Three new tricholomatoid dark blue Entoloma spp. from temperate forests in Japan, with the redescription of E. cyanonigrum

Abstract

Three species of tricholomatoid dark blue Entoloma spp. from temperate forests in Japan are described as new to science. Entoloma obscurocyaneum and E. purpureobrunneolum show a dull dark blue pileus, with the former distinguished by the purple-brown suprapellis hyphae of the pileipellis and the latter by its light brown color. Entoloma quasicyanonigrum is similar to E. cyanonigrum but exhibits smaller basidiospores. Our phylogenetic analysis placed them within a moderately supported clade that generally shares the characteristics of having a hymeniderm- or palisadoderm-type pileipellis. To date, the sole tricholomatoid dark blue Entoloma documented from Japan is E. cyanonigrum. Because it was confirmed that the type specimen of E. cyanonigrum no longer exists, the original material, the colored drawing by Tsuguo Hongo, was designated as the lectotype, and E. cyanonigrum was redescribed based on Hongo’s observation notes and our examination of herbarium specimens.

Entoloma s.l. (Entolomataceae, Agaricales, Basidiomycota) is a large genus with approximately 1,500 described species (Noordeloos & Gates, 2012). It is primarily characterized by pink spore prints and angular basidiospores (Horak, 1980; Noordeloos, 1992). Basidiomata are highly diverse in morphology, ranging from pluteoid to collybioid, omphalioid, tricholomatoid, mycenoid, and pleurotoid, with some sequestrated species. From a micromorphological point of view, the structure and pigmentation of the pileipellis and the presence or absence of clamp connections and cystidia are of particular importance for species identification (Noordeloos, 1992; Noordeloos & Gates, 2012; Noordeloos et al., 2022).

Several tricholomatoid dark blue Entoloma spp. have been reported from different regions worldwide, including E. bloxamii (Berk. & Broome) Sacc., E. nitidum Quél., E. haastii G. Stev., and E. hymenidermum Largent (Largent et al., 2014; Noordeloos, 1992). Species such as E. azureosquamulosum Xiao L. He & T.H. Li, E. coeruleoviride Corner & E. Horak, E. marinum Corner & E. Horak, and E. simillimum Corner & E. Horak are known from Asia and have similar macromorphology (He et al., 2012; Horak, 1980). In Japan, only one tricholomatoid dark blue Entoloma species, E. cyanonigrum (Hongo) Hongo (Japanese name: Kon-iro-ippon-shimeji), has been recorded (Katsumoto, 2010) and is considered endemic to Japan (Hosoya et al., 2016).

From 2019 to 2022, some members of the Kansai Mycological Club conducted mushroom surveys in temperate forests in Nara and Shiga, Japan, resulting in several dark blue Entoloma collections. These were superficially similar to E. cyanonigrum but were distinguished by the basidiospore size and the structure of the pileipellis. Herein, we describe them as new and report the phylogenetic analysis results of the internal transcribed spacer (ITS) regions of the nuclear rDNA and the D1/D2 domain of the large subunit of the nuclear rDNA (nrLSU). Because the holotype of E. cyanonigrum was already lost, the original material (Tsuguo Hongo’s color drawing) was designated as the lectotype, and E. cyanonigrum is redescribed here.

Anatomical observations and measurements of newly collected materials are based on fresh material mounted in water, except as noted below. The dimensions of basidia were measured based on fresh material mounted in 3% (w/v) potassium hydroxide solution (KOH), and those of cheilocystidia were measured based on dried material mounted in 3% KOH. For the dried specimens of E. cyanonigrum in OSA, basidia and basidiospores were measured in 3% KOH, and other tissues were measured in water. For the observation of pileipellis, ammonium hydroxide (NH₄OH) was used when necessary. Congo red (CR) was also used to stain hyaline structures in 3% KOH (KOH/CR). Terminology and microscopic measurements followed Noordeloos et al. (2022). Slide preparations were examined and photographed using a BH2 microscope (Olympus, Tokyo, Japan) equipped with a WRAYCAM-EL510 digital camera (Wraymer, Osaka, Japan). All measurements were made using PhotoRuler version 1.1.3 (http://inocybe.info/). The length of the curved structure was measured along the axis using the polyline tool in the program. The dimensions of basidiospores were measured by placing an imaginary rectangle around the spore, excluding the apiculi, as described by Noordeloos et al. (2022). The size range of basidiospores was described as (a-)b-c(-d): the range b-c represented ≥ 90% or more of the measured values (b = mean − SD, c = mean + SD), and “a” and “d” were the extreme values. The length/width ratio (Q) of each basidiospore was reported as a range of Q values. The number of basidiospores examined was expressed as “n.” The examined specimens were oven-dried and deposited in the Osaka Museum of Natural History (OSA; Osaka, Japan).

Genomic DNA was extracted as described by the method of Izumitsu et al. (2012). The cycling conditions of the polymerase chain reaction (PCR) using KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan) followed the protocol described by Masumoto and Degawa (2020). For PCR amplification of the nuclear rRNA gene, the primer pair ITS1F (Gardes & Bruns, 1993) and LR5 (Vilgalys & Hester, 1990) was used for ITS and nrLSU. The PCR products were purified via polyethylene glycol precipitation and sequenced by the Fasmac sequencing service (Kanagawa, Japan) using the following primers: ITS1F and ITS4 (White et al., 1990) for ITS; and LR0R (Rehner & Samuels, 1994) and LR5 for nrLSU. The sequences generated in this study have been deposited with the International Nucleotide Sequence Database Collaboration via the DNA Data Bank of Japan.

The sequence data generated in this study were analyzed with closely related taxa retrieved from the GenBank database based on BLAST searches and recent publications (Table 1). The sequence datasets for each region were aligned using the MAFFT version 7 online service (Katoh et al., 2019) with default parameters and manually refined in SeaView version 5.0.4 (Gouy et al., 2010). For the phylogenetic analysis, the aligned ITS and nrLSU sequences were concatenated in SeaView. A maximum likelihood (ML) phylogenetic analysis was performed using RAxML-NG version 1.0.3 (Kozlov et al., 2019) with 1000 bootstrap replicates for the bootstrap analysis for each branch. The best-fitting substitution models were estimated using ModelTest-NG version 0.2.0 (Darriba et al., 2020), with the HKY+I+G4 model was selected for ITS and TrN+I+G4 for nrLSU based on the Bayesian information criterion. The resulting ML tree was visualized using FigTree version 1.4.4 (https://github.com/rambaut/figtree/releases) and edited in Inkscape version 1.3 (https://inkscape.org/). The alignment and tree were submitted to TreeBASE (http://www.treebase.org; accession no. 31246).

Table 1. Taxa included in molecular phylogenetic analysis and their GenBank accession numbers. Accession numbers in bold indicate newly generated sequence in this study.

Taxon	Voucher/strain	ITS	nrLSU	Reference
Calliderma fibulatum	SP393751	-	FJ973677	Karstedt & Capelari (2010)
Clitocella fallax	CBS 129.63	AF357017	AF223166	Hofstetter et al. (2002)
Clitopilopsis hirneola	ME Noordeloos 199956	KC710132	GQ289211	Morgado et al. (2013)
“Entocybe trachyospora”	ZRL20151426	LT716037	KY418852	Zhao et al. (2017)
Entoloma bloxamii	MEN 200442	KC710087	GQ289154	Morgado et al. (2013)
Entoloma callidermum	Stubbe 06252	KC710115	KC710153	Morgado et al. (2013)
Entoloma coeruleogracilis	MEN 2004055	KC710107	GQ289167	Morgado et al. (2013)
Entoloma coeruleoviride	Stubbe 06236	KC710057	KC710134	Morgado et al. (2013)
Entoloma gracilior	G. Gates E1220	KC710112	GQ289169	Morgado et al. (2013)
Entoloma griseolazulinum	i11	-	GQ289166	Co-David et al. (2009)
Entoloma haastii	MEN 2006617	KC710089	KC710145	Morgado et al. (2013)
Entoloma “haastii”	BY21	-	AF261309	Moncalvo et al. (2002)
Entoloma hymenidermum	DLL10025	-	JQ793648	Largent et al. (2014)
Entoloma hymenidermum	DLL10054 (holotype)	-	JQ793649	Largent et al. (2014)
Entoloma kermandii	G. Gates E227 (holotype)	-	GQ289173	Co-David et al. (2009)
Entoloma “kujuense”	N.K.Zeng2943	-	MT829098	Zeng & Jiang (2020)
Entoloma madidum	MEN 2004030	KC710127	KC710158	Morgado et al. (2013)
Entoloma nitidum	MEN 200324	KC710122	GQ289175	Morgado et al. (2013)
Entoloma obscurocyaneum	OSA-MY-9554 (holotype)	LC796882	LC796875	this study
Entoloma obscurocyaneum	OSA-MY-9565	LC796883	LC796876	this study
Entoloma prunuloides	MEN 200340	KC710073	GQ289184	Morgado et al. (2013)
Entoloma purpureobrunneolum	OSA-MY-9553 (holotype)	LC796880	LC796873	this study
Entoloma purpureobrunneolum	OSA-MY-9564	LC796881	LC796874	this study
Entoloma quasicyanonigrum	OSA-MY-9552 (holotype)	LC796877	LC796870	this study
Entoloma quasicyanonigrum	OSA-MY-9563	LC796878	LC796871	this study
Entoloma quasicyanonigrum	OSA-MY-9560	LC796879	LC796872	this study
Entoloma sinuatum	J. Wisman 2003-09-19	-	GQ289193	Morgado et al. (2013)
Entoloma subsinuatum	MEN 2005624	KC710067	KC710138	Morgado et al. (2013)
Entoloma trachyosporum	H. den Bakker 1153	KC710088	GQ289198	Morgado et al. (2013)
Entoloma violaceotinctum	DLL10088 (holotype)	-	JQ793650	Largent et al. (2014)
Entoloma sp.	D582	OR066305	OR066355	Parnmen et al. (unpub.)
Entoloma sp.	HKAS 52713	JQ410336	JQ320134	He et al. (2012)
Entoloma sp.	TJ Baroni 9895	-	GQ289156	Co-David et al. (2009)
Entoloma sp.	LAM 0474	-	KY091066	Peay & Lim (unpub.)
Entoloma sp.	TH9118	-	KT339288	Smith & Henkel (unpub.)
Uncultured Entoloma	HKAS:50504	-	KJ648463	He et al. (unpub.)

None of the specimens of dark blue Entoloma spp. collected in this study matched the description of E. cyanonigrum. The morphology of these specimens was divided into three groups according to the combinations of basidiospore size and pileipellis structure, but they did not match the description of any known species. They differ in the pigmentation of the hyphal elements forming the pileipellis. In Group 1 (= E. quasicyanonigrum), the pileipellis has only dark blue pigmentation. In Groups 2 (= E. obscurocyaneum) and 3 (= E. purpureobrunneolum), the pileipellis is bilayered. In addition to the dark blue pigmentation of the subpellis, the suprapellis hyphae of the former and the latter have brown and purple-brown pigmentation, respectively. The type of pileipellis is hymeniderm in Groups 1 and 3, and palisadoderm in Group 2. The basidiospores of Groups 1 and 2 overlap slightly in size, but those of Group 3 are larger than those of Groups 1 and 2.

The three dark blue Entoloma spp. collected in our study were placed in the Prunuloides clade according to Co-David et al. (2009) and also located in clade B2 as defined by Morgado et al. (2013) (Fig. 1). However, they were not in the well-supported clade containing E. bloxamii and E. prunuloides (Fr.) Quél. but in a well-supported monophyletic clade with E. kermandii G.M. Gates & Noordel. as the basal clade. Furthermore, the three new species formed a weakly supported monophyletic lineage within it, suggesting that they are closely related. The well-supported monophyletic clade sister to E. kermandii (hereafter referred to as the Calliderma clade) included Calliderma fibulatum Karstedt & Capelari, E. callidermum (Romagn.) Noordel. & Co-David, E. coeruleoviride, E. griseolazulinum Manim. & Noordel., E. hymenidermum, and E. violaceotinctum Largent. All are either trichoderm-, hymeniderm-, or palisadoderm-type pileipellis, rather than cutis-type (Eyssartier et al., 2012; Horak, 1980; Karstedt & Capelari, 2010; Largent et al., 2014; Manimohan et al., 2006). The pileipellis of the three Entoloma spp. is also of a hymeniderm or palisadoderm type. As already noted by Morgado et al. (2013) and Noordeloos & Gates (2012), the pileipellis composed of erect hyphal elements is a representative character of the Calliderma clade.

Fig. 1 - Maximum likelihood (ML) phylogenetic reconstruction of the Prunuloides clade in Entolomataceae based on the combined dataset (ITS+nrLSU). ML bootstrap support (BS) values ≥ 50% are indicated above and below the branches. Newly obtained sequences are shown in bold. The scale bar represents the number of substitutions per site.

Taxonomy

Entoloma cyanonigrum Hongo (Hongo), in Katsumoto, List of Fungi Recorded in Japan: 308 (2010)

MycoBank no.: 540115.　Figs. 2, 3, 5D.

Fig. 2 - Entoloma cyanonigrum. A: Dried basidioma. B: Basidioma from the bottom side. C: Pileipellis (in water). Note that the hyphae retain their dark blue pigmentation. D: Hymeniderm-type pileipellis drawn by Tsuguo Hongo. E: Detail of hyphae of pileipellis (in water). F: Hymenium and hymenial trama (in water). G: A clamp connection in the hymenial trama (in KOH/CR). H: Basidiospores (in water). I: Basidiospores (in KOH/CR). A-C, E-I: OSA-MY-941. D: Hongo Herb. No. 6196 (= OSA-MY-104661). Bars: C 300 µm; D 20 µm; F 50 µm; E, G-I 10 µm.

Fig. 3 - Color and line drawings of Entoloma cyanonigrum by Tsuguo Hongo. A: Original drawing of E. cyanonigrum (Tsuguo Hongo no. 100; cited as Rhodophyllus nitidus Quél.; lectotype). The depicted specimen itself is unfortunately lost. B: Drawing of Tsuguo Hongo No. 6196 (= OSA-MY-104661) [cited as Rhodophyllus cyanoniger (Hongo) Hongo]. The scientific name correction in double lines was made by Tsuguo Hongo himself. Source: Tsuguo Hongo Collection: Color drawings of Japanese Fungi by Osaka Museum of Natural History.

Basionym: Entoloma subnitidum S. Imai f. cyanonigrum Hongo, J. Jpn. Bot. 26: 145 (1951) [MycoBank no.: 346748]

≡ Rhodophyllus subnitidus (S. Imai) Hongo f. cyanoniger (Hongo) Hongo, J. Jpn. Bot. 29: 92 (1954) [MycoBank no.: 352474]

= Rhodophyllus nitidus sensu Imazeki & Hongo, Col. Ill. Jpn. Fungi 1: 78 (1957), non (Quél.) Quél., Enchir. fung. (Paris): 58 (1886) [MycoBank no.: 493193]

= Rhodophyllus cyanoniger (Hongo) Hongo, nom. nud., Col. Ill. Mushr. Jpn. 1: 263 (1987)

Typification: Holotype not located (KYO). Lectotype designated here (MBT 10026267): Water color painting by Hongo, Tsuguo Hongo Collection: Color drawing of Japanese Fungi, unpublished: plate no. 100, as Rhodophyllus nitidus Quél. (original plate kept in OSA) (Fig. 3A).

Basidiomata: tricholomatoid. Pileus: 30-70 mm, convex to subumbonate, dark blue (22F3-22F5; Ridgway, 1912), velutinous, radially wrinkled, not hygrophanous, margin often slightly upturned; context whitish. Lamellae: sinuate, subdistant to subcrowded, up to 8 mm wide, incarnate, edge serrate. Stipe: 40-100 × 4-12 mm, almost equal, occasionally incrassate, fibrillose, dull blue (21D4) to bluish-gray (21D3), whitish at both ends, context white with grayish-blue tint near the surface, solid or stuffed. Odor: mild. Taste: indistinct. Basidiospores: 8.8-11.1 × 7.9-9.6 µm (n = 50; in KOH/CR; based on OSA-MY-941), Q = 1.0-1.3, isodiametric to subisodiametric, 5-6-angled in side-view. Basidia: 29-41 × 10-14 μm (in KOH/CR; based on OSA-MY-941), 4-spored, clavate. Lamella edge: fertile. Cheilocystidia: absent. Hymenophoral trama: subparallel. Pileipellis: a hymeniderm of 2-3 inflated clavate hyphae in a row, terminal cells subclavate, 15-50 × 8-15 μm, with bluish-gray contents. Stipitipellis: not observed. Clamp connections: present in hymenophoral trama.

Specimens examined: JAPAN. Shiga Pref., Otsu, Seta, gregarious on the ground in mixed forest of Pinus densiflora Siebold et Zucc. and Chamaecyparis obtusa (Siebold et Zucc.) Endl., 5 Oct 1958, coll. T. Hongo, Hongo Herb. No. 1908 (= OSA-MY-104659); Kyoto Pref., Sakyo, Iwakura, in woods of P. densiflora, 9 Oct 1959, coll. M. Hamada, Hongo Herb. No. 2031 (= OSA-MY-104700); Shiga Pref., Otsu, Nango-Imodani, in oak forest, 12 Sep 1980, coll. T. Hongo, Hongo Herb. No. 6196 (= OSA-MY-104661); Hyogo Pref., Takarazuka, Nagatani, Iyadani, in Pinus-deciduous mixed forest, 23 Sep 1983, coll. M. Okamoto, det. T. Hongo (OSA-MY-941).

Habitat: Solitary on the ground in mixed forest of P. densiflora and oak with occasional C. obtusa.

Japanese name: Kon-iro-ippon-shimeji (Hongo, 1951)

Notes: The authors have spent the past few years searching for fresh material of E. cyanonigrum in the areas where Tsuguo Hongo had collected it previously. This effort led to the discovery of E. obscurocyaneum, E. purpureobrunneolum, and E. quasicyanonigrum, as described here; however, E. cyanonigrum itself could not be found. According to the protologue of E. cyanonigrum (originally described as E. subnitidum f. cyano-nigrum), the type material was deposited at “Phytopatholog. Univ. Kyoto” (Hongo, 1951). Because the type specimens deposited there by Hongo have since been transferred to the Kyoto University Museum (KYO; Sakuma, 2019), the authors visited KYO to search for the holotype of E. cyanonigrum, but it could not be located. Additionally, because most of Hongo’s type collection is housed in the herbarium of the National Science Museum, Tokyo (TNS), this study reviewed the lists of Hongo’s type materials published by TNS (Doi, 1991, 1993, 1996, 1998), but none of them included the type of E. cyanonigrum. Thus, the type specimen of E. cyanonigrum appears to be lost. The first author examined specimens from the Hongo herbarium housed at OSA, which included several specimens identified by Hongo as E. cyanonigrum, though none were type material. The redescription of E. cyanonigrum in this study was based on the Hongo’s observation notes (Supplementary Figs. S1, S3-S6; Supplementary Table S1) housed at OSA [Tsuguo Hongo’s personal manuscript: “Notes on Japanese Fungi Nos. 1 (1950-1951), 21 (Sep 1958-Oct 1958), 22 (Oct 1958-Jul 1960), and 56 (Jul 1980-Sep 1980)”] and on our re-examination of the specimens. Specifically, the size of basidiospores and basidia was based on the relatively well-preserved specimen OSA-MY-941. Unfortunately, attempts to sequence the ITS and nrLSU regions of OSA-MY-941 failed, likely due to DNA degradation.

As mentioned above, the holotype of E. cyanonigrum was already lost. However, the colored drawing (Tsuguo Hongo Collection: Color drawing of Japanese Fungi, plate no. 100; Fig. 3A), which served as the basis for the line drawing in the original description [see Figure 2 in Hongo (1951)] is kept at OSA. The authors regard this drawing as the original material and designate it as the lectotype of E. cyanonigrum Hongo (Hongo) [International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code), Arts. 9.3, 9.4, & 9.12. Retrieved May 4, 2025, from https://www.iapt-taxon.org/nomen/main.php].

Many dark blue Entoloma species have been reported as E. cyanonigrum in Japan (e.g., Ikeda, 2013; Imazeki et al., 2011; Yamamoto & Ohmae, 2024). These entities should be re-examined based on basidiospore size, pileipellis type, and DNA sequences. Entoloma quasicyanonigrum was collected in areas where E. cyanonigrum was previously found, and its characteristics closely match those of E. cyanonigrum, except for differences in basidiospore size. Pinus densiflora was abundant in these areas in the 1950s, but with the spread of pine wilt disease, most of the P. densiflora has been lost, and the vegetation is now dominated by Quercus and Castanopsis. If E. cyanonigrum is associated with P. densiflora and E. quasicyanonigrum is associated with Quercus and Castanopsis, this may explain why E. cyanonigrum is no longer found there. In the future, E. cyanonigrum should be rediscovered and designated as an epitype, and molecular data should be obtained for comparison with the morphologically similar E. quasicyanonigrum.

Entoloma obscurocyaneum Ohkubo & H. Masumoto, sp. nov.

MycoBank no.: 855060.　Figs. 4, 5C, 6C.

Fig. 4 - Entoloma obscurocyaneum. A: Basidioma. B: Sectioned basidioma. C: Another sectioned basidioma. D: Higher magnification of Fig. 4C, showing the slight umbo at center. E: Bilayered pileipellis (in water). F: Hyphae of suprapellis of pileipellis (in NH₄OH). A, B, E, F: OSA-MY-9554 (holotype). C, D: OSA-MY-9565. Bars: A-C 1 cm; D 5 mm; E 20 µm; F 10 µm.

Fig. 5 - Basidiospores of tricholomatoid dark blue Entoloma species described/redescribed here. A: E. quasicyanonigrum (OSA-MY-9552, holotype). B: E. purpureobrunneolum (OSA-MY-9553, holotype). C: E. obscurocyaneum (OSA-MY-9554, holotype). D: E. cyanonigrum (OSA-MY-941). Bars: 5 µm.

Fig. 6 - Cheilocystidia of tricholomatoid dark blue Entoloma species described here. A: E. quasicyanonigrum (OSA-MY-9552, holotype). B: E. purpureobrunneolum (OSA-MY-9564). C: E. obscurocyaneum (OSA-MY-9554, holotype). Bars: 10 µm.

Diagnosis: This species is characterized by the bilayered pileipellis consisting of brown suprapellis and dark blue subpellis, giving the pileus a dull dark blue color.

Holotypus: JAPAN. Shiga Pref., Otsu, Ryukoku-no-Mori (Ryukoku Forest), on soil in broad-leaved forest, 7 Aug 2022, coll. Y. Ohkubo (specimen ID: OSA-MY-9554), GenBank accession no.: ITS (LC796882) and nrLSU (LC796875).

Etymology: The epithet refers to the dull dark blue pileus.

Basidiomata: tricholomatoid. Pileus: 40-50 mm, plano-convex to depressed, sometimes with slight umbo at center, grayish-blue, nearly black in the center and blue at the margin, scabrulose, not hygrophanous, margin crenulate, indistinctly radially rugulose; context white. Lamellae: sinuate, subdistant to close, up to 6 mm wide, incarnate, edge serrulate. Stipe: 46-50 × 6-7 mm, cylindrical, almost equal, grayish-blue and fibrillose-striate in the lower part, becoming paler in the upper part, with white base; context white, solid. Odor: none. Taste: acrid. Basidiospores: (6.3-)6.6-7.3(-7.9) × (5.2-)5.8-6.8(-7.5) μm (n = 27), Q = 1.0-1.2, isodiametric to subisodiametric, 5-6-angled in side-view. Basidia: 36-47 × 10-12 μm (in KOH/CR), 4-spored, clavate, not clamped. Lamella edge: heterogeneous. Cheilocystidia: 22-40 × 4.6-8.2 µm (in KOH/CR; based on dried material), irregularly cylindrical, occasionally with a swollen apex or papilla. Hymenophoral trama: subparallel, composed of cylindrical hyphae, 13-22 µm wide. Pileipellis: bilayered; suprapellis a palisade of cylindrical to clavate hyphae, 27-74 × 9-18 μm wide, brownish red to grayish brown; subpellis composed of cylindrical hyphae, 20-37 × 5.3-9.6 µm wide, dark blue, slightly interwoven. Pileitrama: composed of cylindrical hyphae, hyaline, 14-30 µm wide. Stipitipellis: a cutis of cylindrical hyphae, 2.8-4.5 µm wide, hyphae hyaline or faintly grayish-blue, terminal cells often inflated. Clamp connections: abundant in all tissues, rarely double.

Habitat and distribution: Solitary on the ground in a sparse forest of broad-leaved shrubs mixed with C. obtusa. Currently known only from Japan (Shiga).

Other specimen examined: JAPAN. Shiga, Otsu, Gennai pass, on soil in a broad-leaved forest, 4 Sep 2022, coll. Y. Ohkubo (OSA-MY-9565).

Japanese name: Kusumi-kon-iro-gasa

Notes: Entoloma obscurocyaneum is similar to E. purpureobrunneolum but differs in having smaller basidiospores (< 8 µm in length; Fig. 5), thicker cheilocystidia (> 9 µm wide; Fig. 6), and brown pigment in the suprapellis of the pileipellis, as opposed to purple-brown. BLAST search revealed that the ITS sequence of E. obscurocyaneum (LC796882) matched well (99%) with the three sequences labeled as “Entocybe trachyospora” (LT716037, OL616146, and ON794415). However, because genuine Entocybe trachyospora (Largent) Largent, T.J. Baroni & V. Hofst. (= Entoloma trachyosporum Largent) is located in a distant lineage (Fig. 1; Baroni, 2011; Morgado et al., 2013), these sequences appear to be based on misidentified samples. One of these sequences (ON794415) is from China, but further comparisons based on specimens are needed to determine their identity. The nrLSU sequence of E. obscurocyaneum (LC796875) showed 99.28% homology (= 832/838 bp) with the sequence labeled “Entoloma kujuense voucher N.K.Zeng2943” (MT829098), and phylogenetic analysis indicated that they form a strongly supported monophyletic clade (BS = 100; Fig. 1). However, the purple coloration of the velvety pileus and the larger heterodiametric basidiospores (10-12.5 × 7-8.5 µm) of this specimen (FHMU1912; Zeng & Jiang, 2020) suggest that it is a distinct species from E. obscurocyaneum.

Entoloma purpureobrunneolum Ohkubo & H. Masumoto, sp. nov.

MycoBank no.: 855061.　Figs. 5B, 6B, 7, 8.

Fig. 7 - Entoloma purpureobrunneolum. A: Basidioma with slight depression. B: Sectioned basidioma with small umbo. C: Bottom side of Fig. 7A. D: Bottom side of Fig. 7B. E: Bilayered pileipellis (in water). F: Hyphae of suprapellis of pileipellis (in NH₄OH). A, C: OSA-MY-9564. B, D-F: OSA-MY-9553 (holotype). Bars: A, C, D 1 cm; B 5 mm; E 20 µm; F 10 µm.

Fig. 8 - Connections between suprapellis (arrow) and subpellis (arrowhead) hyphae in the pileipellis of Entoloma purpureobrunneolum (in KOH/CR; OSA-MY-9564). Note that one to four clavate to cylindrical hyphae vertically or obliquely connected to each inflated cell of subpellis. Bar: 10 µm.

Diagnosis: This species is characterized by the bilayered pileipellis with one to four purple-brown suprapellis hyphae connected to a dark blue subpellis hypha (Fig. 8), resulting in a dull dark blue pileus.

Holotypus: JAPAN. Shiga Pref., Otsu, Gennai pass, on soil in a sparse forest of broad-leaved shrubs mixed with C. obtusa, 11 Sep 2022, coll. Y. Ohkubo (specimen ID: OSA-MY-9553), GenBank accession no.: ITS (LC796880) and nrLSU (LC796873).

Etymology: The epithet refers to the purplish-brown pigmentation of the suprapellis of the pileipellis.

Basidiomata: tricholomatoid. Pileus: 25-30 mm, subumbonate to plano-convex with a slightly depressed center, dark blue near black, uniformly colored, velutinous, indistinctly radially rugulose, not hygrophanous, margin faintly striate, crenulate; context white. Lamellae: sinuate, subdistant, 3-4.5 mm wide, incarnate, edge serrulate. Stipe: 28-37 × 4-5 mm, cylindrical, almost equal, fibrillose-striate, grayish blue, paler at the top, with white base; context white, solid. Odor: none. Taste: bitter to acrid. Basidiospores: (8.2-)8.5-9.5(-10.0) × (7.0-)7.6-8.5(-9.0) μm (n = 30), Q = 1.1-1.2, subisodiametric, 5-6-angled in side-view. Basidia: 36-48 × 11-13 μm (in KOH/CR), 4-spored, clavate to cylindrical, rarely with a basal clamp. Lamella edge: heterogeneous. Cheilocystidia: 22-44 × 9.2-14.3 µm (in KOH/CR; based on dried material), clavate, with a swollen apex, often clamped. Hymenophoral trama: subparallel, composed of cylindrical hyphae, 12-33 µm wide. Pileipellis: bilayered; suprapellis a hymeniderm of clavate hyphae, 25-61 × 11-29 µm, one to four clavate to cylindrical hyphae vertically or obliquely connected to per inflated cell of subpellis, hyphae light brown to blackish-brown with purplish tints; subpellis composed of inflated hyaline hyphae, 19-47 × 13-25 µm, containing dark blue granular to irregularly aggregated pigments. Pileitrama: composed of cylindrical hyphae, 12-32 µm wide, hyaline with some dark blue pigments. Stipitipellis: a cutis of cylindrical hyphae, 2.3-6.5 µm wide, hyphae pale blue with aggregated dark blue pigments. Clamp connections: abundant in stipe trama, scarce in other tissues.

Habitat and distribution: Solitary on the ground in a mixed forest with C. obtusa. Currently known only from Japan (Shiga). BLAST search revealed that the nrLSU sequence of E. purpureobrunneolum (LC796873) matched well with an environmental sequence (KJ648463; 99.76% = 831/833 bp) derived from soil in China. The distribution in China will require further examination based on specimens.

Other specimen examined: JAPAN. Shiga Pref., Otsu, Gennai pass, on soil in a mixed forest with C. obtusa, 21 Sep 2021, coll. Y. Ohkubo (OSA-MY-9564).

Japanese name: Gennai-kon-iro-gasa

Notes: Entoloma purpureobrunneolum is similar to E. obscurocyaneum in having a dull dark blue pileus and a bilayered pileipellis. Entoloma purpureobrunneolum differs from E. obscurocyaneum based on its larger basidiospores (> 8 µm; Fig. 5), wider cheilocystidia (> 9 µm; Fig. 6), and the presence of a purple-brown pigment in the suprapellis of the pileipellis.

Entoloma quasicyanonigrum Ohkubo, D. Sakuma & H. Masumoto, sp. nov.

MycoBank no.: MB 855062.　Figs. 5A, 6A, 9.

Fig. 9 - Entoloma quasicyanonigrum. A: Basidioma in situ. B: Sectioned basidioma of Fig. 9A, showing upturned margin and fistulose stipe. C: Basidioma with a convex pileus. D: Sectioned basidioma of Fig. 9C. E: Pileipellis (in water). F: Hyphae of pileipellis (in water). A, B, E, F: OSA-MY-9552 (holotype). C, D: OSA-MY-9560. Bars: B-D 1 cm; E 100 µm; F 10 µm.

Diagnosis: This species is characterized by a dark blue pileus, a hymeniderm pileipellis, and basidiospores usually ≤ 8.5 µm in length.

Holotypus: JAPAN. Nara Pref., Owada-chou, Yata-Momiji-Tani, on soil in a mixed forest of Q. serrata Murray and Q. variabilis Blume, 28 Jul 2020, coll. Y. Ohkubo (specimen ID: OSA-MY-9552), GenBank accession no.: ITS (LC796877) and nrLSU (LC796870).

Etymology: “Quasi-” means “just as.” The epithet refers to its macromorphological resemblance to E. cyanonigrum.

Basidiomata: tricholomatoid. Pileus: 36-75 mm, convex to subumbonate, dark blue, velutinous, radially rugulose over disc, not hygrophanous, margin often slightly upturned; context white. Lamellae: sinuate, subdistant to close, up to 10 mm wide, incarnate, edge serrate. Stipe: 55-70 × 4.5-7.5 mm, almost equal, occasionally incrassate, fibrillose-striate, dark blue, paler at the top, with white base; context white with grayish-blue tint near the surface, fistulose or occasionally stuffed. Odor: slightly fungoid. Taste: bitter, slightly acrid. Basidiospores: (6.7-)7.0-7.9(-8.5) × (5.8-)6.2-6.9(-7.6) μm (n = 50), Q = 1.1-1.2, subisodiametric, 5-6-angled in side-view. Basidia: 39-48 × 10.4-12.8 μm (in KOH/CR), 4-spored, clavate, occasionally with a basal clamp. Lamella edge: heterogeneous, with cystidia mixed with basidia. Cheilocystidia: 15-50 × 5.5-8.5 µm (KOH/CR; based on dried material), irregularly cylindrical. Hymenophoral trama: subparallel, composed of cylindrical hyphae, 13-34 µm wide. Pileipellis: a hymeniderm of 2-3 inflated clavate hyphae in a row, slightly interwoven, terminal hyphae inflated, occasionally pointed, 28-62 × 14-31 μm, hyphae dark blue or hyaline with irregular to granular dark blue pigments. Pileitrama: composed of cylindrical hyphae, hyaline with granular dark blue pigment, 10-29 µm wide. Stipitipellis: a cutis of cylindrical hyphae, 4.4-13 µm wide, hyaline just below the pileus, pale blue to dark blue or hyaline with granular to irregular dark blue pigments in other parts, the ends of hyphae rise in bundles in some places, terminal cells occasionally inflated, apex obtuse. Clamp connections: abundant in hymenium and stipe, scattered in other tissues.

Habitat and distribution: Solitary on the ground in forests dominated by Q. serrata and Q. variabilis. Currently known only from Japan (Nara and Shiga). BLAST search revealed that the ITS sequence of E. quasicyanonigrum (LC796877) was almost identical (99.83% = 587/588 bp) to that of a specimen labeled as “Entolomataceae sp. 1” (LC667087) collected in Ome City, Tokyo, Japan (Shirakawa et al., 2022), suggesting that E. quasicyanonigrum can be widely distributed in the temperate areas of Japan.

Other specimens examined: JAPAN. Nara Pref., Ikoma, Kurondo-no-Mori, on soil covered with fallen leaves of Cryptomeria japonica (L.f.) D.Don, 5 Sep 2021, coll. C. Nomura (OSA-MY-9563); Shiga Pref., Otsu, Gennai pass, on soil in a forest sparsely planted with C. obtusa, 21 Sep 2021, coll. Y. Ohkubo (OSA-MY-9560); ditto, coll. Y. Ohkubo (OSA-MY-9561); ditto, coll. by Y. Ohkubo (OSA-MY-9562).

Japanese name: Kon-iro-ippon-shimeji-modoki

Notes: Entoloma quasicyanonigrum is morphologically similar to E. cyanonigrum, but the former is distinguished from E. cyanonigrum based on its smaller basidiospores (≤ 8.5 µm in length; Fig. 5) and thicker hyphae in the pileipellis (14-31 µm). There are also several other Entoloma spp. with a dark blue pileus. Entoloma quasicyanonigrum differs from E. haastii and E. nitidum, which have a cutis-type pileipellis (Noordeloos, 1992; Noordeloos & Gates, 2012). Entoloma hymenidermum differs from E. quasicyanonigrum in the absence of cheilocystidia, the formation of larger basidiospores (6.8-10.0 × 6.2-8.8 µm) (Largent et al., 2014), and the nrLSU DNA sequence. Entoloma simillimum shares many characteristics with E. quasicyanonigrum but differs in smaller basidia (20-25 × 5-7 µm) and slenderer pileipellis hyphae (4-10 µm wide) (Horak, 1980). The nrLSU sequence of E. quasicyanonigrum showed 97.49% (815/836 bp) homology to the sequence labeled “Entoloma haastii BY21” and formed a moderately supported monophyletic clade in our phylogenetic analysis (Fig. 1). Unfortunately, the specimen information for this sequence is not provided; therefore, we could not compare it to E. quasicyanonigrum.

Key to the species of dark blue tricholomatoid Entoloma known from Japan

1a. Pileipellis unilayered, contains only dark blue pigment ...... 2

1b. Pileipellis bilayered, suprapellis contains brownish pigment, subpellis contains dark blue pigment ...... 3

2a. Basidiospores lager than 8.5 µm in length ...... E. cyanonigrum

2b. Basidiospores up to 8.5 µm in length ...... E. quasicyanonigrum

3a. Suprapellis brownish red to grayish brown, basidiospores up to 8.0 µm in length ...... E. obscurocyaneum

3b. Suprapellis brownish with purplish tints, basidiospores larger than 8.0 µm in length ...... E. purpureobrunneolum

Disclosures

The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.