Mycoscience VOL.62 (2021) 217-223

Short communication longipilum sp. nov. (, ) from Japan Yukito Tochiharaa, b, *, Tomoya Hiraoc, Muneyuki Ohmaed, Kentaro Hosakab, and Tsuyoshi Hosoyab a Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan b Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan c Mycologist Group in Okayama, 554-10 Tsubue, Kurashiki, Okayama 710-0034, Japan d Edible Mushrooms Institute, Hokken Co. Ltd., 1296-4, Oyamadashimogo, Nakagawa, Nasu, Tochigi 324-0602, Japan

ABSTRACT Microstoma longipilum sp. nov. collected from two localities in Japan is described. It is characterized by long apothecial hairs and salmon pink discs. Molecular phylogenetic analyses supported the novelty of the . We additionally reported the overlooked morphology of hyphal mats, conidiogenous cells produced directly from , and conidia. With the addition of M. longipilum, now six species of Microstoma are documented in Japan.

Keywords: ITS-5.8S, mycobiota, new species, phylogeny

Article history: Received 4 November 2020, Revised 14 March 2021, Accepted 15 March 2021, Available online 20 July 2021.

The Microstoma Bernstein (Sarcoscyphaceae, Pezizales) is To obtain isolates, a piece of an apothecium was pasted under characterized by long stipes, white-hairy receptacles, orange to red the lid of a Petri dish so that ascospores could be freely discharged discs, gelatinized ectal excipulum and ascospores with smooth sur- onto potato dextrose agar (PDA; Nissui, Tokyo, Japan). Germinated faces (Otani, 1980). Seven species have been differentiated in the ascospores were transferred to PDA slants to establish pure isolates. genus: M. aggregatum Otani, M. apiculosporum Yei Z. Wang, M. Isolates were deposited in the NITE Biological Resource Center camerunense Douanla-Meli, M. floccosum (Sacc.) Raitv., M. macros- (NBRC), Kisarazu, Japan. To observe germination, asco- porum (Y. Otani) Y. Harada & S. Kudo, M. protractum (Fr.) were also discharged onto corn meal agar (CMA; Nissui), Kanouse, and M. radicatum T.Z. Liu, Wulantuya & W.Y. Zhuang stored at 20 °C, and observed after 12 h. Pieces of medium with (Liu, Wulantuya, & Zhuang, 2018; Ohmae, Yamamoto, & Orihara, ascospores were then cut out using sterilized scalpels, transferred 2020). In Japan, M. aggregatum, M. apiculosporum, M. floccosum, into new Petri dishes, immersed in tap water at 20 °C, and exam- M. macrosporum, and M. protractum have been reported (Katumo- ined after 12 h. to, 2010; Ohmae et al., 2020). The germination of ascospores and micromorphological charac- In 2014, the second author of the present study collected a spec- teristics of the apothecia were examined using cotton blue (Wako imen of Microstoma characterized by unique morphological fea- Pure Chemical Industries, Osaka, Japan) dissolved in water (CBW) tures in a primeval forest of Fagus crenata Blume in Maniwa, or tap water as a mounting fluid in the living state using a BX51 Okayama. Thereafter specimens of the same fungus were collected microscope equipped with a Nomarski interference contrast device yearly from the same locality. In 2020, a new locality of the fungus (Olympus, Tokyo, Japan). To check the ascal iodine reaction, Mel- was found in a forest dominated by broad-leaved trees (not includ- zer’s reagent (MLZ) was used. To confirm the swelling of asco- ing Fagus spp.) in Yamakita, Kanagawa. The fungus resembled M. spores and dissolution of glassy materials of hairs, 3% or 10% (w/v; aggregatum in that it had aggregated apothecia like colonial corals the same applied for all ‘%’ described below for concentrations of but differed from it by having much longer excipular hairs. In this solutions) potassium hydroxide (KOH) aqueous solution was used. study, we report the fungus as a new species to science based on Ascospore sizes were recorded both in the living state (= just after morphology and molecular phylogeny. immersed in CBW) and in the dead state (= 6 h after immersed in Some fresh specimens of the fungus were used to establish cul- MLZ) and were described in the following order: variation of tures, and others were air-dried for 1 wk at 20 °C and deposited in length and width (arithmetic mean of length and width ± standard the mycological herbarium of the National Museum of Nature and deviation), variation of Q (arithmetic mean of Q ± standard devia- Science, Tsukuba, Japan (TNS) and the Kanagawa Prefectural Mu- tion). Q is the ratio of length/width. seum of Natural History, Odawara, Japan (KPM). Molecular phylogenetic analyses were conducted including oth- er species of the genus Microstoma using the internal transcribed * Corresponding author. spacer region of nuclear ribosomal DNA containing partial ITS1- E-mail: [email protected] 5.8S-ITS2 (ITS-5.8S). For the five species of Microstoma known in

This is an open-access paper distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivative 4.0 international license (CC BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.47371/mycosci.2021.03.003 ― 217 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223

Japan, sequences were derived from specimens at the TNS herbari- est-NG 0.1.6 (Darriba et al., 2019) based on Akaike’s information um. DNA was extracted from mycelia cultivated on 2% (w/v) malt criterion. MP analysis was conducted using MEGA X (Kumar, extract broth (BactoTM Malt Extract; Thermo Fisher Scientific, Stecher, Li, Knyaz, & Tamura, 2018). Gaps and missing data were Waltham, MA, USA) following the modified CTAB method (Hosa- eliminated. A heuristic search was carried out under the tree bisec- ka & Castellano, 2008; Tochihara & Hosoya, 2019). When isolates tion reconnection branch swapping (TBR) algorithm with search were not available, DNA was extracted from pieces of dried apoth- level 2, in which the initial trees were obtained by the random ad- ecia using the same method. The ITS-5.8S region was then ampli- dition of sequences (10 replicates). Branch support was evaluated fied, sequenced, and aligned following procedures described by by 1,000 bootstrap replications. Phylogenetic trees were illustrated Tochihara and Hosoya (2019). Aligned sequences were deposited using FigTree 1.4.4 (Rambaut, 2018) based on the ML and MP anal- in the DNA Data Bank of Japan (DDBJ) (Table 1). Additionally, yses. The ultimate sequence matrix and ML best-scored tree were sequences > 400 bp derived from non-Japanese Microstoma sam- registered to TreeBase (http://purl.org/phylo/treebase/phylows/ ples were obtained from GenBank and added to the phylogenetic study/TB2:S26996). analyses. occidentalis (Schwein.) Sacc. and S. tataken- sis Yei Z. Wang & Cheng L. Huang were selected as the outgroup (Table 1). The obtained sequences (Table 1) were aligned using MAFFT 7 Microstoma longipilum Tochihara, T. Hirao & Hosoya, sp. nov. (Katoh & Standley, 2013) under the Q-INS-i option and manually Figs. 1, 2. edited. Molecular phylogenetic analyses were performed based on MycoBank no.: MB 836783. the maximum likelihood (ML) and the maximum parsimony (MP) methods. ML analysis was conducted using RAxML-NG 0.9.0 (Ko- Diagnosis: Characterized by aggregated apothecia, long acute zlov, Darriba, Flouri, Morel, & Stamatakis, 2019) with 1,000 boot- hairs, and salmon pink to pale orange discs. strap replications after suitable model estimation using Modelt- Holotype: JAPAN, Okayama, Maniwa, on rotten wood of Fagus

Table 1. Taxa analyzed in molecular phylogenetic analyses. Isolates ITS GenBank Taxon Specimen no. Locality Coll. Date (NBRC) Accession no. Microstoma aggregatum TNS-F-61614 JAPAN, Fukushima, Kawauchi 2005/10/1 - LC584234 M. aggregatum TNS-F-80795 JAPAN, Fukushima, Kawauchi 2017/10/6 - LC584235 M. aggregatum TNS-F-81070 JAPAN, Hokkaido, Asahikawa, Mt. Tosshozan (Type 2017/10/6 - LC584236 Locality) M. aggregatum TNS-F-81149 JAPAN, Fukushima, Kawauchi 2017/9/9 - LC584237 M. aggregatum TNS-F-88858 JAPAN, Hokkaido, Chitose 2019/9/22 - LC584238 M. apiculosporum TNS-F-37021 JAPAN, Ehime, Ozu 2010/10/12 114763 LC584239 M. apiculosporum TNS-F-45127 JAPAN, Miyazaki, Kobayashi 2011/10/24 - LC584240 M. apiculosporum KPM-NC 28117 JAPAN, Ibaraki, Kasama 2019/11/4 114652 LC584241 M. floccosum FLAS-F-65620 USA, Minnesota, Rice, Nerstrand Big Woods State Park 2013/6/29 - MT3739221 M. floccosum 420526MF0271 CHINA, Hubei - - MH1420201 M. floccosum FH K. Griffith MEXICO - - AF3940451 M. floccosum FH K. Griffith MEXICO - - AF3940461 M. floccosum - USA, Pennsylvania - - AF0263091 M. floccosum TNS-F-56039 JAPAN, Nagano, Ueda 1991/6/21 - LC584242 M. floccosum TNS-F-56212 JAPAN, Kanagawa, Yokosuka 1992/5/29 - LC584243 M. floccosum TNS-F-56670 JAPAN, Aomori, Aomori 1994/5/7 - LC584244 M. floccosum TNS-F-41525 JAPAN, Ibaraki, Daigo 2011/7/16 - LC584245 M. floccosum TNS-F-66316 JAPAN, Fukushima, Iwaki 2014/6/22 - LC584246 M. floccosum TNS-F-88714 JAPAN, Saga, Kashima 2019/6/14 - LC584247 M. longipilum TNS-F-61424 JAPAN, Okayama, Maniwa 2014/7/19 110694 LC584248 M. longipilum TNS-F-61946 JAPAN, Okayama, Maniwa 2015/7/22 114764 LC584249 (holotype) M. longipilum TNS-F-65705 JAPAN, Okayama, Maniwa 2016/7/10 114765 LC584250 M. longipilum TNS-F-60527 JAPAN, Okayama, Maniwa 2020/7/8 - LC584251 M. longipilum TNS-F-60530/ JAPAN, Kanagawa, Yamakita 2020/7/15 - LC584252 KPM-NC 28281 M. radicatum CFSZ 10833 CHINA, Inner Mongolia 2016/5/26 - MG8452301 M. radicatum CFSZ 10833 CHINA, Inner Mongolia 2016/5/27 - MG8452311 M. radicatum CFSZ 10833 CHINA, Inner Mongolia 2016/5/27 - MG8452321 M. macrosporum TNS-F-15609 JAPAN, Fukushima, Yanaizu 2007/4/10 114761 LC584253 M. macrosporum TNS-F-13589 JAPAN, Hyogo, Shiso 2007/3/31 - LC584254 M. macrosporum TNS-F-39247 JAPAN, Hokkaido, Kamikawa 2011/5/11 - LC584255 M. macrosporum TNS-F-57413 JAPAN, Fukushima, Kitakata 2000/4/8 - LC584256 M. macrosporum TNS-F-80334 JAPAN, Niigata, Yahiko 2017/4/1 - LC584257 M. macrosporum TNS-F-80822 JAPAN, Hokkaido, Tohma 2017/4/26 - LC584258 FLAS-F-61366 USA, Georgia, Rabun, Appalachian Mountains 2017/7/21 - MT3740261 NKR-56 S. tatakensis TNM F0993 TAIWAN - - NR_1566011 1Downloaded sequences from GenBank. 2Outgroup doi: 10.47371/mycosci.2021.03.003 ― 218 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223 crenata, 22 Jul 2015, leg. T. Hirao (TNS-F-61946). µm (including the glassy portion), elongating percurrently; glassy DNA sequence ex-holotype: LC584249 (ITS). portion 2.5–8 µm thick (usually 5 µm thick), not stained by MLZ or Etymology: Referring to the long hairs of the apothecia. CBW, instantly dissolved in 10% KOH. Subhymenium 30–40 µm Japanese name: Karasake-kitsune-no-sakazuki (karasake = ob- thick, composed of interwoven hyphae; hyphae 1.9–4.0 µm wide, solete term meaning ‘salmon pink’, kitsune-no-sakazuki = cup for containing red or orange droplets containing carotenoids as seen in fox meaning Microstoma spp.) paraphyses. Ectal excipulum ca. 150 µm thick, composed of thick- Description: Hyphal mats scattered on rotten wood of broad- walled refractive cells easily separated from medullary excipulum leaved tree, dark brown, composed of complexly interwoven hy- in squash mounts, consisting of two layers: inner layer composed phae; hyphae resembling the apothecial hairs, hyaline to dark of irregular-shaped cells 2.5–18 × 2.5–8 µm, thick-walled (up to 2 brown, 6–10 µm wide, glassy walled; glassy parts instantly dis- µm thick), becoming smaller and thinner-walled near the margin; solved in 10% KOH. Apothecia usually aggregated, deeply cupu- outer layer composed of globular cells; cells 2.5–10 × 2.5–9 µm, late, 4–16 mm diam, subsessile to stipitate, up to 15 mm high thick-walled (up to 2 µm thick), becoming perhaps thinner-walled (usually 10 mm high); outside totally covered by long, acute and near the margin. Medullary excipulum 50–75 µm thick; upper part white hairs usually bundled together; stipes up to 5 × 1.5–3 mm, textura porrecta composed of thick hyphae (up to 7 µm wide) fre- integrated with each other near bases, not arising from rhizoids quently branching; lowermost part composed of thinner hyphae (pseudorhiza). Disc concave, dull pink to pale orange when fresh, (up to 3 µm wide) toward the ectal excipulum. Apothecial margin becoming rather more intense when dry. Hairs arising from outer thick, composed of two-layered ectal excipulum (referred to above) and inner ectal excipular cells, cylindrical, acute toward the apices, and medullary excipulum; medullary excipulum textura prismatica with thick and glassy walls, up to 3000 (usually over 1500) × 10–20 to t. angularis, composed of irregular-shaped cells; cells hyaline,

Fig. 1. Morphology of Microstoma longipilum (A, B: Apothecia in nature, 10 Jul 2016 in the type locality; C, U, V: TNS-F-60530; D–T: TNS-F-60527). A: Immature apothecia occurring on Fagus crenata log. B: Fresh apothecia relatively faded. C: Fresh apothecia occurring on brown hyphal mats (arrowhead). D: Vertical section at the middle of apothecium. D1: . D2: Subhymenium. D3: Medullary excipulum. D4: Ectal excipulum. E: Marginal part of vertical section of apothecium. F: Magnified cross section of apothecium at the marginal part. The outermost layer (arrowhead) is composed of relatively thick-walled cells. G: Hyphae of subhymenium. H: Ectal excipular cells. I: Hyphae of medullary excipulum. J: Anastomosis of paraphyses (arrowhead). K: . L: Ascal tips with eccentric opercula. M: Basal part of ascus. N: Hair. O: Hair bases. P: Hair apices. Parts of percurrent proliferation are shown by the arrowhead. Q: Hairs mounted in 10% KOH whose glassy materials are dissolved. R: from a hyphal mat. The wall, dissolved in 10% KOH, is shown on the left side of the arrowhead. S: Ascospore. T: Ascospore in 3% KOH. U: Ascospore producing a germ tube on CMA. V: Conidiogenous cells and conidia produced from an ascospore. Bars: B, C 10 mm; D, K 100 µm; E, N, U 50 µm; F, M, P 20 µm; G–J, L, O, Q–T, V 10 µm. Mounted in tap water (D–K, N, P, U); CBW (L, M, O, S, T, V); 10% KOH (Q, R (left side)); 3% KOH (T). doi: 10.47371/mycosci.2021.03.003 ― 219 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223

Fig. 2. Line drawings of Microstoma longipilum (TNS-F-60530). A: Apothecia. B: Ascospores. C: Paraphyses. D: Hairs. D1: Hair with percurrent elongation. D2: Basal part of hair arising from outermost layer of ectal excipulum. E: Ascus. E1: Tip of eccentrically operculate ascus. E2: Basal parts of asci. F: Schematic diagram of vertical section of apothecium. F1: Hyphae of medullary excipulum. F2: Ectal excipulum. F3: Cells of marginal sterile parts of apothecium. Bars: 10 µm. thin-walled, 1.2–38 × 1.2 ×17 µm, becoming more cubical and dark-brown rhizoids at the basal parts of stipes. In the Microstoma more thick-walled toward the upper layer. Asci 275–350 × 10–17.5 species lacking rhizoids, M. longipilum resembles M. aggregatum in µm (n = 40), maturing simultaneously, clavate, operculate with producing aggregated apothecial clusters but can easily be distin- eccentric opercula, inamyloid, thick-walled (up to 2.5 µm thick), guished by the existence of much longer hairs, larger asci, anasto- especially thicker-walled below the opening where the inner layer mosed paraphyses, and their occurrence in different seasons (Table becoming abruptly thickened toward the opening while the outer 2). Microstoma longipilum is also distinguishable from other spe- layer becomes thinner, arising from simple septa, abruptly thinner cies of the genus by aggregated apothecia, ascospores without near bases and becoming filiform or irregularly incurving, ending apiculi, and pale-colored discs (Table 2). up furcate or swollen. Ascospores ovoid to ellipsoid without apicu- Ecology: Forming apothecia from early Jun to Jul (rainy season li, hyaline, smooth, (20–)21.9–26.1(–27.5) × 11–12.5 µm (24 ± 2.12 in Japan) on rotten wood of broad-leaved trees. × 11.6 ± 0.48 µm), Q = (1.8–)1.9–2.3 (–2.5) (2.1 ± 0.2) in the dead Other specimens examined: JAPAN, Okayama, Maniwa, on state (mounted in MLZ, n = 30), (20–)24–28.6(–30) × (11.5–)12.5– rotten wood of Fagus crenata, 19 Jul 2014, leg. T. Hirao 13.7(–14) µm (26.3 ± 2.3 × 13.1 ± 0.60 µm), Q = (1.7–)1.8–2.2(–2.3) (TNS-F-61424). Locality as above, on rotten wood of an unidenti- (2.0 ± 0.17) in the living state (mounted in CBW, n = 30), contain- fied tree (probablyF . crenata), 30 Jun 2015, leg. T. Hirao ing numerous globose lipid bodies, encased in gelatinous sheath (TNS-F-61948). Locality as above, on rotten wood of F. crenata, 10 that swells and immediately detaches from the in KOH solu- Jul 2016, leg. T. Hirao (TNS-F-65705). Locality as above, on rotten tions, not germinating within asci, germinating by elongating sin- wood of F. crenata, 8 Jul 2020, leg. T. Hirao (TNS-F-60527; 60528). gle germ tube from either polar end on PDA or CMA; conidioge- JAPAN, Kanagawa, Yamakita, on rotten wood of a broad-leaved nous cells produced directly from ascospores when immersed in tree (not F. crenata) lying near small swamp surrounded by Alnus tap water within 12 h, almost spherical with opening, 3–4.5 µm at japonica (Thunb.) Steud. and Cornus controversa Hemsl. ex Prain, the widest parts, blastically producing spherical conidia; conidia 15 Jul 2020, leg. T. Orihara (TNS-F-60530, duplicate KPM-NC 2–2.5 µm wide; ascospores producing conidia directly never pro- 28281). ducing germ tubes. Paraphyses filiform with apices sometimes Notes: The glassy wall materials of the hairs of M. longipilum swelling or branched irregularly like fingers, thin-walled, 1.5–3 µm are instantly dissolved with 10% KOH (Fig. 1Q). The same phenom- wide, arising from subhymenium, almost equal to asci, anastomo- enon is also known in glassy-haired members of sed at the base, containing numerous red or orange droplets con- (), such as the genera Mollisina Höhn. ex Weese and Ur- taining carotenoids. ceolella Boud. (Hosoya & Otani, 1997). In Microstoma, the phenom- Distinguishing characteristics: Microstoma longipilum is charac- enon was reported in M. floccosumby Pant and Tewari (1973), M. terized by extremely long hairs (> 2 mm), which are much longer macrosporum (Harada & Kudo, 2000), and M. apiculosporum (Oh- than those of any other member of the genus. Microstoma longipi- mae et al., 2020). Although Harada and Kudo (2000) reported that lum differs from M. protractum and M. radicatum in the absence of hairs were dissolvable in a few hours with 2.5% KOH, hair-dissolu- doi: 10.47371/mycosci.2021.03.003 ― 220 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223

Table 2. Comparison of features of Microstoma spp. lacking rhizoids at the base of stipes.

Occurring Special Distribution References Disc color Asci (µm) Ascospores (µm) Paraphyses Hairs (µm) season remarks M. aggregatum Japan, China1 Otani (1990) autumn pink to 214.4–272.0 24.0–32.0 × not 252–378 × 12.8 Apothecia coral color × 12.8–14.4 9.6–12.8 anastomosed are aggregated. M. apiculosporum Taiwan, Japan2 Wang (2004) autumn orange red 325–335 × 25–30 × 9–10 anastomosed 270–1200 × (Sep to Nov2) 12–14 (bipolar 12–15 apiculate) M. camerunense Cameroon Douanla-Meli Sep pink 245–335 × (13–)14–16(–17) anastomosed (up to 150 long) & Langer (brownish (6–)7–12 × 3–4(–4.5) (2005) yellow (bipolar when dry) apiculate) M. floccosum USA3, Canada4, Kanouse summer scarlet 300–350 × 20–35 × 14–16 anastomosed (unmentioned) Mexico4, South (1948) (mainly Jun to 18–20 Korea4, Russia5, Aug) Japan6, Taiwan4 Raitviir summer to early dull red 300–350 × 27–31 × 11–14 anastomosed over 1000 × (1965) autumn 18–20 12–18 (Aug to Sep) M. longipilum Japan this study early summer dull pink to 275–350 × 20–30 × 11.5–14 anastomosed up to 3000 Apothecia (Jun to Jul) pale orange 10–17.5 in living, (usually over are 20–27.5 × 1500) × 10–20 aggregated. 11–12.5 in dead M. macrosporum Japan, China1 Harada & autumn and orange red 500–560 × 42–60 × 16–21 anastomosed longer hairs: Margin of Kudo (2000) early spring7 23–26 450–550 × 20–28, apothecia is shorter hairs: split into 30–100 × 5–7 lobes. 1Based on Zhuang & Wang (1997). 2Based on Ohmae et al. (2020). 3Based on Kanouse (1948) and GBIF (https://www.gbif.org/). 4Based only on GBIF. 5Based on Raitviir (1965) and GBIF. 6Based on Otani (1980) and GBIF. 7Overwintering in immature state (Harada & Kudo, 2000) tion should be checked instantly using KOH at higher concentra- was composed of 482 sites. In the ML analysis, a ML tree was yield- tions for efficient observation. ed based on the SYM+I+G4 model. In the MP analysis, 315 sites In Microstoma, the presence of hyphal mats has been reported without gaps and missing data were used, and four most parsimo- in M. apiculosporum (Wang, 2004; Ohmae et al., 2020), M. floccos- nious trees were generated with tree length = 238, consistency in- um (Kanouse, 1948; Wang, 2001), and M. radicatum (Liu et al., dex = 0.600897, and retention index = 0.863077. Since there were 2018). In the present study, for the first time, we confirmed that no topological contradictions between the ML best-scored tree and hyphae consisting of hyphal mats of M. longipilum showed the one of the MP trees, the ML tree was illustrated and ML bootstrap same chemical reaction as hairs using 10% KOH (Fig. 1R). values (MLBP) and MP bootstrap values (MPBP) over 50% were The direct germination of ascospores to produce mitospores in indicated on branches in this order (Fig. 3). Sarcoscyphaceae have been reported in some species of including M. apiculosporum, M. macrosporum, M. radi- Kuntze (Boedijn, 1929; Boedijn, 1933; Paden, 1975), catum, M. aggregatum, and M. longipilum were strongly supported Denison (Pfister, 1973), and Sarcoscypha (Fr.) Boud. (Rosinski, (MLBP/MPBP > 85%), except for MPBP for M. macrosporum, 1953; Baral, 1984; Harrington, 1990; Fenwick, 1994). In conidia which had lower support. Since five sequences of M. longipilum production, while blastoconidia production on germ tubes is wide- formed a strongly supported (MLBP = 87% and MPBP = 94%) ly known in such genera (Boedijn, 1933; Rosinski, 1953; Pfister, and were apparently separated from other members of Microstoma, 1973; Paden, 1975; Baral, 1984; Harrington, 1990; Fenwick, 1994), the novelty of the fungus was strongly supported. Considering that direct production of conidiogenous cells on ascospores is restricted 11 sequences of M. floccosum from the materials from Japan, Chi- to Cookeina sulcipes (Berk.) Kuntze (Boedijn, 1929; Boedijn, 1933; na, USA, and Mexico did not form a strongly supported clade Paden, 1975). Microstoma longipilum is the second report of the (MLBP = 79%, MPBP < 50%), M. floccosum may be heterogeneous. latter type of ascospore germination producing mitospores in Sar- Microstoma longipilum is the sixth reported species of the genus coscyphaceae. in Japan. In Japan, M. aggregatum is distributed in limited areas (a It is most likely that the conidial production in M. longipilum is few localities in Hokkaido and a single locality in Kawauchi, induced by the presence of water, and thus, conidial formation in Fukushima) and strict conservation measures have been taken in other species of Microstoma may be confirmed by immersing fresh each habitat (Ohmae et al., 2020). Since M. longipilum seems to be ascospores in water. Since the conidia of M. longipilum are very restricted to only two localities in primeval forests, these measures small and do not germinate, they probably function as spermatia. should be taken in each habitat.

For the molecular phylogenetic analyses, sequences of M. floc- Disclosure cosum collected in the USA, China, and Mexico, and M. radicatum collected in China were downloaded from GenBank (Table 1). In The authors declare no conflicts of interest. total, 36 sequences were analyzed. The aligned sequence matrix doi: 10.47371/mycosci.2021.03.003 ― 221 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223

Fig. 3. Molecular phylogenetic tree of Microstoma constructed using RAxML-NG 0.9.0 based on ITS-5.8S region. Branch support value was indicated in the following order: MLBP/MPBP. Nodes supported by MLBP or MPBP < 50% was indicated with ＇-＇

Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software Acknowledgements version 7: improvements in performance and usability. Molecular Biology and Evolution, 30, 772–780. https://doi.org/10.1093/molbev/mst010 We are grateful to Dr. Takamichi Orihara at the Kanagawa Pre- Katumoto, K. (2010). List of fungi recorded in Japan (in Japanese). The Kanto fectural Museum of Natural History for collecting samples. Branch of the Mycological Society of Japan. Kozlov, A. M., Darriba, D., Flouri, T., Morel, B., & Stamatakis, A. (2019) RAxML-NG: References a fast, scalable and user-friendly tool for maximum likelihood phylogenetic in- ference. Bioinformatics, 35, 4453–4455. https://doi.org/10.1093/bioinformatics/ Baral, H. O. (1984). Taxonomische und ökologische Studien über Sarcoscypha coc- btz305 cinea agg., Zinnoberrote Kelchbecherlinge. (Kurzfassung) (in German). Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018) MEGA X: Molecular Zeitschrift für Mykologie, 50, 117–145. evolutionary genetics analysis across computing platforms. Molecular Biology Boedijn, K. B. (1929). Beitrag zur Kenntnis der Pilzflora von Sumatra. Recueil des and Evolution, 35, 1547–1549. https://doi.org/10.1093/molbev/msy096 travaux botaniques néerlandais, 26, 396–439. Liu, T. Z., Wulantuya, & Zhuang, W. Y. (2018). A new species of Microstoma (Sarco- Boedijn, K. B. (1933). The genera and Cookeina in Netherlands India. scyphaceae) from China with a key to known species of the genus (in Chinese). Bulletin du Jardin Botanique de Buitenzorg, 13, 57–76. Mycosystema, 37, 555–558. https://doi.org/10.13346/j.mycosystema.180015 Darriba, D., Posada, D., Kozlov, A. M., Stamatakis, A., Morel, B., & Flouri, T. (2019). Ohmae, M., Yamamoto, K., & Orihara, T. (2020). First report of Microstoma apiculo- ModelTest-NG: A new and scalable tool for the selection of DNA and protein sporum (Sarcoscyphaceae, Pezizales) from Japan (in Japanese). Japanese Jour- evolutionary models. Molecular Biology and Evolution, 37, 291–294. https://doi. nal of , 61, 27–32. https://doi.org/10.18962/jjom.jjom.R01-10 org/10.1093/molbev/msz189 Otani, Y. (1980). Sarcoscyphineae of Japan. Transactions of the Mycological Society of Douanla-Meli, C., & Langer, E. J. (2005). Notes on discomycetes (Helotiales, Peziza- Japan, 21, 149–179. les): New species and new records from Cameroon. Mycotaxon, 92, 223–237. Otani, Y. (1990). Miscellaneous notes on Japanese discomycetes (in Japanese). Re- Fenwick, G. A. (1994). Some observations on the germination of Sarcoscypha austri- ports of the Tottori Mycological Institute, 28, 251–265. aca ascospores in the laboratory. Mycologist, 8, 124–127. https://doi. Paden, J. W. (1975). Ascospore germination, growth in culture, and imperfect spore org/10.1016/S0269-915X(09)80158-4 formation in Cookeina sulcipes and Phillipsia crispata. Canadian Journal of Harada, Y., & Kudo, S. (2000). Microstoma macrosporum stat. nov., a new taxonomic Botany, 53, 56–61. https://doi.org/10.1139/b75-008 treatment of a vernal discomycete (Sarcoscyphaceae, Pezizales). Mycoscience, Pant, D. C., & Tewari, V. P. (1973). Chemical reaction of the hairs of Microstoma 41, 275–278. https://doi.org/10.1007/BF02489683 floccosum. Mycologia, 65, 199–201. https://doi.org/10.1080/00275514.1973.1201 Harrington, F. A. (1990). Sarcoscypha in North America (Pezizales, Sarcoscyphace- 9418 ae). Mycotaxon, 38, 417–458. Raitviir, A. (1965). Sarcoscyphaceae in the Far East (in Russian). Eesti NSV Teaduste Hosaka, K., & Castellano, M. A. (2008). Molecular of Geastrales with Akadeemia toimetised. Bioloogia, 14, 529–535. special emphasis on the position of Sclerogaster. Bulletin of the National Science Rambaut, A. (2018). FigTree. Molecular evolution, phylogenetics and epidemiology, Museum. Series B, Botany, 34, 161–173. Edinburgh. Retrieved Sep 1, 2020, from http://tree.bio.ed.ac.uk/software/fig- Hosoya, T., & Otani, Y. (1997). Hyaloscyphaceae in Japan (1): Non-glassy-haired tree/ members of the tribe Hyaloscypheae. Mycoscience, 38, 171–186. https://doi. Pfister, D. H. (1973). Notes on discomycetes. III. Ascospore germination org/10.1007/BF02460853 and growth in culture of Nanoscypha tetraspora (Pezizales, Sarcoscyphineae). Kanouse, B. B. (1948). The genus Plectania and its segregates in North America. Mycologia, 65, 952–956. https://doi.org/10.1080/00275514.1973.12019510 Mycologia, 40, 482–497. https://doi.org/10.2307/3755155 Rosinski, M. A. (1953). Two types of spore germination in doi: 10.47371/mycosci.2021.03.003 ― 222 ― Y. Tochihara et al. / Mycoscience VOL.62 (2021) 217-223

(Scop. ex Fr.) Lambotte. Mycologia, 45, 302–306. https://www.jstor.org/sta- ble/4547697 Tochihara, Y., & Hosoya, T. (2019). Three new species of Incrucipulum (Lachnaceae, Helotiales, ) from Japan, Phytotaxa, 403, 25–38. https://doi. org/10.11646/phytotaxa.403.1.2 Wang, Y. -Z. (2001). Discomycetes of the Sarcoscyphaceae in Taiwan. Mycotaxon, 79, 329–336. Wang, Y. -Z. (2004). A new species of Microstoma from Taiwan. Mycotaxon, 89, 119–122. Zhuang, W. -Y., & Wang, Z. (1997). Some new species and new records of discomy- cetes in China. VII. Mycotaxon, 63, 307–321.

doi: 10.47371/mycosci.2021.03.003 ― 223 ―