Accepted Manuscript Taxonomic reconsideration of Tricholoma foliicola (Agaricales, Basidiomycota) based on basidiomata morphology, living culture characteristics, and phylogenetic analyses Naoki Endo, Shuji Ushijima, Eiji Nagasawa, Ryo Sugawara, Yudai Okuda, Kozue Sotome, Akira Nakagiri, Nitaro Maekawa PII: S1340-3540(19)30055-5 DOI: https://doi.org/10.1016/j.myc.2019.07.002 Reference: MYC 458 To appear in: Mycoscience Received Date: 1 February 2019 Revised Date: 23 July 2019 Accepted Date: 24 July 2019 Please cite this article as: Endo N, Ushijima S, Nagasawa E, Sugawara R, Okuda Y, Sotome K, Nakagiri A, Maekawa N, Taxonomic reconsideration of Tricholoma foliicola (Agaricales, Basidiomycota) based on basidiomata morphology, living culture characteristics, and phylogenetic analyses, Mycoscience (2019), doi: https://doi.org/10.1016/j.myc.2019.07.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Short communications 2 3 Taxonomic reconsideration of Tricholoma foliicola (Agaricales, Basidiomycota) based on 4 basidiomata morphology, living culture characteristics, and phylogenetic analyses RI PT 5 6 Naoki Endo a, b*, Shuji Ushijima c, Eiji Nagasawa c, Ryo Sugawara b, Yudai Okuda b, Kozue 7 Sotome a, b, Akira Nakagiri a, b, Nitaro Maekawa a, b 9 a M AN US C 8 Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University, 10 4-101 Koyama, Tottori 680-8553, Japan 11 b Faculty of Agriculture, Tottori University, 4-101 Koyama, Tottori 680-8553, Japan 12 c The Tottori Mycological Institute, Kokoge 211, Tottori 689-1125, Japan 13 * Corresponding author: Fungus/Mushroom Resource and Research Center, Faculty of 15 Agriculture, Tottori University, 4-101, Koyama, Tottori, 680-8553, Japan Tel: +81-857-31-5882 17 Fax: +81-857-31-5888 19 E-mail address: endo_nao@tottori-u.ac.jp (N. Endo) AC C 18 EP 16 TE D 14 20 Text 18 pages; Figures 4. 21 Supplementary materials; 3 Supplementary Tables 1 ACCEPTED MANUSCRIPT 1 ABSTRACT 2 Tricholoma foliicola was taxonomically reevaluated based on analyses of the holotype and 4 newly collected materials. Basidiospores of T. foliicola were irregularly ellipsoid, showing a 5 cyanophilic reaction with or without tubercles. Phylogenetic analyses of the internal 6 transcribed spacer (ITS) and large subunit 28S regions of the fungal nuclear ribosomal RNA 7 gene demonstrated that T. foliicola is a species of Gerhardtia characterized by irregularly 8 shaped basidiospores. Thus, we transferred this species to Gerhardtia foliicola comb. nov. 9 Cultured mycelia of G. foliicola on malt extract agar medium produced cystidia covered with 10 granules and abundant thallic conidia (arthroconidia), with both schizolytic and rhexolytic 11 secession. M AN US C RI PT 3 12 Keywords: 14 Arthroconidia, Lyophyllaceae, rDNA phylogeny, Saprotrophic fungi AC C EP TE D 13 2 ACCEPTED MANUSCRIPT The genus Tricholoma (Fr.) Staude is characterized by a tricholomatoid basidioma; white, 2 smooth, and subglobose to oblong basidiospores; and mostly ectomycorrhizal associations 3 with woody plants (Singer, 1986). However, Tricholoma foliicola Har. Takah., originally 4 described from Japan without molecular phylogenetic analyses, exhibits saprotrophic habits 5 (Takahashi, 2001). Recently, we found that basidiospores of this species had an irregular 6 outline with or without tubercles, and that living mycelium stored at the culture collection of 7 the Fungus/Mushroom Resource and Research Center (FMRC), Faculty of Agriculture, 8 Tottori University, produced numerous thallic conidia; neither feature has ever been observed 9 in the genus Tricholoma. Reschke et al. (2018) also documented that “T. foliicola” specimens 10 sampled from Japan have verrucose basidiospores, and they subsequently suggested that it 11 should be transferred to the genus Gerhardtia; however, they only examined two specimens, 12 without either type materials or sufficient phylogenetic analyses. In the present study, 13 therefore, we taxonomically reevaluated T. foliicola based on basidiomata morphology, living 14 culture characteristics, and molecular phylogeny by analyzing the holotype and newly 15 collected materials. M AN US C D TE 16 RI PT 1 Basidiomata specimens of T. foliicola were sampled from Japan in 2013–2017 (Supplementary Table S1). 18 KPM-NC0007429 were borrowed from the Kanagawa Prefectural Museum of Natural 19 History, (KPM), Japan. Sampled basidiomata were photographed, and mycelia were isolated 20 from the inner tissue of several specimens using 2% malt extract agar (MA) medium. Five 21 established isolates were deposited in the FMRC as Tottori University fungal culture (TUFC) 22 strains, and were used for the microscopic observations described below (Supplementary 23 Table S1). Sampled specimens were then freeze- or air-dried at 45 °C for 1–2 d and deposited 24 as vouchers at the mushroom herbarium of the FMRC (TUMH). Several specimens were also 25 deposited at the Tottori Mycological Institute (TMI). The specimens KPM-NC0007428 (holotype) and AC C EP 17 3 ACCEPTED MANUSCRIPT The surface structure of basidiospores and conidia were observed using a scanning 2 electron microscope (SEM). A small portion of the spore-prints of SU20171026 (TUMH 3 63262) moistened with 50 mM of phosphate-buffered saline (PBS) and an agar plug of 4 isolates were fixed with 1% OsO4 solution, and then substituted with ethanol solution and 5 isoamyl acetate. Subsequently, fixed samples were dehydrated using a critical-point dryer 6 (JCPD-5, JEOL, Tokyo) and coated with Pt using a magnetron sputter coater (MSP-1S, 7 Vacuum Device Inc., Tokyo). The coated samples were inspected under SEM (SU1510, 8 Hitachi, Tokyo). M AN US C RI PT 1 The length (L), width (W), length/width ratio (Q), and mean length (Lm), mean width 10 (Wm), and mean length/width ratio (Qm) of 30 basidiospores from each specimen were 11 measured under a differential interference contrast (DIC) microscope (Eclipse 80i, Nikon 12 Imaging, Tokyo) using 40× and 100× immersion objective lenses. The cyanophilic and 13 siderophilic reactions of basidia were observed under DIC and light microscopy (Eclipse 80i) 14 by staining with cotton-blue dissolved in lactic acid and acetocarmine with Fe3+ (Singer, 15 1986). We also observed the basidiole, hyphal system, pileipellis, stipitipellis, hymenophoral 16 trama, conidia and cultured mycelia to inform species descriptions. Color names in double 17 quotation marks are from the Online Auction Color Chart, i.e., "oac" (Anonymous, 2004) and 18 the Methuen Handbook of Colour (Kornerup & Wanscher, 1967). EP TE D 9 DNA extraction from basidiomata specimens and cultured mycelia, PCR amplification, 20 and cycle sequencing followed Endo et al. (2014, 2015) with minor modification. The ITS 21 and large subunit (LSU) regions of fungal nrDNA were amplified by PCR using the primers 22 ITS1-F/LB-W or CTB6/LR5F (Gardes & Bruns, 1993; Tedersoo et al., 2008). The PCR 23 products were purified using the QIAquick PCR Purification Kit (Qiagen Inc., Hilden, 24 Germany). Cycle sequencing reactions were performed on both forward and reverse strands 25 using a BigDye Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., MA, AC C 19 4 ACCEPTED MANUSCRIPT USA) and the same primer pair with PCR. The reaction products (10 µL) were purified with 2 ethanol and then sequenced using the ABI Prism 3100 Genetic Analyzer (Thermo Fisher 3 Scientific Inc.). The nucleotide sequences obtained for each strand were assembled with 4 Clustal W (Thompson, Higgins, & Gibson, 1994), and the complementarity between the 5 strands was confirmed. The full sequences have been deposited in the DNA Data Bank of 6 Japan (DDBJ; see also Supplementary Table S2). RI PT 1 Molecular phylogenetic analyses were performed using MEGA v7.02 software (Kumar, 8 Stecher, & Tamura, 2016). In the LSU dataset, sequence data of the family Lyophyllaceae 9 from GenBank were included in the analysis (Supplementary Table S2; Moncalvo, Lutzoni, 10 Rehner, Johnson, & Vilgalys, 2000; Hofstetter, Clémençon, Vilgalys, & Moncalvo, 2002; 11 Cooper 2014; Latha, Raj, Cherolil, Sharafudheen, & Manimohan, 2016; Bellanger et al., 12 2015; Vizzini et al., 2015, 2017; Li, Li, Wang, Deng, & Song, 2017; Matheny et al., 2017; 13 Yang, Huang, Zhao, Zeng, & Tang, 2018), because the results of a nucleotide BLAST search 14 of T. foliicola sequences in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed high 15 homology with the species of this family. In the ITS dataset, sequence data of the genus 16 Gerhardtia and related taxa (Calocybella) were included (Supplementary Table S2), because 17 T. foliicola sequences were included in a monophyletic clade with species of Gerhardtia in 18 the LSU phylogeny (see also Results section and Fig. 3). Both the ITS and LSU datasets were 19 aligned using MUSCLE (Edgar, 2004), and were manually refined using Seaview software 20 (http://pbil.univ-lyon1.fr/software/seaview3). The refined alignment was submitted to 21 TreeBase (http://www.treebase.org; Accession No.: S23865 for ITS, S23866 for LSU). 22 Phylogenetic trees, i.e., maximum likelihood (ML) trees, were constructed with MEGA 23 software using a general time-reversible (GTR) + gamma-distributed and invariant site (GI) 24 model for the LSU dataset, and a GTR + gamma-distributed (G) model for the ITS dataset. 25 Model selection was conducted with MEGA software based on values of the Akaike AC C EP TE D M AN US C 7 5 ACCEPTED MANUSCRIPT information criterion (AIC). The gaps/missing data treatment was employed for all sites. The 2 ML bootstrap support (MLBS) values were obtained using nonparametric bootstrapping with 3 1,000 replicates. The generated trees were rooted by Entoloma species in the LSU dataset, 4 and by Myochromella boudieri in the ITS dataset as outgroup taxa (Vizzini et al., 2015; 5 Matheny et al., 2017). The bootstrap support of the neighbor joining bootstrap support (NJBS) 6 tree was also obtained using MEGA for both the LSU and ITS datasets. The Bayesian 7 posterior probability (BPP) of each node was calculated by Bayesian inference (BI) analysis 8 using MrBayes v.3.2.1 software (Ronquist et al., 2012), with the same substitution model as 9 used for the ML analysis (GTR+GI for LSU dataset and GTR+G for ITS dataset, 10 respectively). Two runs with four Markov chain Monte Carlo iterations were performed for 11 1,000,000 generations when the average standard deviation of the split frequencies was below 12 0.01 (the first 25% of generations were treated as burn-in). Trees were kept for every 100 13 generations, and the latter 75% of trees were used to calculate the 50% majority-rule 14 consensus topology, and to determine the BPP for individual branches. The generated ML 15 trees were viewed using MEGA and edited with Microsoft PowerPoint (Microsoft, Redmond, 16 Washington) and Adobe Photoshop (Adobe, San Jose, California). TE D M AN US C RI PT 1 19 Taxonomy AC C 18 EP 17 20 Gerhardtia foliicola (Har. Takah.) N. Endo, S. Ushijima, Nagas., Sotome, Nakagiri, & N. 21 Maek. comb. nov. 22 MycoBank no.: MB 829624. Figs. 1, 2. 23 24 ≡ Tricholoma foliicola Har. Takah. Mycoscience 42(4): 358, 2001 (basionym). 6 ACCEPTED MANUSCRIPT Diagnosis: This species is characterized by a collybioid basidioma, ellipsoid to 2 cylindrical basidiospores sized 3.5–5.5 × 2–3 µm with an irregular outline, fruiting on leaf or 3 bark litter and humus of both coniferous and broad-leaved trees, and production of 4 arthroconidia sized 3.5–10 × 2–5 µm and rare cystidia covered with granules on living 5 cultured mycelium. The morphologically and phylogenetically closest species, G. borealis, is 6 distinguished by slightly distant lamellae and slightly longer basidiospores. RI PT 1 Macromorphology of basidiomata: Basidiomata collybioid (Fig. 1A, B). Pileus 25–40 8 mm in diam., slightly hemispherical to convex when young, margin involute when young, 9 convex to plano convex in aged basidiomes. Pileus surface smooth, not viscid, not glabrous, 10 hygrophanous, not striate at margin; dark brown (oac720 and 638) to brown (oac645) at 11 center, pale brown (oac653) toward margin. Context white, thin. Taste mild. Lamellae white, 12 thin, sinuate to somewhat free or emarginate, densely crowded (3–4 pieces/mm), edges 13 slightly toothed, concolorous. Stipe 40–60 × 5–10 mm, surface white, smooth, silky, clavate, 14 hollow at middle part. D M AN US C 7 Micromorphology of basidiomata: Basidiospores [10 collections and 300 spores] L × W 16 = 3.5–5.5 × 2–3 µm, Q = 1.5–2.5, Lm × Wm = 4–5 × 2.5 µm, Qm = 1.5–2, ellipsoid to 17 subcylindrical or subfusiform with or without tubercles in irregular outline, non-amyloid, 18 thin-walled to slightly thick-walled, cyanophilic, hyaline (Fig. 1C–E). Basidia 15–30 × 3.5– 19 6.5 µm, cylindrical to subclavate with siderophilic granules, 4-spored, smooth, thin-walled to 20 slightly thick-walled, cyanophilic, hyaline (Fig. 1F, G). Basidiole 15–25(–30) × 3–5.5 µm, 21 cylindrical to subclavate with siderophilic granules, smooth, thin-walled to slightly 22 thick-walled, cyanophilic, hyaline (Fig. 1F, G). Pileipellis an ixocutis, hyphae 2–12 µm wide, 23 irregularly entangled, cylindrical, smooth, including intercellular brown pigment (Fig. 1H). 24 Hyphae of pileitrama 9–20 µm wide, irregularly entangled, cylindric to inflated, branched, 25 thin-walled, smooth, hyaline. Stipitipellis a cutis, hyphae 2–5 µm wide, thin-walled, hyaline, AC C EP TE 15 7 ACCEPTED MANUSCRIPT 1 smooth. Stipititrama consisting of parallel, smooth, hyphae 1–13 µm wide, longitudinally 2 running, smooth, thin-walled, hyaline. Hymenophoral trama regular or somewhat parallel, 3 hyphae 5–13 µm wide, smooth, thin-walled, hyaline (Fig. 1I). All septa lack clamp 4 connections. Cultural morphology: Cultured mycelia whitish to pale brownish on MA medium, 6 dense, sometimes loose, aerial hyphae present (Fig. 2A–C). Arthroconidia [five strains and 7 150 conidia] (3–)3.5–10(–12.5) × 2–5(–5.5) µm, length/width ratio 1–5, globose to ellipsoid, 8 sometimes resembling kernels of corn, or rectangle to cylindrical with schizolytically or 9 rhexolytically secession, thin-walled to thick-walled, smooth, hyaline, abundant (Fig. 2D–G). 10 Cystidia 14–23 × 6.5–12.5 µm, clavate to subclavate, covered with granules, thin-walled, 11 hyaline, rare (4 to 5 in each plate; Fig. 2H). Hyphae 1.5–5 µm width, thin-walled to slightly 12 thick-walled, smooth, sometimes forming a bundle, all septa lack clamp connections, hyaline 13 (Fig. 2I). 16 M AN US C D 15 Habitat and distribution: Temperate region of Japan in Jul to Nov, on humus or leaf or bark litter of coniferous or broad-leaved trees. TE 14 RI PT 5 Materials examined: JAPAN, Kanagawa Pref., Yamato-shi, 13 Oct 2000, coll. H. Takahashi (KPM-NC0007428, 18 (KPM-NC0007429); Tottori Pref., Tottori-shi, Kokoge, on humus in Quercus spp. forest, 7 19 Nov 2013, coll. E. Nagasawa (Specimen: TUMH 60927, Culture: TUFC 100780); Hamasaka, 20 on litter in Pinus thunbergii Parl. forest, 01 Nov 2016, coll. S. Ushijima (Specimen: TUMH 21 62816); Karo, on litter in Pinus thunbergii forest, 26 Oct 2017, coll. S. Ushijima (Specimen: 22 TUMH 63262); Houki-cho, on litter in P. thunbergii forest, 26 Sep 2017, coll. S. Ushijima 23 (Specimen: TUMH 62882, Culture: TUFC 101301); Hino-cho, on litter in P. thunbergii forest, 24 10 Oct 2017, coll. S. Ushijima (Specimen: TUMH 63021, Culture: TUFC 101394); Miyagi 25 Pref., Sendai-shi, Aoba-ku, Dainohara, on litter in P. densiflora Sieb. et Zucc. forest, 01 Sep holotype); 02 Jul 2000, coll. H. Takahashi AC C EP 17 8 ACCEPTED MANUSCRIPT 2017, coll. R. Sugawara (Specimen: TUMH 63019, Culture: TUFC 101392); Rifu-cho, on 2 litter in P. densiflora forest, 03 Sep 2017, coll. R. Sugawara (Specimen: TUMH 63020, 3 Culture: TUFC 101393); Yamanashi Pref., Minamitsuru-gun, Narusawa-mura, on litter in 4 Tsuga diversifolia (Maxim.) Mast. and Pinus parviflora Sieb. et Zucc. forest, 06 Sep 2018, 5 coll. R. Sugawara (Specimen: TUMH 63314 and TUMH 63315). RI PT 1 Note: This is the first report of a species of the genus Gerhardtia from Japan. 7 Measurement data of basidiospores and basidia for each specimen and conidia from each 8 culture are shown in Supplementary Table S3. Basidiospores and basidia of G. foliicola 9 specimens examined in this study corresponded in size with the original descriptions of “T. 10 foliicola” published by Takahashi (2001); however, our specimens showed irregularly shaped 11 spore-walls with a cyanophilic reaction, and basidia with both cyanophilic and siderophilic 12 reactions were reported for the genus Gerhardtia in family Lyophyllaceae (Bon, 1994; Vizzini 13 et al., 2015, 2017; Matheny et al., 2017). Although the generic concept of Gerhardtia has 14 been refined as having smooth or verruculose basidiospores, clampless hyphae, and a cutis, 15 trichodermium, or hymeniderm pileipellis (Vizzini et al., 2015), due to the discovery of G. 16 pseudosaponacea J.A. Cooper & P. Leonard, which has smooth basidiospores (Cooper, 2014), 17 the morphological characteristics of G. foliicola fully correspond to this generic concept. EP TE D M AN US C 6 The phylogenetic tree inferred from the LSU dataset (Fig. 3) demonstrated that Japanese 19 G. foliicola specimens and cultures formed a single clade and grouped with the species of 20 Gerhardtia among family Lyophyllaceae with strong support (MLBS/NJBS/BPP: 96/97/0.97). 21 Gerhardtia borealis (Fr.) Contu & A. Ortega, the type species of the genus, formed a 22 monophyletic clade with G. foliicola with strong support (93/96/1). Gerhardtia highlandensis 23 (Hesler & A.H. Sm.) Consiglio & Contu, and G. sinensis T.H. Li, T. Li & W.Q. also grouped 24 with G. foliicola and G. borealis with strong support (93/96/1). The phylogenetic tree inferred 25 from the ITS dataset (Fig. 4) also strongly supported both groupings of Japanese G. foliicola AC C 18 9 ACCEPTED MANUSCRIPT (94/99/1) and species of Gerhardtia (80/79/1). Gerhardtia borealis formed a sister clade with 2 Japanese G. foliicola, but showed distance. Other Gerhardtia species, G. highlandensis, G. 3 sinensis, G. cibaria (Singer) Matheny, Sánchez-García & T.J. Baroni, and G. citrinolobata 4 Angelini & Vizzini were distinct from Japanese G. foliicola. Reschke et al. (2018) also 5 suggested that “T. foliicola” should be transferred to the genus Gerhardtia, their supposition 6 lacked sufficient molecular analyses and was founded on examination of limited specimens. 7 In contrast, our present results strongly demonstrated that T. foliicola belongs in the genus 8 Gerhardtia. M AN US C RI PT 1 Gerhardtia borealis [= G. incarnatobrunnea (Ew. Gerhardt) Bon], originally described 10 from Germany, is the morphologically and phylogenetically closest species to G. foliicola; 11 this species exhibits slightly distant lamellae and longer basidiospores compared to G. 12 foliicola (Gerhardt, 1982). Other Gerhardtia species, G. cibaria, G. sinensis, G. 13 highlandensis, G. leucopaxilloides (H.E. Bigelow & A.H. Sm.) Consiglio & Contu, G. 14 piperata (A.H. Sm.) Bon, G. pseudosaponacea and G. suburens (Clémençon) Consiglio & 15 Contu, are macromorphologically and micromorphologically distinct from G. foliicola 16 (Bigelow & Smith, 1969; Consiglio & Contu, 2004; Mešić & Tkalčec, 2009; Cooper, 2014; Li 17 et al., 2017; Matheny et al., 2017). EP TE D 9 This is the first report of pure cultures of G. foliicola. Pure culture isolates and parent 19 basidioma exhibited identical sequences. We found numerous arthroconidia on cultured 20 mycelia of G. foliicola, a characteristic that has never been observed in the genus Tricholoma 21 (Walther et al., 2005). On the other hand, various Lyophyllaceae genera, i.e., Arthromyces, 22 Asterophora, Blastosporella, Calocybe, Hypsizygus, Fibulochlamys, Ossicaulis, Sagaranella, 23 Sphagnurus, and Termitomyces have anamorphic life cycles at the stages of vegetative 24 mycelium or basidiomata (Singer, 1986; Brunner & Miller, 1988; Nagasawa & Arita, 1988; 25 Moncalvo, Rehner, & Vilgalys, 1993; Walther et al., 2005; Baroni et al., 2007; Madrid, Cano, AC C 18 10 ACCEPTED MANUSCRIPT Stchigel, Gené, & Guarro, 2010). In the genus Gerhardtia, an anamorphic stage has been 2 reported in G. suburens and G. leucopaxilloides cultures (Clémençon, 1968; presented as 3 Lyophyllum suburens and L. leucopaxilloides). Although arthroconidia of G. suburens and G. 4 leucopaxilloides cultures have been reported as “chlamydospores” by Clémençon (1968), 5 their morphological characteristics and production systems, as illustrated by line drawings, 6 clearly indicated that they are thick-walled arthroconidia. Because we also found thick-walled 7 arthroconidia in G. foliicola in the present study, the presence of thick-walled arthroconidia on 8 cultured hyphae may be a significant character of the genus Gerhardtia. M AN US C RI PT 1 The arthroconidia of G. foliicola showed both schizolytic and rhexolytic secession. The 10 line drawing by Clémençon (1968) indicated that the arthroconidia of both G. suburens and G. 11 leucopaxilloides also exhibited both schizolytic and rhexolytic secession. In contrast, the 12 arthroconidia of Hypsizygus and Ossicaulis showed only schizolytic secession (Nagasawa & 13 Arita, 1988 and our unpublished data). We will perform further studies to determine if these 14 differences in the secession mode of arthroconidia in Lyophyllaceae relate to the taxonomic 15 affiliation (e.g., subfamily, genus, or species) of targeted fungal species. TE D 9 Cultures of G. foliicola produced cystidia covered with granules. To our knowledge, the 17 production of cystidia on cultured mycelia has not been reported for other species. Because 18 the frequency of cystidia was relatively low in cultures of G. foliicola, it may have been 19 overlooked in other Lyophyllaceae species. Thus, we will perform further comprehensive 20 study of the presence of cystidia, as well as anamorphic conidia productivity and its 21 conidiogenesis on living cultures of other Lyophyllaceae species, including other species of 22 Gerhardtia. AC C EP 16 23 24 Disclosure 11 ACCEPTED MANUSCRIPT 1 The authors declare no conflicts of interest. All the experiments undertaken in this study 2 comply with the current laws of the country where they were performed. 3 Acknowledgments 5 This study is supported in part large research grant from Institute for Fermentation, Osaka 6 (IFO). We great thank Takamichi Orihara, Kanagawa Prefectural Museum of Natural History 7 for loaning holotype specimen. We also thank the staff of the Division of Instrumental 8 Research, Research Center for Supports to Advanced Science, Shinshu University, for 9 technical support with DNA sequencing. 10 M AN US C RI PT 4 11 References 12 Anonymous. (2004). The Online Auction Color Chart. Stanford: The Online Auction Color 13 Chart Company. Baroni, T. J., Franco-Molano, A. E., Lodge, J., Linder, D. L., Horak, E., & Hofstetter, V. 15 (2007). Arthromyces and Blastosporella, two new genera of conidia-producing 16 lyophylloid agarics (Agaricales, Basidiomycota) from neotropics. Mycological Research, 17 111, 572–580. http://doi.org/10.1016/j.mycres.2007.03.007. EP TE D 14 Bellanger, J. M., Moreau, P. A., Corriol, G., Bidaud, A., Chalange, R., Dudova, Z., et al. 19 (2015). Plunging hands into the mushroom jar: a phylogenetic framework of 20 Lyophyllaceae 21 http://doi.org/10.1007/s10709-015-9823-8. 22 23 24 25 AC C 18 (Agaricales, Basidiomycota). Genetica, 143, 169–194. Bigelow, H. E., & Smith, A. H. (1969). The status of Lepista–a new section of Clitocybe. Brittonia, 21, 144–177. http://doi.org/10.2307/2805523. Bon, M. (1994). Deux Lyophylloideae intéressantes et le genere Gerhardtia st. et nom. nov. (in French). Documents Micologiques, 24, 65–68. 12 ACCEPTED MANUSCRIPT 1 2 Brunner, I. L., & Miller Jr., O. K. (1988). Calocybe fallax: its ecology and cultural behavior. Mycotaxon, 33, 41–50. Consiglio, G., & Contu, M. (2004). The genus Gerhardtia (Lyophyllaceae) in Europe, with 4 notes on the remaining known species. Micologia e vegetazione mediterranea 19, 151– 5 162. RI PT 3 Cooper, J. A. (2014). New species and combinations of some New Zealand agarics belonging 7 to Clitopilus, Lyophyllum, Gerhardtia, Clitocybe, Hydnangium, Mycena, Rhodocollybia 8 and Gerronema. Mycosphere, 5, 263–288. http://doi.org/10.5943/mycosphere/5/2/2. 9 Clémençon, H. (1968). Contributions to the knowledge of the genera Lyophyllum and 10 Calocybe (Agaricales, Basidiomycetes) III. Two chlamydospore forming Lyophyllum 11 species. Nova Hedwigia, 16, 417–427. 12 M AN US C 6 Edgar, R. C. (2004). MUSCLE: a multiple sequence alignment method with reduced time and 13 space complexity. 14 http://doi.org/10.1186/1471-2105-5-113. Bioinformatics, 5, 113. D BMC Endo, N., Kawamura, F., Kitahara, R., Sakuma, D., Fukuda, M., & Yamada, A. (2014). 16 Synthesis of Japanese Boletus edulis ectomycorrhizae with Japanese red pine. 17 Mycoscience, 55, 405–416. http://doi.org/10.1016/j.myc.2013.11.008. EP TE 15 Endo, N., Dokmai, P., Suwannasai, N., Phosri, C., Horimai, Y., Hirai, N., et al. (2015). 19 Ectomycorrhization of Tricholoma matsutake with Abies veitchii and Tsuga diversifolia 20 in 21 https://doi.org/10.1016/j.myc.2014.12.004. AC C 18 the subalpine forests of Japan. Mycoscience, 56, 402–412. 22 Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes 23 –application to the identification of mycorrhizae and rusts. Molecular Ecology, 2, 113– 24 118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x. 13 ACCEPTED MANUSCRIPT 1 Gerhardt, E. (1982). Über zwei neue Tricholomataceen: Collybia hebelomoides und 2 Lyophyllum incarnatobrunneum, gefunden in Berlin. Zeitschrift für Mykologie, 48, 239– 3 243. Hofstetter, V., Clémençon, H., Vilgalys, R., & Moncalvo, J. M. (2002). Phylogenetic analyses 5 of the Lyophylleae (Agaricales, Basidiomycota) based on nuclear and mitochondrial 6 rDNA 7 http://doi.org/10.1017/S095375620200641X. 9 Mycological Research, 106, 1043–1059. Kornerup, A., & Wanscher, J. H. (1967). Methuen handbook of colour, 2nd edn. London: M AN US C 8 sequences. RI PT 4 Methuen Publishing. 10 Kumar, S., Stecher, G., & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics 11 Analysis Version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870– 12 1874. https://doi.org/10.1093/molbev/msw054. Latha, K. P. D., Raj, K. N. A., Cherolil, T., Sharafudheen, S. A., & Manimohan, P. (2016). 14 Three new species of Calocybella from India based on morphology and molecular 15 phylogeny. Phytotaxa, 255, 133–143. http://doi.org/10.11646/phytotaxa.255.2.2. TE D 13 Li, T., Li, T., Wang, C., Deng, W., & Song, B. (2017). Gerhardtia sinensis (Agaricales, 17 Lyophyllaceae), a new species and newly recorded genus for China. Phytotaxa, 332, 18 172–180. http://doi.org/10.11646/phytotaxa.332.2.4. EP 16 Madrid, H., Cano, J., Stchigel, A., Gené, J., & Guarro, J. (2010). Ramophialophora humicola 20 and Fibulochlamys chilensis, two new microfungi from soil. Mycologia, 102, 605–612. 21 http://doi.org/10.3852/09-128. AC C 19 22 Matheny, P. B., Baroni, T. J., Simoni, A., Holgado Rojas, M., E., Sánchez-García, M., & Gates, 23 G. M. (2017). The wild edible mushroom Pleurocollybia cibaria from Peru is a species 24 of Gerhardtia in the Lyophyllaceae (Agaricales). Cryptogamie, Mycologie, 38, 205–212. 25 http://doi.org/10.7872/crym/v38.iss2.2017.205. 14 ACCEPTED MANUSCRIPT 1 2 Mešić, A., & Tkalčec, Z. (2009). Studies on Croatian Basidiomycota 1: Gerhardtia piperata (Agaricales). Mycotaxon, 110, 413–421. http://doi.org/10.5248/110.413. Moncalvo, J. M., Lutzoni, F. M., Rehner, S. A., Johnson, J., & Vilgalys, R. (2000). 4 Phylogenetic relationship of agaric fungi based on nuclear large subunit ribosomal DNA 5 sequences. Systematic Biology, 49, 278–305. http://doi.org/10.1093/sysbio/49.2.278. RI PT 3 Moncalvo, J. M., Rehner, S. A., & Vilgalys, R. (1993). Systematics of Lyophyllum section 7 Difformia based on evidence from culture studies and ribosomal DNA sequences. 8 Mycologia, 85, 788–794. http://www.jstore.org/stable/3760610. 9 10 M AN US C 6 Nagasawa, E., & Arita, I. (1988). A note on Hypsizygus ulmarius and Hypsizygus marmoreus in Japan. Reports of the Tottori Mycological Institute, 26, 71–78. 11 Reschke, K., Popa, F., Yang, Z. L., & Kost, G. (2018). Diversity and taxonomy of Tricholoma 12 species from Yunnan, China, and notes on species from Europe and North America. 13 Mycologia, 110, 1089–1109. http://doi.org/10.1080/00275514.2018.1512295. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., et al. 15 (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice 16 across 17 http://doi.org/10.1093/sysbio/sys029. 19 20 21 TE large model space. Systematic Biology, 61, 539–542. EP a Singer, R. (1986). The Agaricales in modern taxonomy, 4th edn. Koenigstein: Koeltz Scientific Books. AC C 18 D 14 Takahashi, H. (2001). Notes on new Agaricales of Japan 3. Mycoscience, 42, 355–360. http://doi.org/10.1007/BF02461218. 22 Tedersoo, L., Jairus, T., Horton, B. M., Abarenkov, K., Suvi, T., Saar, I. et al. (2008). Strong 23 host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as 24 revealed by DNA barcoding and taxon-specific primers. New Phytologist, 180, 479– 25 490. http://doi.org/10.1111/j.1469-8137.2008.02561.x. 15 ACCEPTED MANUSCRIPT Thompson, D. J., Higgins, D. G., & Gibson, T. (1994). CLUSTALW: Improving the 2 sensitivity of progressive multiple sequence alignment through sequence weighting, 3 position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4 4673–4680. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC308517. Accessed in 6 5 Nov 2018. RI PT 1 6 Vizzini, A., Angelini, C., & Ercole, E. (2017). Is the species diversity in the lyophylloid 7 genera Calocybella and Gerhardtia (Agaricales, Basidiomycota) underestimated? Two 8 new 9 http://doi.org/10.11646/phytotaxa.291.4.1. from the Dominican Republic. Phytotaxa, M AN US C species 291, 241–252. 10 Vizzini, A., Consiglio, G., Setti, L., & Ercole, E. (2015). Calocybella, a new genus for 11 Rugosomyces pudicus (Agaricales, Lyophyllaceae) and emendation of the genus 12 Gerhardtia. IMA Fungus, 6, 1–11. http://doi.org/10.5598/imafungus.2015.06.01.01. 13 Walther, G., Garnica, S., & Weiss, M. (2005). The systematic relevance of conidiogenesis 14 modes the gilled Agaricales. 15 http://doi.org/10.1017/S0953756205002868. Mycological Research, 109, 525–544. TE D in Yang, S. D., Huang, H. Y., Zhao, J., Zeng, N. K., & Tang, L. P. (2018). Ossicaulis 17 yunnanensis sp. nov. (Lyophyllaceae, Agaricales) from southwestern China. 18 Mycoscience, 59, 33–37. http://doi.org/10.1016/j.myc.2017.07.008. 20 21 AC C 19 EP 16 Figure legends 22 Fig. 1. Basidiomata morphology of Gerhardtia foliicola. A: Raw basidiomata (TUMH 63021). 23 B: Dried holotype (KPM-NC0007428). C: Scanning electron micrograph of basidiospore 24 (TUMH 63262). D, E: Differential interference contrast (DIC) micrograph of basidiospores 25 stained with cotton-blue (D: TUMH 63262; E: KPM-NC0007428). F: DIC micrograph of 16 ACCEPTED MANUSCRIPT 1 basidia stained with acetocarmine–Fe3+ (TUMH 63262). H: DIC micrograph of pileipellis 2 (TUMH 62816). I: DIC micrograph of hymenophoral trama (TUMH 62816). Bars: C 5 µm; 3 D–G 10 µm; H 30 µm; I 50 µm. 4 Fig. 2. Culture morphology of Gerhardtia foliicola. A, B: Colony on malt extract agar (MA) 6 plate (TUFC 101301). C: Dissection micrograph of aerial hyphae with producing conidia 7 (TUFC 101301). D: Scanning electron micrograph (SEM) of arthroconidia with schizolytic 8 sessesion (TUFC 101301). E: SEM of arthroconidia with rhezolytic sessesion (TUFC 101392). 9 F: Differential interference contrast (DIC) micrograph of arthroconidia with schizolytic 10 sessesion (TUFC 101301). G: DIC micrograph of arthroconidia with rhezolytic sessesion 11 (TUFC 101301). H: DIC micrograph of cystidia of cultured mycelium (TUFC 101392). I: 12 DIC micrograph of the hyphal bundle of cultured mycelium (TUFC 101392). Bars: A, B 1 13 cm; C 100 µm; D, E 5 µm; F–I 10 µm. D 14 M AN US C RI PT 5 Fig. 3. Maximum likelihood (ML) tree of the family Lyophyllaceae inferred from the LSU 16 sequence. The two values at each node represent the percentage of ML bootstrap support 17 (MLBS)/neighbor joining bootstrap support (NJBS)/Bayesian posterior probability (BPP). 18 The thick node indicates the significantly supported branch in at least two analyses (MLBS ≥ 19 70%, NJBS ≥ 70%, or BPP ≥ 0.95). Each sample name following the species epithet is given 20 as the specimen or culture ID in this study, or the DNA Data Bank of Japan (DDBJ) accession 21 number. AC C EP TE 15 22 23 Fig. 4. Maximum likelihood (ML) tree of the genus Gerhardtia and related taxa inferred by 24 ITS sequence. The two values at each node represent the percentage of ML bootstrap support 25 (MLBS)/neighbor joining bootstrap support (NJBS)/Bayesian posterior probability (BPP). 17 ACCEPTED MANUSCRIPT The thick node indicates the significantly supported branch in at least two analyses (MLBS ≥ 2 70%, NJBS ≥ 70%, or BPP ≥ 0.95). Each sample name following the species epithet is given 3 as the specimen or culture ID in this study, or the DNA Data Bank of Japan (DDBJ) accession 4 number. AC C EP TE D M AN US C RI PT 1 18 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Highlights 2 (1) Tricholoma foliicola forms irregularly-shaped basidiospores. 3 (2) This species phylogenetically grouped with species of genus Gerhardtia. 4 (3) We re-described this species as G. foliicola comb. nov. 5 (4) Cultured mycelia of G. foliicola produced conidia and cystidia on nutrient agar medium. AC C EP TE D M AN US C RI PT 1 1