–9 µm). Its occurrence in oak woods dominated by Q. myrsinifolia (Fagaceae) in early winter is also distinctive.

Type: JAPAN, Tottori Pref., Tottori City, Kokoge, on the ground in a Q. myrsinifolia stand, 18 Nov 2016, coll. E. Nagasawa, EN16-397 (TMI 26360, holotype; TUMH 62973, isotype).

Gene sequence ex-holotype: LC260648 (ITS).

Etymology: “olivaceonigrum” refers to the dark-greenish olivaceous to almost blackish color of the pileus surface.

Japanese name: Kuromidori-shimeji.

Pileus 35–110 mm diam, conico-convex when young, broadly convex to expanded when mature, typically with a pointed conical umbo, ambiguously umbonate, margin somewhat recurved or irregularly torn when old; pileus surface smooth, slightly viscid when wet, innately and radially fibrillose, background color pale greenish to greenish yellow near “4E7–8” to “4D8” often lighter toward the margin, middle part slightly olivaceous brown, with dark olivaceous “5F7–8” to almost blackish fibrils; flesh thick at the center, becoming abruptly thin toward the margin, firm at first, more or less fragile when old, very pale yellow, tinted grayish or slightly smoke-brown “4F2–3” spotted at the umbonate center. Lamellae broad, 6–10 mm wide, adnexed to deeply sinuate, sometimes nearly free, dull white to pale yellow near “oac858” to “oac900”; edges more or less irregular, concolorous with faces, sometimes discolored dark olivaceous brown “5F8” to more or less more greenish. Stipe 50–150 × 8–18 mm, cylindrical, often with a narrowed, subpointed base, at times more or less compressed, often curved near the base, flexuous at times, surface smooth, silky-fibrillose, white often tinted pale yellow “2A2–3” to pale grayish yellow “oac892” to “oac899” in the area exposed to sunlight, sometimes faintly and partially stained reddish “oac655” to “oac794” at the base buried in soil; flesh solid, rarely hollow when old, fibrous-fleshy, dull white or grayish in the upper stipe. Odor farinaceous, taste slightly bitter. KOH test on flesh of the stipe base negative.

Basidiospores subglobose to broadly ellipsoid, 6–10 × 5–9 µm [180 spores from 9 collections (TMI 26351, 26353, 26354, 26355, 26356, 26357, 26358, 26359 and 26360)], range of mean = 7.2–8.2 × 5.3–6.8 µm, L_m = 7.93 µm, W_m = 6.08 µm, Q = 1.07–1.63, Q_m = 1.31, range of Q_m = 1.2–1.38, smooth, thin-walled. Basidia 30.5–44.5 × 6.5–10 µm, clavate, with 2–4–(5) sterigmata, not clamped at the base. Cystidia absent. Pileipellis an ixocutis consisting of tubular hyphae, 3.5–22 µm wide, slightly interwoven to more or less running parallel, thin-walled; walls incrusted with pale olive brown to dark blackish brown pigments in water and 2.5% KOH, without clamps. Hymenophoral trama parallel, consisting of cylindrical, thin-walled, colorless, smooth, up to 20 µm wide hyphae, without clamps. Stipitipellis a cutis of parallel, cylindrical, thin-walled, colorless, smooth, up to 7 µm wide hyphae, without clamps. Stipe trama of parallel, smooth, cylindrical, up to 21 µm wide, colorless, thin-walled hyphae, slightly ampullate at the septa, without clamps.

Habit and habitat: Gregarious or in small groups, rarely solitary; on soil in a lowland oak woods dominated by Q. myrsinifolia; early winter (Nov to early Dec).

Additional specimens examined: JAPAN, Tottori Pref., Tottori City, Kokoge, 13 Nov 2000, coll. S. Ushijima (TMI 26351); 18 Nov 2004, coll. Y. Nishio, EN04-146 (TMI 37389); 19 Nov 2009, coll. E. Nagasawa and Y. Nishio, EN09-454 (TMI 26354); 26 Nov 2009, coll. E. Nagasawa and Y. Nishio, EN09-458 (TMI 26353); 14 Nov 2012, coll. E. Nagasawa, EN12-434 (TMI 26355); 29 Nov 2012, coll. E. Nagasawa, EN12-443 (TMI 26356); 28 Nov 2013, coll. E. Nagasawa, EN13-487 (TMI 26357); 5 Dec 2013, coll. E. Nagasawa, EN13-488 (TMI 26358); 10 Nov 2014, coll. E. Nagasawa, EN14-530 (TMI 26359); 22 Nov 2016, coll. E. Nagasawa, EN16-403 (TMI 26352); 2 Dec 2017, coll. S. Ushijima (TMI 26367).

Note: Tricholoma olivaceonigrum is morphologically very similar to some members of the section Tricholoma, such as T. sejunctum, T. viridilutescens M. M. Moser, T. olivaceoluteolum Reschke, Popa, Zhu L. Yang & G. Kost, T. portentosum, and T. sinoportentosum Zhu L. Yang, K. Reschke, F. Popa & G. Kost (Christensen & Heilmann-Clausen, 2013; Reschke, Popa, Yang, & Kost, 2018). Tricholoma sejunctum, originally described as Agaricus sejunctus Sowerby from England (Sowerby, 1799), has a pileus colored lighter greenish yellow overall, with dark brown to blackish fibrils and slightly smaller broadly ellipsoid basidiospores, and associates with deciduous members of Fagaceae (Table 2). Tricholoma viridilutescens, originally described by Moser (1978) from Austria, has a yellowish brown pileus with blackish sparse fibrils and associates with coniferous trees of Picea and Abies (Table 2; Christensen & Heilmann-Clausen, 2013). Tricholoma olivaceoluteolum, originally described from Yunnan, China, differs in having a yellow to olive brown pileus and shorter basidiospores with lower Q values, sized 5.5–6.5 × 5–6 µm (Reschke et al., 2018).

Table 2-Morphological and ecological differences between Tricholoma olivaceonigrum and other similar species of sect. Tricholoma.

Scientific name	Pileus		Lamella edge	Basidiospore			Host plante
	Surface colorc	Fibril colorc		Shaped	Ave. Size (µm)	Ave. Q value
T. olivaceonigrum	GrY–OBr	Sparse, DO–B	Irregular	BE–E	7–8 × 5.5–7	1.2–1.38	EB
T. portentosum	PG–GBr, B	Dense, DG, B	Smooth	E–NE	6–7 × 4–4.5	1.38–2	DB–C
(T. portentosum)a	G–DGBr	Dense, DG–B	Smooth	E–O	5.4–7 × 3.7–4.5	1.33–1.69	C
T. sejunctuma	Y–OGr	Dense, DOGr	Smooth	BE–E	5.9–6.9 × 4.9–5.5	1.15–1.42	DB
T. viridilutescensa	LY–YBr	Sparse, DOGr–B	Smooth	BE	6.3–8 × 5.3–6.6	1.16–1.35	C
T. sinoportentosumb	YBr–DBr	Dense, Br–B	Smooth	BE	5.5–7 × 5–5.5	1.1–1.4	C
T. olivaceoluteolumb	Y–BrO	Dense, Dark color	Smooth	SG–BE	5.5–6.5 × 5–6	1.1–1.2	DB

^a Morphological characteristics were referred from Christensen and Heilmann-Clausen (2013).

^b Referred from Reschke et al. (2018).

^c B: Black, Br: Brown, BrO: Brownish olive, DBr: Dark brown, DG: Dark gray, DGBr: Dark grayish brown, DO: Dark olivaceous; DOGr: Dark olivaceous green, G: Gray, GrY: Greenish Yellow, GBr: Grayish brown, LY: Lemon yellow, OBr: Olive brown, OGr: Olivaceous green, PG: Pale gray, Y: Yellow, YBr: Yellowish brown.

^d SG: Subglobose, BE: Broadly ellipsoid, E: Ellipsoid, NE: Narrowly ellipsoid, O: Oblong.

^e EB: Evergreen broad-leaved tree, DB: Deciduous broad-leaved tree, C: Coniferous tree.

Tricholoma portentosum and T. sinoportentosum are also quite similar to this new species morphologically. The former, an old species well-known in the literature and widely distributed in the Northern Hemisphere, is different mainly in having a dark grayish brown to nearly blackish silky-fibrillose pileus and narrower ellipsoid basidiospores (Christensen & Heilmann-Clausen, 2013). However, the latter species, recently described from Yunnan, China, seems to be more similar to T. olivaceonigrum in its color of basidiomata but apparently different in the clamped basidia and the habitat in coniferous forests (Reschke et al., 2018).

The phylogenetic tree inferred from ITS sequences (Fig. 3) supported that T. olivaceonigrum belongs to the section Tricholoma (Heilmann-Clausen, Christensen, Frølev, & Kjøller, 2017). The eight T. olivaceonigrum specimens newly sequenced in this study formed a single clade with strong support, i.e., maximum likelihood tree with maximum likelihood bootstrap/Bayesian posterior probabilities (MLBS/BPP: 99/1.00), showing a sister relationship to the T. portentosum clade (Fig. 3). This result supported the morphological distinctness of T. olivaceonigrum from other similar species. To our knowledge, T. olivaceonigrum has only been found in the type locality in Tottori Prefecture, western Japan.Corner (1994)presented a monograph of tropical Asian Tricholoma, however, there is no information on related species of the section Tricholoma. Further research in evergreen broad-leaved forests of the family Fagaceae in warm-temperate regions of Japan and neighboring countries might unravel additional localities where this species occurs.

Fig. 3– Maximumlikelihood (ML) tree inferred from the ITS sequence of Tricholoma olivaceonigrum and related species in the section Tricholoma. Support values greater than 50/0.5 are indicated on the branches (maximum likelihood tree with maximum likelihood bootstrap/Bayesian posterior probabilities; MLBS/BPP). Bold branches are significantly supported by both MLBS (≥ 70%) and BPP (≥0.95). The scale bar indicates nucleotide substitutions per site. AU: Austria; CA: Canada; CN: China; DE: Germany; DK: Denmark; FI: Finland; FR: France; IT: Italy; JP: Japan; NO: Norway; SL: Slovenia; SW: Sweden; US:United States of America.

Disclosure

The authors declare no conflicts of interest.

Acknowledgments

We would like to thank Editage (www.editage.jp) for English language editing.

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