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Microstoma Longipilum Sp. Nov. (Sarcoscyphaceae, Pezizales) from Japan

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Microstoma Longipilum Sp. Nov. (Sarcoscyphaceae, Pezizales) from Japan Mycoscience: Advance Publication doi: 10.47371/mycosci.2021.03.003 Short Communication (Received November 4, 2020; Accepted March 15, 2021) J-STAGE Advance Published Date: May 7, 2021 Short communication Microstoma longipilum sp. nov. (Sarcoscyphaceae, Pezizales) from Japan Yukito Tochihara a, b, *, Tomoya Hirao c, Muneyuki Ohmae d, Kentaro Hosaka b, and Tsuyoshi Hosoya b 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 *E-mail: [email protected] 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 The genus Microstoma Bernstein (Sarcoscyphaceae, Pezizales) is characterized by long stipes, white-hairy receptacles, orange to red discs, gelatinized ectal excipulum and ascospores with smooth surfaces (Otani, 1980). Seven species have been differentiated in the genus: M. aggregatum Otani, M. apiculosporum Yei Z. Wang, M. camerunense Douanla-Meli, M. floccosum (Sacc.) Raitv., M. macrosporum (Y. Otani) Y. Harada & S. Kudo, M. protractum (Fr.) Kanouse, and M. radicatum T.Z. Liu, Wulantuya & W.Y. Zhuang (Liu, Wulantuya, & Zhuang, 2018; Ohmae, Yamamoto, & Orihara, 2020). In Japan, M. aggregatum, M. apiculosporum, M. floccosum, M. macrosporumAdvance, and M. protractum have been Publication reported (Katumoto, 2010; Ohmae et al., 2020). In 2014, the second author of the present study collected a specimen of Microstoma characterized by unique morphological features in a primeval forest of Fagus crenata Blume in Maniwa, Okayama. Thereafter specimens of the same fungus were collected yearly from the same locality. In 2020, a new locality of the fungus was - 1 - Mycoscience: Advance Publication found in a forest dominated by broad-leaved trees (not including Fagus spp.) in Yamakita, Kanagawa. The fungus resembled M. aggregatum in that it had aggregated apothecia like colonial corals but differed from it by having much longer excipular hairs. In this study, we report the fungus as a new species to science based on morphology and molecular phylogeny. Some fresh specimens of the fungus were used to establish cultures, and others were air-dried for 1 wk at 20 °C and deposited in the mycological herbarium of the National Museum of Nature and Science, Tsukuba, Japan (TNS) and the Kanagawa Prefectural Museum of Natural History, Odawara, Japan (KPM). To obtain isolates, a piece of an apothecium was pasted under the lid of a Petri dish so that ascospores could be freely discharged onto potato dextrose agar (PDA; Nissui, Tokyo, Japan). Germinated ascospores were transferred to PDA slants to establish pure isolates. Isolates were deposited in the NITE Biological Resource Center (NBRC), Kisarazu, Japan. To observe ascospore germination, ascospores were also discharged onto corn meal agar (CMA; Nissui), stored at 20 °C, and observed after 12 h. Pieces of medium with ascospores were then cut out using sterilized scalpels, transferred into new Petri dishes, immersed in tap water at 20 °C, and examined after 12 h. The germination of ascospores and micromorphological characteristics of the apothecia were examined using cotton blue (Wako Pure Chemical Industries, Osaka, Japan) dissolved in water (CBW) or tap water as a mounting fluid in the living state using a BX51 microscope equipped with a Nomarski interference contrast device (Olympus, Tokyo, Japan). To check the ascal iodine reaction, Melzer’s reagent (MLZ) was used. To confirm the swelling of ascospores and dissolution of glassy materials of hairs, 3% or 10% (w/v; the same applied for all ‘%’ described below for concentrations of solutions) potassium hydroxide (KOH) aqueous solution was used. Ascospore sizes were recorded both in the living state (= just after immersed in CBW) and in the dead state (= 6 h after immersed in MLZ) and were described in the following order: variation of length and width (arithmetic mean of length and width ± standard deviation), variation of Q (arithmetic mean of Q ± standard deviation). Q is the ratio of length/width.Advance Publication Molecular phylogenetic analyses were conducted including other species of the genus Microstoma using the internal transcribed spacer region of nuclear ribosomal DNA containing partial ITS1-5.8S-ITS2 (ITS-5.8S). For the five species of Microstoma known in Japan, sequences were derived from specimens at the TNS herbarium. DNA was extracted from mycelia cultivated on 2% (w/v) malt extract broth (BactoTM Malt - 2 - Microstoma longipilum sp. nov. (Sarcoscyphaceae, Pezizales) from Japan Extract; Thermo Fisher Scientific, Waltham, MA, USA) following the modified CTAB method (Hosaka & Castellano, 2008; Tochihara & Hosoya, 2019). When isolates were not available, DNA was extracted from pieces of dried apothecia using the same method. The ITS-5.8S region was then amplified, sequenced, and aligned following procedures described by Tochihara and Hosoya (2019). Aligned sequences were deposited in the DNA Data Bank of Japan (DDBJ) (Table 1). Additionally, sequences > 400 bp derived from non-Japanese Microstoma samples were obtained from GenBank and added to the phylogenetic analyses. Sarcoscypha occidentalis (Schwein.) Sacc. and S. tatakensis Yei Z. Wang & Cheng L. Huang were selected as the outgroup (Table 1). The obtained sequences (Table 1) were aligned using MAFFT 7 (Katoh & Standley, 2013) under the Q-INS-i option and manually edited. Molecular phylogenetic analyses were performed based on the maximum likelihood (ML) and the maximum parsimony (MP) methods. ML analysis was conducted using RAxML-NG 0.9.0 (Kozlov, Darriba, Flouri, Morel, & Stamatakis, 2019) with 1,000 bootstrap replications after suitable model estimation using Modeltest-NG 0.1.6 (Darriba et al., 2019) based on Akaike’s information criterion. MP analysis was conducted using MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). Gaps and missing data were eliminated. A heuristic search was carried out under the tree bisection reconnection branch swapping (TBR) algorithm with search level 2, in which the initial trees were obtained by the random addition of sequences (10 replicates). Branch support was evaluated by 1,000 bootstrap replications. Phylogenetic trees were illustrated using FigTree 1.4.4 (Rambaut, 2018) based on the ML and MP analyses. The ultimate sequence matrix and ML best- scored tree were registered to TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S26996). Taxonomy Microstoma longipilum Tochihara, T. Hirao & Hosoya, sp. nov. Figs. 1, 2. MycoBank no.: MB 836783. AdvanceDiagnosis: Characterized by aggregated Publication apothecia, long acute hairs, and salmon pink to pale orange discs. Holotype: JAPAN, Okayama, Maniwa, on rotten wood of Fagus crenata, 22 Jul 2015, leg. T. Hirao (TNS-F-61946). DNA sequence ex-holotype: LC584249 (ITS). Etymology: Referring to the long hairs of the apothecia. - 3 - Mycoscience: Advance Publication Japanese name: Karasake-kitsune-no-sakazuki (karasake = obsolete term meaning ‘salmon pink’, kitsune-no-sakazuki = cup for fox meaning Microstoma spp.) Description: Hyphal mats scattered on rotten wood of broad-leaved tree, dark brown, composed of complexly interwoven hyphae; hyphae resembling the apothecial hairs, hyaline to dark brown, 6–10 µm wide, glassy walled; glassy parts instantly dissolved in 10% KOH. Apothecia usually aggregated, deeply cupulate, 4–16 mm diam, subsessile to stipitate, up to 15 mm high (usually 10 mm high); outside totally covered by long, acute and white hairs usually bundled together; stipes up to 5 × 1.5–3 mm, integrated with each other near bases, not arising from rhizoids (pseudorhiza). Disc concave, dull pink to pale orange when fresh, becoming rather more intense when dry. Hairs arising from outer and inner ectal excipular cells, cylindrical, acute toward the apices, with thick and glassy walls, up to 3000 (usually over 1500) × 10–20 µm (including the glassy portion), elongating percurrently; glassy portion 2.5–8 µm thick (usually 5 µm thick), not stained by MLZ or CBW, instantly dissolved in 10% KOH. Subhymenium 30–40 µm thick, composed of interwoven hyphae; hyphae 1.9–4.0 µm wide, containing red or orange droplets containing carotenoids as seen in paraphyses. Ectal excipulum ca. 150 µm thick, composed of thick-walled refractive cells easily separated from medullary excipulum in squash mounts, consisting of two layers: inner layer composed of irregular-shaped cells 2.5–18 × 2.5–8 µm, thick-walled (up to 2 µm thick), becoming smaller and thinner-walled near the margin; outer layer composed of globular cells; cells 2.5–10 × 2.5–9 µm, thick-walled (up to 2 µm thick), becoming perhaps thinner-walled near the margin. Medullary excipulum 50–75 µm thick; upper part textura porrecta composed of thick hyphae (up to 7 µm wide) frequently branching; lowermost part composed of thinner
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