Microstoma longipilum sp. nov. (Sarcoscyphaceae, Pezizales) from 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.

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. macrosporum, and M. protractum have been 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 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.

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 (Bacto^TM Malt 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).

Table 1. Taxa analyzed in molecular phylogenetic analyses.

Taxon	Specimen no.	Locality	Coll. Date	Isolates (NBRC)	ITS GenBank 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 Locality)	2017/10/6	-	LC584236
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 (holotype)	JAPAN, Okayama, Maniwa	2015/7/22	114764	LC584249
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/KPM-NC 28281	JAPAN, Kanagawa, Yamakita	2020/7/15	-	LC584252
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
Sarcoscypha occidentalis	FLAS-F-61366 NKR-56	USA, Georgia, Rabun, Appalachian Mountains	2017/7/21	-	MT3740261
S. tatakensis	TNM F0993	TAIWAN	-	-	NR_1566011

¹Downloaded sequences from GenBank.

²Outgroup

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.

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: Hymenium. 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: Ascus. 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: Hypha 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).

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.

Diagnosis: Characterized by aggregated 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.

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 hyphae (up to 3 µm wide) toward the ectal excipulum. Apothecial margin thick, composed of two-layered ectal excipulum (referred to above) and medullary excipulum; medullary excipulum textura prismatica to t. angularis, composed of irregular-shaped cells; cells hyaline, thin-walled, 1.2–38 × 1.2 ×17 µm, becoming more cubical and more thick-walled toward the upper layer. Asci 275–350 × 10–17.5 µm (n = 40), maturing simultaneously, clavate, operculate with eccentric opercula, inamyloid, thick-walled (up to 2.5 µm thick), especially thicker-walled below the opening where the inner layer becoming abruptly thickened toward the opening while the outer layer becomes thinner, arising from simple septa, abruptly thinner near bases and becoming filiform or irregularly incurving, ending up furcate or swollen. Ascospores ovoid to ellipsoid without apiculi, hyaline, smooth, (20–)21.9–26.1(–27.5) × 11–12.5 µm (24 ± 2.12 × 11.6 ± 0.48 µm), Q = (1.8–)1.9–2.3 (–2.5) (2.1 ± 0.2) in the dead state (mounted in MLZ, n = 30), (20–)24–28.6(–30) × (11.5–)12.5–13.7(–14) µm (26.3 ± 2.3 × 13.1 ± 0.60 µm), Q = (1.7–)1.8–2.2(–2.3) (2.0 ± 0.17) in the living state (mounted in CBW, n = 30), containing numerous globose lipid bodies, encased in gelatinous sheath that swells and immediately detaches from the spore in KOH solutions, not germinating within asci, germinating by elongating single germ tube from either polar end on PDA or CMA; conidiogenous cells produced directly from ascospores when immersed in tap water within 12 h, almost spherical with opening, 3–4.5 µm at the widest parts, blastically producing spherical conidia; conidia 2–2.5 µm wide; ascospores producing conidia directly never producing germ tubes. Paraphyses filiform with apices sometimes swelling or branched irregularly like fingers, thin-walled, 1.5–3 µm wide, arising from subhymenium, almost equal to asci, anastomosed at the base, containing numerous red or orange droplets containing carotenoids.

Distinguishing characteristics: Microstoma longipilum is characterized by extremely long hairs (> 2 mm), which are much longer than those of any other member of the genus. Microstoma longipilum differs from M. protractum and M. radicatum in the absence of dark-brown rhizoids at the basal parts of stipes. In the Microstoma species lacking rhizoids, M. longipilum resembles M. aggregatum in producing aggregated apothecial clusters but can easily be distinguished by the existence of much longer hairs, larger asci, anastomosed paraphyses, and their occurrence in different seasons (Table 2). Microstoma longipilum is also distinguishable from other species of the genus by aggregated apothecia, ascospores without apiculi, and pale-colored discs (Table 2).

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

	Distribution	References	Occurring season		Disc color	Asci (µm)	Ascospores (µm)	Paraphyses	Hairs (µm)	Special remarks
M. aggregatum	Japan, China1	Otani (1990)	autumn		pink to coral color	214.4–272.0 × 12.8–14.4	24.0–32.0 × 9.6–12.8	not anastomosed	252–378 × 12.8	Apothecia are aggregated.
M. apiculosporum	Taiwan, Japan2	Wang (2004)	autumn (Sep to Nov2)		orange red	325–335 × 12–14	25–30 × 9–10 (bipolar apiculate)	anastomosed	270–1200 × 12–15
M. camerunense	Cameroon	Douanla-Meli & Langer (2005)	Sep		pink (brownish yellow when dry)	245–335 × (6–)7–12	(13–)14–16(–17) × 3–4(–4.5) (bipolar apiculate)	anastomosed	(up to 150 long)
M. floccosum	USA3, Canada4, Mexico4, South Korea4, Russia5, Japan6, Taiwan4	Kanouse (1948)	summer (mainly Jun to Aug)		scarlet	300–350 × 18–20	20–35 × 14–16	anastomosed	(unmentioned)
		Raitviir (1965)	summer to early autumn (Aug to Sep)		dull red	300–350 × 18–20	27–31 × 11–14	anastomosed	over 1000 × 12–18
M. longipilum	Japan	this study	early summer (Jun to Jul)		dull pink to pale orange	275–350 × 10–17.5	20–30 × 11.5–14 in living, 20–27.5 × 11–12.5 in dead	anastomosed	up to 3000 (usually over 1500) × 10–20	Apothecia are aggregated.
M. macrosporum	Japan, China1	Harada & Kudo (2000)	autumn and early spring7		orange red	500–560 × 23–26	42–60 × 16–21	anastomosed	longer hairs: 450–550 × 20–28, shorter hairs: 30–100 × 5–7	Margin of apothecia is split into 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)

Ecology: Forming apothecia from early Jun to Jul (rainy season in Japan) on rotten wood of broad-leaved trees.

Other specimens examined: JAPAN, Okayama, Maniwa, on rotten wood of Fagus crenata, 19 Jul 2014, leg. T. Hirao (TNS-F-61424). Locality as above, on rotten wood of an unidentified tree (probably F. crenata), 30 Jun 2015, leg. T. Hirao (TNS-F-61948). Locality as above, on rotten wood of F. crenata, 10 Jul 2016, leg. T. Hirao (TNS-F-65705). Locality as above, on rotten wood of F. crenata, 8 Jul 2020, leg. T. Hirao (TNS-F-60527; 60528). JAPAN, Kanagawa, Yamakita, on rotten wood of a broad-leaved tree (not F. crenata) lying near small swamp surrounded by Alnus japonica (Thunb.) Steud. and Cornus controversa Hemsl. ex Prain, 15 Jul 2020, leg. T. Orihara (TNS-F-60530, duplicate KPM-NC 28281).

Notes: The glassy wall materials of the hairs of M. longipilum are instantly dissolved with 10% KOH (Fig. 1Q). The same phenomenon is also known in glassy-haired members of Hyaloscyphaceae (Helotiales), such as the genera Mollisina Höhn. ex Weese and Urceolella Boud. (Hosoya & Otani, 1997). In Microstoma, the phenomenon was reported in M. floccosum by Pant and Tewari (1973), M. macrosporum (Harada & Kudo, 2000), and M. apiculosporum (Ohmae et al., 2020). Although Harada and Kudo (2000) reported that hairs were dissolvable in a few hours with 2.5% KOH, hair-dissolution should be checked instantly using KOH at higher concentrations for efficient observation.

In Microstoma, the presence of hyphal mats has been reported in M. apiculosporum (Wang, 2004; Ohmae et al., 2020), M. floccosum (Kanouse, 1948; Wang, 2001), and M. radicatum (Liu et al., 2018). In the present study, for the first time, we confirmed that hyphae consisting of hyphal mats of M. longipilum showed the same chemical reaction as hairs using 10% KOH (Fig. 1R).

The direct germination of ascospores to produce mitospores in Sarcoscyphaceae have been reported in some species of Cookeina Kuntze (Boedijn, 1929; Boedijn, 1933; Paden, 1975), Nanoscypha Denison (Pfister, 1973), and Sarcoscypha (Fr.) Boud. (Rosinski, 1953; Baral, 1984; Harrington, 1990; Fenwick, 1994). In conidia production, while blastoconidia production on germ tubes is widely known in such genera (Boedijn, 1933; Rosinski, 1953; Pfister, 1973; Paden, 1975; Baral, 1984; Harrington, 1990; Fenwick, 1994), direct production of conidiogenous cells on ascospores is restricted to Cookeina sulcipes (Berk.) Kuntze (Boedijn, 1929; Boedijn, 1933; Paden, 1975). Microstoma longipilum is the second report of the latter type of ascospore germination producing mitospores in Sarcoscyphaceae.

It is most likely that the conidial production in M. longipilum is induced by the presence of water, and thus, conidial formation in other species of Microstoma may be confirmed by immersing fresh ascospores in water. Since the conidia of M. longipilum are very small and do not germinate, they probably function as spermatia.

 

For the molecular phylogenetic analyses, sequences of M. floccosum collected in the USA, China, and Mexico, and M. radicatum collected in China were downloaded from GenBank (Table 1). In total, 36 sequences were analyzed. The aligned sequence matrix was composed of 482 sites. In the ML analysis, a ML tree was yielded based on the SYM+I+G4 model. In the MP analysis, 315 sites without gaps and missing data were used, and four most parsimonious trees were generated with tree length = 238, consistency index = 0.600897, and retention index = 0.863077. Since there were no topological contradictions between the ML best-scored tree and one of the MP trees, the ML tree was illustrated and ML bootstrap values (MLBP) and MP bootstrap values (MPBP) over 50% were indicated on branches in this order (Fig. 3).

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 '-'

Clades including M. apiculosporum, M. macrosporum, M. radicatum, M. aggregatum, and M. longipilum were strongly supported (MLBP/MPBP > 85%), except for MPBP for M. macrosporum, which had lower support. Since five sequences of M. longipilum formed a strongly supported clade (MLBP = 87% and MPBP = 94%) and were apparently separated from other members of Microstoma, the novelty of the fungus was strongly supported. Considering that 11 sequences of M. floccosum from the materials from Japan, China, USA, and Mexico did not form a strongly supported clade (MLBP = 79%, MPBP < 50%), M. floccosum may be heterogeneous.

Microstoma longipilum is the sixth reported species of the genus in Japan. In Japan, M. aggregatum is distributed in limited areas (a few localities in Hokkaido and a single locality in Kawauchi, Fukushima) and strict conservation measures have been taken in each habitat (Ohmae et al., 2020). Since M. longipilum seems to be restricted to only two localities in primeval forests, these measures should be taken in each habitat.

Disclosure

The authors declare no conflicts of interest.

Acknowledgements

We are grateful to Dr. Takamichi Orihara at the Kanagawa Prefectural Museum of Natural History for collecting samples.

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