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Microstoma Longipilum Sp. Nov. (Sarcoscyphaceae, Pezizales)

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Microstoma Longipilum Sp. Nov. (Sarcoscyphaceae, Pezizales) Mycoscience VOL.62 (2021) 217-223 Short communication Microstoma longipilum sp. nov. (Sarcoscyphaceae, Pezizales) 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 fungus. We additionally reported the overlooked morphology of hyphal mats, conidiogenous cells produced directly from ascospores, 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 genus 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 ascospore germination, asco- porum (Y. Otani) Y. Harada & S. Kudo, M. protractum (Fr.) spores 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. Sarcoscypha occidentalis (Schwein.) Sacc. and S. tataken- sis Yei Z. Wang & Cheng L. Huang were selected as the outgroup Taxonomy (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.
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