Mycoscience
Online ISSN : 1618-2545
Print ISSN : 1340-3540
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Two new Mycena section Calodontes species: One newly discovered and the other new to Japan
Kosuke Nagamune, Kentaro Hosaka, Shiro Kigawa, Ryo Sugawara, Kozue Sotome, Akira Nakagiri, Naoki Endo
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Keywords: molecular phylogeny, morphology, Mycenaceae, new to Japan, 1 new taxon
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2024 Volume 65 Issue 3 Pages 111-122

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Abstract

In 2017, two candidate species of Mycena were reported from Japan, with the Japanese names “Togari-sakura-take” and “Mitsuhida-sakura-take”. However, to date, no taxonomic study or formal description has been undertaken for these two species. In the present study, we conducted comprehensive morphological and molecular phylogenetic examinations of “Togari-sakura-take” and “Mitsuhida-sakura-take”, and compared them to known species within the genus Mycena. We performed phylogenetic analyses on a concatenated dataset, including the internal transcribed spacer region of ribosomal RNA, RNA polymerase II largest subunit, and translation elongation factor-1 alpha genes. “Togari-sakura-take” formed a clade with Mycena subulata, which was recently described from China, whereas “Mitsuhida-sakura-take” formed a distinct independent clade. We identified the former as M. subulata based on molecular phylogenetic analyses and morphological observations. However, the Japanese specimens displayed dextrinoid cheilocystidia and caulocystidia as well as the inamyloidity of basidiospores, which differed from the original description of M. subulata based on the materials from China. “Mitsuhida-sakura-take” was characterized by its remarkably dense lamellae and could be distinguished from known Mycena species by the combination of absent pleurocystidia and presence of bowling pin-shaped cheilocystidia. Here, we describe “Mitsuhida-sakura-take” as a new species, named Mycena densilamellata, in the section Calodontes.

1. Introduction

The genus Mycena (Pers.) Roussel belongs to the family Mycenaceae Overeem and order Agaricales Underw. (Wijayawardene et al., 2020). This genus is one of the species-rich taxa within Agaricales, with approximately 600 valid species, according to the Index Fungorum database (http://www.indexfungorum.org/; accessed on Dec 5 2023) and recent studies on the phylum Basidiomycota (e.g., He et al., 2019). In Japan, approximately 70 Mycena species have been reported to date (Cha et al., 2010; Katumoto, 2010; Shirayama, 2010; Terashima et al., 2016). Mycena typically produces small to medium-sized mycenoid, omphalinoid, or collybioid basidioma, and possesses smooth or branched-cheilocystidia and pleurocystidia, diverticulate, and less frequently smooth hyphae of pileipellis. In addition, the lamella trama appears vinaceous to purplish brown (dextrinoid) upon staining with Melzer's reagent, whereas basidiospores are generally amyloid (Maas Geesteranus, 1980). Similar to other agarics, the taxonomy of Mycena is undergoing a transition from conventional research methods relying heavily on the morphological characteristics of basidiomata (Maas Geesteranus, 1980) to approaches focusing more on molecular phylogenetic analyses (Harder et al., 2010; Liu et al., 2022).

Mycena species have diverse lifestyles, primarily as saprophytes, while certain species are recognized as orchid mycorrhizal symbionts or plant pathogens (Kitahara et al., 2022; Krishnan, 2017). Some Mycena species are known to invade and associate with mosses (Davey et al., 2013). Recent insights into the role of Mycena species as symbionts of plants have been provided through the inoculation of cultured strains onto their host plants (Thoen et al., 2020) as well as field observations (Harder et al., 2023). Therefore, further taxonomic studies incorporating ecology are needed to understand the species diversity of the genus Mycena.

Mycena section Calodontes (Fr. ex Berk.) Quél. sensu Maas Geesteranus (1989) generally produces basidioma with raphanoid odor and diverse colors (frequently with purplish or violaceous tints), as well as smooth cheilocystidia, pleurocystidia, and smooth hyphae of pileipellis (Maas Geesteanus, 1989). This section is divided into four subsections according to their morphological characteristics (Maas Geesteranus, 1989; Maas Geesteranus & de Meijer, 1997). Subsection Violacellae Sing. ex Maas Geest. lectotypified by M. violacella (Speg.) Singer [=Poromycena violacella (Speg.) Singer], is unique in having inamyloid basidiospores and lacking pleurocystidia. Subsection Purae (Konr. and Maubl) Maas Geest., lectotypified by M. pura (Pers.) P. Kumm., is characterized by the presence of amyloid basidiospores, cheilocystidia, and commonly has pleurocystidia with colorless contents. Subsection Marginatae J.E. Lange, lectotypified by M. pelianthina (Fr.) Quél., also has amyloid basidiospores but is unique among the various validated subsections in forming cheilocystidia and pleurocystidia with purplish brown contents. Subsection Generosae Maas Geest. & de Meijer, type species, M. generosa, also produces amyloid basidiospores but is distinct as it lacks pleurocystidia and has cheilocystidia with much narrow necks, which are not broadly rounded at the apex.

Harder et al. (2010) attributed almost 30 species to sect. Calodontes. Since then, several taxonomic studies from Asia have proposed taxonomic revisions, and around 10 taxa have been added to this section: M. cahaya A.L.C. Chew & Desjardin, M. polycystidiata Z.W. Liu, Y.P. Ge, L. Zou & Q. Na, M. rufobrunnea Z.W. Liu, Y.P. Ge & Q. Na, M. seminau A.L.C. Chew & Desjardin, M. shengshanensis Z.W. Liu, Y.P. Ge & Q. Na, M. sinar A.L.C. Chew & Desjardin, M. sirayuktha Aravind. & Manim., M. subulata Z.W. Liu, Y.P. Ge & Q. Na, M. yuezhuoi Z.W. Liu, Y.P. Ge & Q. Na, and a new variety M. sinar var. tangkaisinar A.L.C. Chew & Desjardin (Aravindakshan & Manimohan, 2015; Chew et al., 2014; Liu et al., 2021, 2022). Despite the description of multiple new species from Asia, only four, i.e., M. pelianthina, M. pura, M. rosea Gramberg, and M. subaquosa A.H. Sm., have been reported from Japan (Hongo, 1953; Imai, 1938; Kudo & Nagasawa, 2009, 2017; Murata, 1979). Therefore, additional species are expected to be found in Japan.

Kigawa (2017) introduced two Mycena species, “Mycena sp.”-1 and “Mycena sp.”-2, which likely belong to sect. Calodontes based on their morphology. Kigawa (2017) suggested that these were new species and gave “Mycena spp.”- 1 and 2 the Japanese names of “Togari-sakura-take” due to its awl-shaped cheilocystidia and “Mitsuhida-sakura-take” due to its crowded lamellae, respectively. The taxonomic positions of “Togari-sakura-take” and “Mitsuhida-sakura-take” are unclear.

The present study evaluated the taxonomic position of “Togari-sakura-take” (“Mycena sp.”-1) and “Mitsuhida-sakura-take” (“Mycena sp.”-2) described by Kigawa (2017) from Japan at the species and subsection levels. To this end, we used the two specimens examined by Kigawa (2017), which are stored at the Hiratsuka City Museum, and our newly collected specimens from various areas in Japan. We compared the morphology between these two species and other species of the Mycena section Calodontes. We performed phylogenetic analyses using the DNA sequences of the internal transcribed spacer (ITS) region of ribosomal RNA, RNA polymerase II largest subunit (RPB1), and translation elongation factor-1 alpha (TEF1) genes.

2. Materials and methods

2.1. Collection of basidiomata specimens and cultures

Each three basidiomata of “Mycena spp.”- 1 and 2 were collected from five areas of Japan between Sep and Oct of 2019-2021 (Table 1). Fresh basidiomata were examined macroscopically, air-dried at 45 °C for 1-2 d, and stored. To preserve specimens in a good condition for a long period with a minimum damage, we obtained five cultures that can be used repeatedly for destructive DNA analyses on 1.5% malt extract agar medium containing 1.5% malt extract (Oriental Yeast Co., Ltd., Tokyo, Japan) and 1.5% agar (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) from each fresh basidioma. The dried specimens and cultures were deposited in the Fungus/Mushroom Resource and Research Center (FMRC), Tottori University (Table 1). Fungal strains deposited were cryopreserved in a vapor-phase liquid nitrogen tank at −190 °C. Deposition and utilization of Tottori University Fungal Culture Collection (TUFC) strains were supported by FMRC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (http://nbrp.jp). We also examined two specimens housed at the fungal herbarium of National Museum of Nature and Science (TNS; TNS-F-75029 and TNS-F-75058) for morphological and phylogenetic analyses. Furthermore, we assessed the morphology of the two specimens examined by Kigawa (2017) that were housed at the Hiratsuka City Museum (HCM-58-6667 and HCM-58-6665) (Table 1).

Table 1 - Mycena specimens examined in this study.

SpeciesSpecimen number in herbariumStrain number in culture collectionLocality in JapanCollection dateVegetation a
“Mycena sp.”-1TUMH 65483TUFC 102001Daisen Town, Tottori Pref.11 Sep 2021Fagus crenata
TUMH 65484TUFC 102002Tottori City, Tottori Pref.19 Sep 2021Chamaecyparis obtusa, Betula grossa, Acer sp.
TUMH 65485TUFC 102003Kotoura Town, Tottori Pref.20 Sep 2021Aesculus turbinata, F. crenata, Acer sp.
TNS-F-75058Chichibu City, Saitama Pref.28 Sep 2015ND
HCM-58-6665Gotenba City, Shizuoka Pref.13 Sep 2009ND
“Mycena sp.”-2TUMH 65481TUFC 101999Ebetsu City, Hokkaido14 Sep 2019Picea spp., Betulaceae
TUMH 65486Narusawa Village, Yamanashi Pref.8 Oct 2020Quercus crispula, F. crenata, C. obtusa
TUMH 65482TUFC 102000Tottori City, Tottori Pref.19 Sep 2021C. obtusa, Quercus serrata, Carpinus sp.
TNS-F-75029Chichibu City, Saitama Pref.28 Sep 2015ND
HCM-58-6667Gotenba City, Shizuoka Pref.13 Sep 2009ND

a ND: No data

2.2. Morphological observations of basidiomata

The fresh basidiomata in the fields were photographed, and their forest habitats were recorded. The Online Auction Color Chart (Kramer, 2004) was used as the color standard. Microscopic characters of dried specimens were observed in Melzer's reagent, 3% potassium hydroxide or distilled water using a differential interference contrast microscope (Eclipse 80i, Nikon Co., Tokyo, Japan). The measurements of basidia [not including sterigmata (apical processes for basidiospore production)], basidioles, basidiospores, and cheilocystidia were shown as “(a)b-c(d)”, where (a) indicates the 5th percentile, (b) indicates the average - standard deviation (SD), (c) indicates the average + SD, and (d) indicates the 95th percentile. We measured the minimum and maximum values of sterigmata and caulocystidia. The hyphae length/width are shown as ‘minimum-maximum. We measured 50 basidiospores and 30 basidia, basidioles, and cheilocystidia each. Because certain Mycena species in sect. Calodontes showed a weak amyloid reaction on their basidiospores, we determined the dyeability of basidiospores under differential interference contrast and normal mode light microscopes after treatment with Melzer's reagent for 20 min. Q and its average value (Qm) were calculated from the length/width ratio of basidiospores. We also observed pileipellis, stipitipellis, and structures of hymenophoral trama.

2.3. DNA extraction, PCR amplification, and sequencing

Genomic DNA for PCR amplification was extracted from dried basidioma or living cultures following the method described by Hosaka and Nam (2023) or the microwave method described by Izumitsu et al. (2012). We amplified the full length of the ITS (ITS1-5.8S-ITS2) region and portions of the RPB1 and TEF1 genes. We used Dream Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) for PCR amplification with the following primer pairs: ITS1F and LR5 for ITS region (Gardes & Bruns, 1993; Vilgalys & Hester, 1990), RPB1Mp_f1 and RPB1Mp_r1 for RPB1 gene (Harder et al., 2013), and tEFMp_f1 and tEFMp_r1 or EF526f/EF595f and EF1567r for TEF1 gene (Harder et al., 2013; Kauserud & Schumacher, 2001; Rehner & Buckley, 2005). PCR was performed using the standard protocol: 35 cycles at 95 °C for 30 s, 49.5 °C for 30 s, and 72 °C for 100 s in the ITS1F and LR5 primer pair; 35 cycles at 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 60 s in the RPB1MP_f1 and RPB1MP_r1 primer pair; 35 cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s in tEFMP_f1 and tEFMP_r1 primer pair; 35 cycle of 95 °C for 30 s, 51 °C for 30 s, and 72 °C for 60 s in the EF526f and EF1567r primer pair. For the PCR amplifications of TEF1 gene using the primer pair EF595f and EF1567r, we used touchdown protocols (annealing temperature lowered by 1 °C per cycle), with the following PCR protocol: 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and directly sequenced using the same pair of bidirectional primers for TEF1 and RPB1 genes or ITS5 and ITS4 primers (White et al., 1990) for the ITS region. The nucleotide sequences were analyzed by FASMAC Co., Ltd. (http://fasmac.co.jp), and subsequent phylogenetic analyses of the sequencing data were performed using Bioedit v. 7.2.5 (Hall, 1999). The new sequences were deposited into the GenBank database with accession numbers (LC777686-LC777693, LC777726-LC777741) through the DNA Data Bank of Japan (Table 2).

Table 2 - DNA sequences used for phylogenetic analyses.

Taxon name aSpecimen/Isolate numbersLocalityTypeGenBank accession numbers bReference b
ITSRPB1TEF1ITSRPB1, TEF1
M. aff. holoporphyraDED 8334AfricaMH414547NDNDCooper, 2018ND
M. aff. puraTL8052EcuadorFN394623KF723687KF723641Harder et al., 2010Harder et al., 2013
M. aff. puraTL9433EcuadorFN394622KF723688KF723642Harder et al., 2010Harder et al., 2013
M. aff. puraTL9450EcuadorKJ144653KF723689KF723643Harder et al., 2010Harder et al., 2013
M. aff. puraTL9678EcuadorFN394621KF723690KF723644Harder et al., 2010Harder et al., 2013
M. brunneaCBH386DenmarkFN394565KF723679KF723633Harder et al., 2010Harder et al., 2013
M. brunneaCBH187SwedenFN394564KF723678KF723632Harder et al., 2010Harder et al., 2013
M. brunneaM0160144SwedenHolotypeKT222187NDNDOlariaga et al., 2015ND
M. brunneoviolaceaBAP594AfricaHolotypeMH414546NDNDCooper, 2018ND
M. cahayaKLU M 1221MalaysiaHolotypeKF537248NDNDChew et al., 2014ND
M. densilamellataTUFC 101999JapanEx-holotypeLC777686LC777726LC777734This studyThis study
M. densilamellataTUMH 65486JapanLC777687LC777727LC777735This studyThis study
M. densilamellataTUMH 65482JapanLC777688LC777728LC777736This studyThis study
M. densilamellataTNS-F-75029JapanLC777689LC777729LC777737This studyThis study
M. diosmaLK1191/2002GermanyFN394619KF723700KF723654Harder et al., 2010Harder et al., 2013
M. diosmaCBH400DenmarkFN394617KF723699KF723653Harder et al., 2010Harder et al., 2013
M. dura10315AustriaHolotypeFN394560KF723694KF723648Harder et al., 2013Harder et al., 2013
M. duraARAN-Fungi 1387SpainKT222189NDNDOlariaga et al., 2015ND
M. duraARAN-Fungi 1966SpainKT222190NDNDOlariaga et al., 2015ND
M. duraARAN-Fungi 2950SpainKT222188NDNDOlariaga et al., 2015ND
M. lammiensis165927FinlandFN394552KF723697KF723651Harder et al., 2010Harder et al., 2013
M. luteovariegataTL5614DenmarkFN394602KF723666KF723620Harder et al., 2010Harder et al., 2013
M. luteovariegataCBH226DenmarkEpitypeFN394604KF723664KF723618Harder et al., 2010Harder et al., 2013
M. luteovariegataDB2005/152DenmarkFN394603KF723665KF723619Harder et al., 2010Harder et al., 2013
M. pearsonianaJV06890DenmarkFN394612KF723692KF723646Harder et al., 2010Harder et al., 2013
M. pearsonianaLK880/2002GermanyFN394613KF723693KF723647Harder et al., 2010Harder et al., 2013
M. pearsonianaCBH068GermanyFN394614KF723691KF723645Harder et al., 2010Harder et al., 2013
M. pearsonianaFCME25817USAEpitypeJN182198NDNDHarder et al., 2012ND
M. pearsoniana_IITL3966DenmarkFN394615NDNDHarder et al., 2013ND
M. pearsoniana_IITENN61865USAJN182201NDNDHarder et al., 2012ND
M. pelianthinaCBH016DenmarkFN394547KF723696KF723650Harder et al., 2010Harder et al., 2013
M. pelianthinaCBH015DenmarkFN394549KF723695KF723649Harder et al., 2010Harder et al., 2013
M. polycystidiataFFAAS0417ChinaHolotypeON427731ON468456ON468469Liu et al., 2022Liu et al., 2022
M. polycystidiataFFAAS0418ChinaON427732ON468457ON468470Liu et al., 2022Liu et al., 2022
M. polycystidiataFFAAS0421ChinaON427733ON468458ON468471Liu et al., 2022Liu et al., 2022
M. polycystidiataFFAAS0422ChinaON427734ON468459ON468472Liu et al., 2022Liu et al., 2022
M. pura_ICBH039DenmarkFN394588KF723680KF723634Harder et al., 2010Harder et al., 2013
M. pura_IICBH366DenmarkFN394572KF723673KF723627Harder et al., 2010Harder et al., 2013
M. pura_IICBH404DenmarkFN394566KF723674KF723628Harder et al., 2010Harder et al., 2013
M. pura_IICBH169DenmarkFN394579KF723672KF723626Harder et al., 2010Harder et al., 2013
M. pura_IICBH105DenmarkFN394581KF723671KF723625Harder et al., 2010Harder et al., 2013
M. pura_IIICBH019DenmarkFN394605KF723675KF723629Harder et al., 2010Harder et al., 2013
M. pura_IIICBH022DenmarkFN394574KF723676KF723630Harder et al., 2010Harder et al., 2013
M. pura_IIIKKSlovakiaFN394606KF723677KF723631Harder et al., 2010Harder et al., 2013
M. pura_IVJV06979DenmarkFN394585KF723668KF723622Harder et al., 2010Harder et al., 2013
M. pura_IVCBH410DenmarkFN394595KF723667KF723621Harder et al., 2010Harder et al., 2013
M. pura_IVTL12786SwedenFN394591KF723670KF723624Harder et al., 2010Harder et al., 2013
M. pura_IVTL4571DenmarkFN394583KF723669KF723623Harder et al., 2010Harder et al., 2013
M. pura_VIBAP132USAFN394561KF723660KF723614Harder et al., 2010Harder et al., 2013
M. pura_VIIIS/10/11/2000USAFN394611KF723661KF723615Harder et al., 2010Harder et al., 2013
M. pura_VIIICBH402DenmarkFN394599KF723663KF723617Harder et al., 2010Harder et al., 2013
M. pura_VIIICBH216DenmarkFN394598KF723662KF723616Harder et al., 2010Harder et al., 2013
M. pura_IXCBH166DenmarkFN394607KF723701KF723655Harder et al., 2010Harder et al., 2013
M. pura_IXCBH358DenmarkFN394608KF723702KF723656Harder et al., 2010Harder et al., 2013
M. pura_IXCBH371DenmarkKF913023KF723704KF723658Harder et al., 2013Harder et al., 2013
M. pura_IXCBH367DenmarkKF913022KF723703KF723657Harder et al., 2013Harder et al., 2013
M. pura_XBAP165AUSAFN394563KF723698KF723652Harder et al., 2010Harder et al., 2013
M. roseaTL12393DenmarkFN394555KF723684KF723638Harder et al., 2010Harder et al., 2013
M. roseaCBH409GermanyFN394551KF723683KF723637Harder et al., 2010Harder et al., 2013
M. roseaUP2UKFN394550KF723686KF723640Harder et al., 2010Harder et al., 2013
M. roseaCBH383DenmarkFN394553KF723682KF723636Harder et al., 2010Harder et al., 2013
M. roseaCBH097DenmarkFN394556KF723681KF723635Harder et al., 2010Harder et al., 2013
M. roseaTL12409DenmarkFN394557KF723685KF723639Harder et al., 2010Harder et al., 2013
M. rufobrunneaFFAAS0414ChinaON427728ON468453ON468466Liu et al., 2022Liu et al., 2022
M. rufobrunneaFFAAS0415ChinaON427729ON468454ON468467Liu et al., 2022Liu et al., 2022
M. rufobrunneaFFAAS0416ChinaHolotypeON427730ON468455ON468468Liu et al., 2022Liu et al., 2022
M. seminauKLU M 1223MalaysiaHolotypeKF537250NDNDChew et al., 2014ND
M. seminauKLU M 1226MalaysiaKF537252NDNDChew et al., 2014ND
M. shengshanensisFFAAS0424ChinaHolotypeON427739ON468464ON468477Liu et al., 2022Liu et al., 2022
M. shengshanensisFFAAS0425ChinaON427740ON468465ON468478Liu et al., 2022Liu et al., 2022
M. sinarKLU M 1222MalaysiaHolotypeKF537249NDNDChew et al., 2014ND
M. sinarKLU M 1220MalaysiaKF537247NDNDChew et al., 2014ND
M. sinar var. tangkaisinarKLU M 1225MalaysiaHolotypeKF537251NDNDChew et al., 2014ND
M. subulataFFAAS0419ChinaON427735ON468460ON468473Liu et al., 2022Liu et al., 2022
M. subulataFFAAS0420ChinaON427736ON468461ON468474Liu et al., 2022Liu et al., 2022
M. subulataFFAAS0423ChinaHolotypeON427737ON468462ON468475Liu et al., 2022Liu et al., 2022
M. subulataFFAAS0426ChinaON427738ON468463ON468476Liu et al., 2022Liu et al., 2022
M. subulataTUMH 65483JapanLC777690LC777730LC777738This studyThis study
M. subulataTUMH 65484JapanLC777691LC777731LC777739This studyThis study
M. subulataTUMH 65485JapanLC777692LC777732LC777740This studyThis study
M. subulataTNS-F-75058JapanLC777693LC777733LC777741This studyThis study
M. yuezhuoiFFAAS0346ChinaMW581492MW868168MW882251Liu et al., 2021Liu et al., 2021
M. yuezhuoiFFAAS0344ChinaMW581490MW868166MW882249Liu et al., 2021Liu et al., 2021
M. yuezhuoiFFAAS0345ChinaHolotypeMW581491MW868169MW882250Liu et al., 2021Liu et al., 2021
M. yuezhuoiFFAAS0347ChinaMW581493MW868167MW882252Liu et al., 2021Liu et al., 2021
M. plumbeaAFTOL-ID 1631USADQ494677DQ447928GU187729Matheny et al., 2006Matheny et al., 2006
Binder et al., 2010
M. rubromarginataJV09362DenmarkFN394624KF723705KF723659Harder et al., 2010Harder et al., 2013
M. zephirus54mItalyJF908462NDNDOsmundson et al., 2013Binder et al., 2010

a Roman numeral of Mycena pura followed Harder et al. (2013). Mycena pura V and M. pura XI were currently renamed to M. luteovariegata and to M. brunnea (Harder et al., 2013; Olariaga et al., 2015).

b ND: No data.

2.4. Phylogenetic analyses

The three loci datasets of ITS, RPB1, and TEF1 were used for phylogenetic analyses. The 64 sequences (Binder et al., 2010; Harder et al., 2013; Liu et al., 2021, 2022) obtained from the National Center for Biotechnology Information GenBank and the eight sequences identified by this study were analyzed (Table 2). From these sequences, we selected Mycena rubromarginata (Fr.) P. Kumm. and M. plumbea P. Karst. as outgroups (Table 2). Mycena rubromarginata was selected as an outgroup due to its previous use in Harder et al. (2013). Mycena plumbea occupies the closest position to M. rubromarginata among the Mycena species available in the GenBank, which possess all-three-loci-sequences. The sizes of DNA sequences of each locus were 645 bp (ITS), 488 bp (RPB1), and 419 bp (TEF1). We used a total of 1,552 bp sequences following alignment (including gaps). The dataset was aligned using the Muscle algorithm (Edgar, 2004) on the workbench in MEGA v. 7.0.26 software (Kumar et al., 2016). The topology of the maximum likelihood (ML) tree was estimated using RAxML GUI v. 2.0 software (Edler et al., 2021; Stamatakis, 2014). To estimate the optimal base substitution model, Model test v. 3.7 (Posada & Crandall, 1998) was carried out by using PAUP* 4 (Swofford, 1998), and the GTR+G model was selected as the best one. We assessed the reliability of branching in the tree using nonparametric bootstrap analysis with 1,000 replicates (MLBS). We also estimated the optimal base substitution model to execute Bayesian analysis. MrModeltest v. 2.3 (Nylander, 2008) was carried out using PAUP* 4 (Swofford, 1998). Following the MrModeltest results, we independently assigned the same substitution model [lset nst = 6, rates = invgamma, and prset statefreqpr = dirichlet (1, 1, 1, 1)] for each locus. We used MrBayes V.3.2.7 software (Ronquist et al., 2012) to construct phylogeny and compute Bayesian inference posterior probability (BPP) for each branch under Bayesian inference. MrBayes analysis involved two sets of four chains of Markov chain Monte Carlo composed of three heated chains and one cold chain, with the topology sampled after every 100 generations. In total, 1,500,000 generations were analyzed until the average standard deviation of the split frequencies was < 0.01. After convergence in the Markov chain Monte Carlo, the first 25% of primary topologies were discarded as burn-in; then the 50% consensus tree was constructed using remaining trees. The tree was visualized using FigTree v.1.4.4 software (tree.bio.ed.ac.uk/software/figtree/).

Because only ITS sequences were available for several species of sect. Calodontes, we created the ITS phylogenetic tree using M. rubromarginata and M. zephirus (Fr.) P. Kumm. as outgroups, similar to Chew et al. (2014). The 79 sequences determined by previous studies (Chew et al., 2014; Cooper, 2018; Harder et al., 2010, 2012, 2013; Liu et al., 2021, 2022; Matheny et al., 2006; Olariaga et al., 2015; Osmundson et al., 2013) and eight sequences identified in our study were analyzed using the GTR+G model and RAxML method (Table 2). In MrBayes analysis, we estimated the optimal base substitution model using KAKUSAN4 (Tanabe, 2011) and GTR+G model was selected. Bayesian analysis was performed using the same method as described previously, except that the dataset was analyzed for 1,000,000 generations.

To conduct species delimitation using genealogical concordance phylogenetic species recognition (GCPSR) (Taylor et al., 2000), we generated independent ITS, TEF1 and RPB1 trees. For molecular phylogenetic analysis of individual regions, we used the same sequences as those used to construct the concatenated three gene tree and the analysis method applied to the concatenated dataset with some minor modifications (Table 2). Briefly, we applied the GTR+G model for ML analysis and the SYM+G model for Bayesian inference in the TEF1 dataset, while the GTR+G model for both was used in the cases of ITS and RPB1. Bayesian analysis was performed for 1,500,000 generations. In the present study, we identified the species boundaries based on the concept of Taylor et al. (2000) that the transition point from concordance to conflict of the tree topologies between separate phylogenetic trees inferred from multiple genetic regions determines the limit of species.

The alignment dataset and resulting trees were deposited in TreeBase (https://treebase.org/) under the accession number TB2:S30700 for the tree in Supplementary Fig. S1, TB2:S30701 for Fig. 1, TB2:S31007 for Supplementary Fig. S3 dataset, TB2:S31008 for Supplementary Fig. S4, and TB2:S30699 for Supplementary Fig. S2.

Fig. 1 - Maximum likelihood (ML) tree of the Mycena sect. Calodontes inferred from concatenated multigene (ITS, RPB1, TEF1) sequences using RAxML. Statistical supports at the nodes are ML bootstrap support (MLBS)/Bayesian posterior probability (BPP). Thick nodes indicate strong support (MLBS≥90 % and BPP≥0.95) and omission of the support value shows less than 60 % in MLBS and 0.90 in BPP. Sequences determined in this study are shown in bold letters and the other sequences were obtained from the NCBI database.

3. Results

3.1. Morphology

Both “Mycena spp.”- 1 and 2 exhibited basidiomata with a reddish tone (pinkish or purplish), smooth cheilocystidia, as well as smooth hyphae of both pileipellis and stipitipellis. These features match the characters of sect. Calodontes sense Maas Geesteranus (1989). “Mycena sp.”-1 had inamyloid basidiospores and lacked pleurocystidia corresponding with characteristics of subsect. Violacellae. This fungus resembles M. subulata reported from China (Liu et al., 2022) in having awl-shaped cheilocystidia, but differs from Chinese specimens having amyloid basidiospores. Moreover, Japanese specimens had dextrinoid cheilocystidia and caulocystidia. “Mycena sp.”-2 had amyloid basidiospores and cheilocystidia with a broadly rounded apex, similar to the characteristics of subsect. Purae sensu Maas Geesteranus and de Meijer (1997). This fungus had 32-44 lamellae reaching the stipe, bowling-pin shaped cheilocystidia, and amyloid basidiospores, but lacked pleurocystidia. These features are unique among species of sect. Calodontes. Morphological comparisons with other phylogenetically related or morphologically similar species are discussed in the “Discussion” part.

3.2. Phylogenetic analyses and species delimitation

Both “Mycena spp.”- 1 and 2 formed clades in the position of sect. Calodontes in all phylogenetic trees inferred from each of the ITS, RPB1, and TEF1 sequences and their concatenated data (Fig. 1; Supplementary Figs. S1-S4). “Mycena sp.”-1 clustered with Chinese M. subulata specimens with high MLBS/BPP values in the concatenated dataset (100/1) and it the individual ITS (98/1), RPB1 (99/1), and TEF1 (100/1) trees. High degrees of DNA homology were observed between Chinese M. subulata and Japanese “Mycena sp.”-1 specimens (ITS, 97.9-99.8%; RPB1, 98.8-100%; TEF1, 98.2-99.5%). According to the species delimitation using the GCPSR concept, we compared tree topologies at the node connecting eight specimens, including Chinese M. subulata and Japanese “Mycena sp.” -1. The topologies coincided within ITS, RPB1, and TEF1 phylogenetic trees (= concordance). However, the topologies below that node were different among the trees (= conflict) (Fig. 2; Supplementary Figs. S2-S4). Consequently, M. subulata and “Mycena sp.”-1 were regarded as conspecific. Although Harder et al. (2013) suggested the possibility of a recombination event occurring in the ITS region in some species of sect. Calodontes, no such traces were detected in the multiple alignment of the ITS sequences of Japanese and Chinese M. subulata specimens (Supplementary Fig. S5). “Mycena sp.”-2 formed a distinct clade clustering without any other species, as indicated by the MLBS/BPP values of 100/1 for the concatenated, 100/1 for ITS, 100/1 for RPB1, and 100/1 for TEF1 trees (Fig. 1; Supplementary Figs. S1-S4). “Mycena sp.”-2 formed a sister clade with European M. diosma in concatenated and TEF1 trees without MLBS/BPP support (Fig. 1; Supplementary Fig. S3). Topological discordance was observed in the relation between “Mycena sp.”-2 and M. diosma in ITS and RPB1 trees (Supplementary Figs. S1, S2, S4). In addition, DNA homology between M. diosma and “Mycena sp.”-2 specimens was 84.1-84.8% in the ITS, 94.3-94.5% in the RPB1, and 89.4% in the TEF1. Therefore, we concluded that “Mycena sp.”-2 and M. diosma are different species. Although we failed to obtain sequences from Kigawa's original specimens, the other sequences derived from specimens with similar morphological characteristics to Kigawa's descriptions (Kigawa, 2017) clustered into the “Mycena sp.”-1 or “Mycena sp.”-2 clade (Fig. 1; Supplementary Figs. S1-S4). In the three regions where GCPSR were performed, almost all species-clades were reproduced in each tree. However, in the ITS region, M. luteovariegata and M. pura IX did not constitute monophyly, which did not match the results of TEF1 and RPB1 trees, as reported by Harder et al. (2013) (Supplementary Figs. S2-S4).

Fig. 2 - Species delimitation based on the genealogical concordance phylogenetic species recognition (GCPSR) concept. Topology of the three-loci-concatenated tree was employed. “Concordance” shows the node that all three independent trees (ITS, TEF1 and RPB1) corresponded to each other, and “Conflict” shows the node that those independent trees did not correspond to each other. Statistical supports at the nodes are ML bootstrap support (MLBS)/Bayesian posterior probability (BPP). (see also Supplementary Figs. S2-S4).

3.3. Taxonomy

Based on the phylogenetic and morphological analyses, we concluded that “Mycena sp.”-1 is M. subulata, which has been described from China, whereas “Mycena sp.”-2 is a new species. The morphological characteristics of these species are described below.

Mycena subulata Z.W. Liu, Y.P. Ge & Q. Na, MycoKeys 93: 46 (2022) Fig. 3

Fig. 3 - Mycena subulata. Macroscopic (A-F) and microscopic (G-N) images. A, B: Basidiomata (A: TUMH 65484; B: TUMH 65483), C: Pileus (TUMH 65485), D, E: Lamellae (D; TUMH 65484, E; TUMH 65485), F: Stipe (TUMH 65483). G: Basidiospore in 3% KOH (TUMH 65484), H: Basidiospores in Melzer's reagent (TUMH 65484), I: Basidium (TUMH 65485), J: awl-shaped cheilocystidia (TUMH 65484), K: Obclavate cheilocystidia (TNS-F-75058). L: awl-shaped cheilocystidia dyeing with Melzer's reagent (TUMH 65483), M: Obclavate cheilocystidia dyeing with Melzer's reagent (TNS-F-75058), N: Caulocystidium dyeing with Melzer's reagent (TUMH 65485). Bars: A, B 15 mm; C-F 5 mm; G, H 5 µm; I-N 10 µm.

Japanese name: Togari-sakura-take (Kigawa, 2017).

Macromorphology: Basidiomata (Fig. 3A, B) small- to medium-sized, mycenoid to collybioid. Pileus (Fig. 3C) 15-55 mm in diam, subumbonate to convex; surface light pink (oac487) to brown-pink (oac730); margin striate when wet, gradually disappear as it dry, glabrous, hygrophanous, whitish (oac909) to light pink (oac550) or pale orange (oac682). Pileal context 2 mm in thickness. Lamellae (Fig. 3D, E) 26-33 reaching the stipe, closed to crowded, adnate to subdecurrent, lateral veins and lamellulae are present, light pink (oac549) to pale orange (oac682). Stipe (Fig. 3F) 20-65 mm long, 2-3 mm diam in center, 3-6 mm diam at basal part, smooth, rarely squarrose, light pink (oac550) to pink (oac486) or orange-brown (oac681); base slightly enlarged, tomentose; mycelial cords present or absent, yellow (oac6) to orange (oac811). Odor raphanoid. Taste unknown.

Micromorphology: Basidiospores (Fig. 3G, H) 6-7.5(-8) × (4-)4.5(-5.5) μm, Q = (1.32-)1.38-1.70(-1.84), Qm = 1.43-1.72 (200 spores from 4 collections), ellipsoid to elongate, rarely pip-shaped, thin-walled, smooth, inamyloid, subhyaline. Basidia (Fig. 3I) (19-)20-24(-26) × (6-)7-8.5 μm, clavate, thin-walled, smooth, inamyloid, 2-4 spored, subhyaline; basal clamps partially present. Sterigmata 1.5-6.5 μm in length, thin-walled, smooth, inamyloid, subhyaline. Basidioles (17.5-)18.5-23(-25) × (5.5-)6-7.5(-8.5) μm, clavate, thin-walled, smooth, inamyloid, subhyaline; basal clamps partially present. Cheilocystidia (Fig. 3J-M) has two shapes; awl-shaped cystidia (33-)39.5-43(-63) × (4.5-)5-8.5(-10.5) μm, thick-walled, smooth, dextrinoid, subhyaline; clavate to obclavate cystidia (26-)27.5-38(-41.5) × (7.5-)8-11.5(-13) μm, thin-walled, smooth, inamyloid, subhyaline; basal clamps partially present. Pleurocysitidia absent. Hymenophoral trama subregular, hyphae 2-8 μm regular thin-walled, smooth, dextrinoid, subhyaline; clamp connections partially present. Pileipellis tomentocutis, hyphae 2-6 μm diam, cylindrical, thin-walled, smooth, weakly dextrinoid, subhyaline; clamp connections partially present. Pileocystidia not observed. Pileitrama pseudoparenchymatous, hyphae 9-25 μm diam, cylindrical, thin-walled, smooth, dextrinoid, subhyaline; clamp connections partially present. Stipitipellis parallel, hyphae 2-4 μm diam, cylindrical, smooth, thin-walled, weakly dextrinoid, subhyaline; clamp connections partially present. Stipitrama parallel, hyphae 2-13 μm diam, cylindrical to ellipsoid, thin-walled, dextrinoid, subhyaline; clamp connections partially present. Caulocystidia (Fig. 3N) rarely present, 36-60 × 5-9 μm, awl-shaped, thick-walled, smooth, dextrinoid, subhyaline; basal clamps partially present.

Ecology: Cool temperate region of Japan, Sep, solitary to gregarious, on litter layer in Cryptomeria japonica (L.f.) D. Don, Fagus crenata Blume, Aesculus turbinata Blume, and/or Acer sp. Heilongjiang Province, China, scattered on the litter layer in Pinus koraiensis Siebold et Zucc., Larix gmelinii (Rupr.) Rupr. ex Kuzen. and Tilia sp. mixed forests.

Materials examined: JAPAN. Tottori Pref., Daisen Town, on the leaf litter in a summer green forest of Qurcus, 11 Sep 2021, leg. K. Nagamune (Specimen: TUMH 65483; Culture: TUFC 102001); Tottori City, Shikano Town, on the ground in a mixed forest of Acer, Chamaecyparis, Cerasus, 19 Sep 2021, leg. K. Nagamune (Specimen: TUMH 65484; Culture: TUFC 102002); Kotoura Town, on the ground in a summer green forest of Fagus, Aesculus, and Acer, 20 Sep 2021, leg. R. Sugawara (Specimen: TUMH 65485; Culture: TUFC 102003): Saitama Pref., Chichibu City, Nakatsugawa, 28 Sep 2015, leg. K. Hosaka (Specimen: TNS-F-75058): Shizuoka Pref. Gotenba City, 13 Sep 2009, leg. S. Kigawa (Specimen: HCM-58-6665).

Note: Many short and branched spinous seta-like structures were formed on the cultured mycelia (Supplementary Fig. S6). These structures resembled the acanthocytes discovered in the cultured mycelia of Stropharia rugosoannulata Farl. ex Murrill as a possible apparatus for defense against nematodes (Yang et al., 2021). A glassy substance covered the seta-like hyphae and could be easily detached and fragmented on slide preparation.

Mycena densilamellata Nagamune, S. Kigawa & N. Endo, sp. nov. Fig. 4

MycoBank no.: MB 850080

Fig. 4 - Mycena densilamellata. Macroscopic (A-F) and microscopic (G-N) images. A, B: Basidiomata (A: TUMH 65481, holotype; B: TUMH 65482), C: Pileus (TUMH 65486), D: Lamellae (TUMH 65482), E: Decurrent lamellae (TUMH 65486), F: Stipes (TUMH 65486). G: Basidiospores (TUMH 65481), H: Pip-shaped basidiospores (TUMH 654826), I: Basidiospores dyeing with Melzer's reagent (TUMH 65481), J: Basidium (TNS-F-75029), K: Cheilocystidium with protruding tip (TUMH 65481). L: Clavate and bowling-pin shaped cheilocystidia (TUMH 65486), M: Hymenium and lamellar edge (TUMH 65482), N: Caulocystidia in Melzer's reagent (TUMH 65486). Bars: A, B 20 mm; C, E 10 mm; D, F 5 mm; G-I 5 µm; J-L, N 10 µm; M 50 µm.

Type: JAPAN, Hokkaido, Ebetsu City, Nopporo Shinrin Kouen Prefectural Natural Park, on the ground of mixed forest where Picea, and Alnus were dominated, 14 Sep 2019, leg. R. Sugawara (holotype, TUMH 65481; isotype, TNS-F-82703)

Ex-holotype culture: TUFC 101999 (polysporic strain)

Gene sequences from ex-holotype culture: LC777686 (ITS), LC777726 (TEF1), LC777734 (RPB1)

Etymology: densus (Latin) + lamellatus (Latin), referring to the densely formed lamella at the gill of the pileus.

Japanese name: Mitsuhida-sakura-take (Kigawa, 2017).

Macromorphology: Basidiomata (Fig. 4A, B) small- to medium-sized, collybioid. Pileus (Fig. 4C) 20-35 mm in diam, umbonate to subumbonate when young, convex with age; surface purplish (oac427) to dark purplish (oac514); margin striate when wet, gradually disappear as it dry, glabrous, hygrophanous, whitish (oac909) to cream (oac900). Pileal context 2 mm thickness. Lamellae (Fig. 4D, E) 32-44 reaching the stipe, crowded, adnexed to adnate, subdecurrent, lateral veins and lamellulae present, pale lilac (oac438). Stipe (Fig. 4F) 30-70 mm long, 2-4 mm diam in center, 4-7 diam at basal part, smooth or slightly longitudinal striate, pinkish (oac473) to reddish brown (oac525); base slightly enlarged, tomentose, concolorous or yellow (oac6); mycelial cords present or absent, whitish (oac909). Odor raphanoid. Taste unknown.

Micromorphology: Basidiospores (Fig. 4G-I) 5.5-6.5(-7) × 3-4(-4.5) μm, Q = (1.50-)1.58-1.89(-2.02), Qm = 1.64-1.80 (200 spores from 4 collections), ellipsoid to cylindrical, rarely pip-shaped, thin-walled, smooth, amyloid, subhyaline. Basidia (Fig. 4J) (17-)19.5-26(-27.5) × (5.5-)6-7(-7.5) μm, clavate or constricted in the middle, thin-walled, smooth, inamyloid, subhyaline, 2-4 spored; basal clamps partially present. Sterigmata 1-6 μm length, thin-walled, smooth, inamyloid, subhyaline. Basidioles (16.5-)18-26(-27.5) × (4-)4.5-6.5(-7.5) μm, clavate, thin-walled, smooth, inamyloid, subhyaline; basal clamps partially present. Cheilocystidia (Fig. 4K-M) (27-)29.5-44(-48) × (7-)8-14(-17) µm, clavate, rarely protruding tip, bowling-pin shaped, thin-walled, smooth, inamyloid, subhyaline; basal clamps partially present. Pleurocystidia absent. Hymenophoral trama subregular, hyphae 3-6 µm diam, thin-walled, smooth, dextrinoid, subhyaline; clamp connections partially present. Pileipellis tomentocutis, hyphae 1-4 μm diam, cylindrical, thin-walled, smooth, inamyloid, subhyaline; clamp connections partially present. Pileocystidia not observed. Pileitrama pseudoparenchymatous, hyphae 4-17 μm diam, cylindrical to ellipsoid, thin-wallled, smooth, dextrinoid, subhyaline; clamp connections partially present. Stipitipellis parallel, hyphae 1-2 μm diam, cylindrical, smooth, thin-walled, weakly dextrinoid, subhyaline; clamp connections partially present. Stipitrama parallel, hyphae 3-10 μm diam, cylindrical to slightly expanded, thin-walled, dextrinoid, subhyaline; hyphae, clamp connections partially present. Caulocystidia (Fig. 4N) rarely present, 24-32.5 × 6-8 μm, obclavate to clavate, thin-walled, smooth, inamyloid, subhyaline; basal clamps partially present.

Ecology: Cool temperate region of Japan, Sep to Oct, gregarious to scattered on litter layer in Quercus serrata Murray, Chamaecyparis obtusa (Siebold et Zucc.) Endl., Fagus crenata, Alnus sp. Picea abies (L.) H. Karst. and Picea glehnii (F. Schmidt) Mast.

Other materials examined: JAPAN. Yamanashi Pref., Minamitsuru-gun, Narusawa Village, on the leaf litter in a mixed forest of Quercus, Fagus, and Chamaecyparis, 8 Oct 2020, leg. R. Sugawara (Specimen: TUMH 65486): Tottori Pref., Tottori City, Shikano Town, on the ground in a mixed forest of Chamaecyparis, Quercus, and Carpinus, 19 Sep 2021, leg. K. Nagamune (Specimen: TUMH 65482; Culture: TUFC 102000): Saitama Pref., Chichibu City, Nakatsugawa, 28 Sep 2015, leg. K. Hosaka (Specimen: TNS-F-75029): Shizuoka Pref. Gotenba City, 13 Sep 2009, leg. S. Kigawa (Specimen: HCM-58-6667).

Note: Many short and branched acanthocyte-like structures were formed on the cultured mycelia (Supplementary Fig. S7) as observed in “Mycena sp.”-1 cultures (see above).

4. Discussion

“Mycena sp.”-1 constituted a clade with the Chinese specimens of M. subulata (Fig. 1; Supplementary Figs. S1-S4) and showed similar characteristics of narrowly fusiform, long, and narrow protuberance of cheilocystidia (Fig. 3). The delimitation of species according to the GCPSR concept (Fig. 2) strongly suggested conspecificity. Mycena subulata has not been reported previously from Japan. Here, we assign the Japanese name “Togari-sakura-take”, to the species, consistent with Kigawa (2017). However, our specimens from Japan did not show the amyloid basidiospores, which differed from the original description by Liu et al. (2022). Although the Japanese specimens showed dextrinoid cheilocystidia, Liu et al. (2022) did not include this characteristic in the original description. It is necessary to observe more specimens (including the type specimen) to determine whether these features exhibit regional differences among the Japanese samples. In the detailed analysis of the molecular data, seven mutation sites in the ITS were detected between Chinese and Japanese M. subulata (Supplementary Fig. S5). However, these base differences did not distinguish between Chinese and Japanese specimens on the phylogenetic trees (Supplementary Figs. S1, S2).

Liu et al. (2022) did not specify the subsection of M. subulata based on the amyloid basidiospores and lack of pleurocystidia. On the other hand, all five of our Japanese specimens demonstrated inamyloid basidiospores, indicating their similarity to the species of subsect. Violacellae. Harder et al. (2012) suggested the phylogenetic position of subsect. Violacellae in the ITS region by designating the epitype of M. pearsoniana Dennis ex Singer. Mycena subulata showed a phylogenetic position close to M. pearsoniana (Liu et al., 2022); our results support that (Fig. 1). Moreover, M. pearsoniana has inamyloid to weakly amyloid basidiospores, suggesting that amyloidity is not a diagnostic feature of species in M. pearsoniana (Harder et al., 2012). Mycena subulata and M. pearsoniana form a highly supported clade (MLBS/BPP = 96/1), which includes the M. shengshanensis described by Liu et al. (2022); a similar pattern was also found in the present study (MLBS/BPP = 100/1) (Fig. 1). Mycena subulata possesses both inamyloid and weakly amyloid basidiospores within the species as M. pearsoniana. Based on these results, we propose that M. subulata should be assigned to subsect. Violacellae. Although the morphological characteristics of subsect. Generosae coincide with those of M. subulata described by Liu et al. (2022), the phylogenetic position of subsect. Generosae has not been clarified. Hence, it is necessary to investigate the phylogenetic position of M. generosa, the type species of subsect. Generosae and the phylogenetic relationship between this subsection and M. subulata.

We assigned the Japanese name “Mitsuhida-sakura-take” to M. densilamellata consistent with Kigawa (2017). This species differs from other species in sect. Calodontes based on the formation of close lamellae, lack of pleurocystidia, and presence of bowling pin-shaped cheilocystidia and amyloid basidiospores. A few specimens had protruding tips of cheilocystidia, the most of which had broadly rounded apices (Fig. 4). These characteristics indicate that M. densilamellata belongs to subsect. Purae according to the definition by Maas Geesteranus and de Meijer (1997). Mycena diosma Krieglst. & Schwöbel from the subsect. Purae resembles M. densilamellata in terms of its pileus color (Harder et al., 2010; Krieglsteiner & Schwöbel, 1982). Despite the lack of significant statistical support and topological incongruence between datasets, our three-loci-concatenated and TEF1 trees suggested a potential sister relationship between the two species (Fig. 1; Supplementary Fig. S3). Mycena diosma has close lamellae (with 24-32 reaching the stipe) (Krieglsteiner & Schwöbel, 1982), whereas M. densilamellata exhibits crowding (with 32-44 reaching the stipe). In addition, M. diosma has few to no pleurocystidia depending on the specimen (Harder et al., 2010; Krieglsteiner & Schwöbel, 1982), whereas pleurocystidia are entirely lacking in M. densilamellata. Mycena densilamellata resembles M. yuezhuoi in terms of amyloid basidiospores and lack of pleurocystidia. However, M. yuezhuoi has slightly closer lamellae (with 21-26 reaching the stipe) emarginated to a stipe (Liu et al., 2021), whereas M. densilamellata exhibits crowded, adnate to decurrent lamellae (with 32-44 reaching the stipe). Furthermore, these two species are categorized in different clades according to the phylogenetic analysis (Fig. 1; Supplementary Figs. S1-S4). Mycena kuehneriana A.H. Sm. resembles M. densilamellata in terms of its amyloid basidiospores and lack of pleurocystidia. However, M. kuehneriana has subfusoid to nearly cylindric cheilocystidia and scattered to rare cheilocystidia (Smith, 1947). Mycena densilamellata is distinguishable from M. kuehneriana by its abundant bowling pin-shaped cheilocystidia. According to Maas Geesteranus (1989), 20 lamellae of M. kuehneriana reached the stipe, with scattered pleurocystidia, whereas M. densilamellata produced 32-44 stipe-reaching lamella without pleurocystidia. Although the crowding of lamellae is a characteristic feature of M. densilamellata, M. rosea, M. sororia Perr.-Bertr., Boissel. & Lambourd. and M. vinacea Cleland also have similar characteristics [with 40, 38-42, and 32-40 lamellae reaching the stipe, respectively (Grgurinovic, 1997; Maas Geesteranus, 1989; Perreau-Bertrand et al., 1996)]. However, M. densilamellata lacks pleurocystidia, whereas the other three have pleurocystidia. In addition, M. densilamellata is distinguishable from the other three based on cheilocystidia shape: those of M. densilamellata are bowling pin-shape, whereas they are fusiform, clavate, or subcylindrical in M. rosea (Maas Geesteranus, 1989); clavate, subcylindrical, or fusiform in M. sororia (Perreau-Bertrand et al., 1996); and cylindrical, cylindro-ventricose, or clavate in M. vinacea (Grgurinovic, 1997). Further detailed comparisons with species, including the remaining sect. Calodontes species, are presented in Supplementary Table S1. The original description of M. sororia is available, but the lack of type specimen and molecular information make it difficult to verify the taxonomic status of this species (Harder et al., 2010). For future revision of the whole sect. Calodontes species, there is an urgent need to redesignate the type specimen to allow molecular phylogenetic analyses of such species.

According to Chew et al. (2014) and Harder et al. (2012), the species of subsect. Marginatae and subsect. Violacellae formed independent monophyly, whereas those of subsect. Purae were separated into multiple clades. The phylogenetic trees constructed by Liu et al. (2021, 2022) based on the three loci (ITS, TEF1, and RPB1) exhibited polyphyly of subsect. Purae. In our phylogeny based on the three loci, monophyly of subsect. Marginatae was observed; however, subsect. Purae was also polyphyletic. M. pearsoniana and M. subulata had a similar morphological appearance to subsect. Violacellae, forming a robust monophyletic clade. However, M. shengshanensis, which cannot be assigned to any subsection due to its morphological characteristics (Liu et al., 2022), occupies an intermediate position between M. pearsoniana and M. subulata (Fig. 1), thereby necessitating the revision of subsect. Violacellae after consideration of the accuracy of the morphological description of M. shengshanensis by Liu et al. (2022). However, we found potential inaccuracies in the original description of M. subulata.

Currently, there is insufficient DNA data to determine the phylogenetic relationships among infrageneric taxa. To date, ITS sequences of 20 species belonging to sect. Calodontes have been registered in the GenBank database, which is < 50% of the known species. Among them, only 15 species, including the novel species, have records of authentic sequences obtained from the type materials. (Chew et al., 2014; Cooper, 2018; Harder et al., 2012, 2013; Liu et al. 2021, 2022; Olariaga et al., 2015). Moreover, the number of available sequences of TEF1 and RPB1 genes is even more limited (Harder et al., 2013; Liu et al., 2021, 2022). The phylogenetic position of subsection Generosae has not been confirmed. Thus, the unavailability of DNA information makes it challenging to determine whether or not the characteristic morphological features of each subsection reflect their phyletic relationship. Harder et al. (2013) suggested that TEF1 may be a more reliable marker for species identification in sect. Calodontes compared to ITS and RPB1. Indeed, only TEF1 indicated potential separation between the Japanese and Chinese populations of M. subulata, although this lacked statistical support (Supplementary Figs. S2-S4). In addition, TEF1 alone suggested a potential sister group relation between M. densilamellata and M. diosma, but this association also lacked statistical support (Supplementary Fig. S3). In future studies, it will be necessary to collect specimens from various parts of the world and obtain additional DNA sequences. This should include not only ITS, TEF1, and RPB1, but also other frequently used regions, such as RPB2, ATP6, etc., along with morphological characteristics. These combined efforts will contribute to the construction of a robust infrageneric classification system accurately reflecting the phylogenetic relations and help to determine the most effective DNA marker for species identification in sect. Calodontes.

The fruiting bodies of M. subulata and M. densilamellata predominantly emerged on litter within the broadleaved forests dominated by oak and beech trees at altitudes exceeding 450 m above sea level across the Japanese mainland. Specimens of M. subulata were collected from Eastern (Saitama Pref.) and Western (Tottori Pref.) regions of Japan. Similarly, specimens of M. densilamellata were collected from Northern (Hokkaido) and Western (Tottori Pref.) regions of Japan. Thus, both species are widely distributed in broadleaved forests in Japan. Mycena species in sect. Calodontes have been well described in Europe since the mid-18th century, whereas few taxonomic studies have been conducted in Asia (Aravindakshan & Manimohan, 2015; Chew et al., 2014; Hennings 1900; Liu et al., 2021, 2022). Further studies on the taxonomy and ecology of Mycena species in Asia are required to gain a thorough understanding of the distribution of these species. On the cultured mycelia of M. subulata and M. densilamellata, we observed acanthocyte-like structures similar to those described by Yang et al. (2021) (Supplementary Figs. S6, S7). On the other hand, Chew et al. (2014) reported inamyloid crystals on mycelia of M. cahaya cultured on MEA. Because we could not verify whether the acanthocyte-like structures covered with glassy substance found in the two Mycena species are identical with one of the previously reported structures, further studies are required to observe their detailed structure and development process. It is also essential to consider their taxonomic importance and ecological function by culture studies of various fungal species of sect. Calodontes.

Disclosure

The authors have no conflicts of interest to declare. All experiments undertaken in this study complied with the current laws of the country where they were performed.

Acknowledgements

We thank Noriko Matsumoto, a curator at the Hiratsuka City Museum, for loaning the specimens of “Mitsuhida-sakura-take” and “Togari-sakura-take”, and Kanagawa mushroom club's members, who collected these samples. We also thank Yamanashi Prefectural Government for generously allowing the sampling of Mycena within the forests of Narusawa Village, Yamanashi Prefecture, Japan; and thank to Hiroko Shirayama and Seiichi Takeda for helping collect these samples; and Eiji Nagasawa to provide information regarding Mycena rosea; and Shuji Ushijima to help literature searching. This work was partially supported by JSPS KAKENHI (grant nos.: 20K06805 and 24680085).

References
 
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