Hydropodia silvae-nipponicae (Porotheleaceae), a new species from evergreen broad-leaved forests in Japan

Abstract

Hydropodia silvae-nipponicae, a new agaric species was described based on the specimens collected from evergreen broad-leaved forests in Japan. The present new species is distinct from H. subalpina, the type species of the genus by morphological characteristics and nuclear rDNA sequences. Hydropodia silvae-nipponicae is characterized by its nodulose cystidium-like terminal elements of pileipellis, cylindrical to clavate pileocystidia with irregular excrescences, and dimorphic pleurocystidia. It is the second species belonging to the genus Hydropodia.

Hydropus subalpinus (Höhn.) Singer has been known as easily identifiable species in the genus Hydropus Kühner ex Singer and it shares clearly distinct morphological features to the other members of the genus as follows: mycenoid or collybioid basidiomata, ochraceous brown pileus, a rooting stipe with pseudorhiza, and allantoid basidiospores (Singer, 1982; Hausknecht, Krisai-Greilhuber, & Klofac, 1997; Malysheva & Svetasheva, 2011; Fernández-Vicente, 2012; Kaygusuz, Ševčíková, Battistin, & Türkekul, 2020; Consiglio, Vizzini, Cooper, Marchetti, Angelini, Brugaletta, & Setti, 2021). Recent molecular phylogenetic studies revealed the strong polyphyly of the current concept of the genus Hydropus and many species previously placed in this genus are distantly related linages in the family Porotheleaceae Murrill and Cyphellaceae Lotsy, which are collectively recognized as “hydropoid fungi” (Kaygusuz et al., 2020; Consiglio et al., 2021; Vizzini, Consiglio, Marchetti, Borovička, Campo, Cooper, Lebeuf, & Ševčíková, 2022). Hydropus subalpinus also was transferred to the recently established genus Hydropodia Vizzini & Consiglio, mainly based on its isolated phylogenetic position in Porotheleaceae (Consiglio et al., 2021).

We continuously obtained several collections of a hydropoid fungus morphologically similar to Hydropodia subalpina (Höhn.) Vizzini, Consiglio & M. Marchetti (≡ Hydropus subalpinus) from evergreen broad-leaved forests in Ibaraki and Kanagawa Prefectures, central Honshu, Japan over the last decade. Our morphological observations and molecular phylogenetic analyses using nuclear ribosomal DNA sequences reveal that the hydropoid fungus is a distinct species from H. subalpina. Because Hydropodia is a monospecific genus with a sole species H. subalpina (Consiglio et al., 2021), here we describe the present hydropoid fungus as a novel species and second member of the genus.

Fresh basidiomata collected from field were photographed and observed macroscopically. Color abbreviations follow Munsell (1961). All descriptions of macroscopic characters were obtained from fresh basidiomata. The following abbreviations are applied to macroscopic descriptions: L = number of lamellae reaching the stipe, l = number of lamellulae between each pair of lamellae. Fresh materials were air-dried using a food dehydrator (Snackmaster Express FD-60; Nesco/American Harvest, Milwaukee, WI, USA) under 46 °C for 46 h. Dried specimens of the hydropoid fungus used in the present study were deposited in herbaria of the National Museum of Nature and Science, Tsukuba, Ibaraki, Japan (TNS) and Ibaraki Nature Museum, Bando, Ibaraki, Japan (INM). Additionally, four dried specimens housed at Tottori Mycological Institute, Tottori, Japan (TMI) which were preliminary reported as Hydropus subalpinus by Nagasawa (1990) were examined.

For light microscopy, hand-cut sections of pileus and stipe surfaces and lamellae from dried specimens were mounted in a drop of water, Melzer's reagent or 3% (w/v) KOH solution after staining with 1% (w/v) Congo red solution on glass slides. The slide preparations were examined and photographed using a OLYMPUS BX51 (Olympus, Tokyo, Japan) with differential interference contrast (DIC) equipment. Dimensions of basidiospores were measured from water-mounted sections, excluding the hilar appendage. Approximately 50 randomly selected basidiospores of each specimen were measured under a light microscope at 1,000 × magnification, using a NIKON ECLIPSE 80i (Nikon, Tokyo, Japan) with image analysis software. Basidiospores dimensions are reported as follows: minimum to maximum of length × minimum to maximum of width; Q, quotient of spore length and spore width in face view; Q_m, the means of Q-values. The notation (n/m/p) indicates that measurements were made on “n” randomly selected spores from “m” basidiomes of “p” collections following Consiglio et al. (2021). Each width of basidia and cystidia was measured at the widest part, and each length of them was measured from the apex (sterigmata were excluded in the basidia) to the basal septum.

DNA extraction, PCR and DNA sequencing of specimens examined in the present study were carried out according to the methods previously introduced by Kasuya, Hosaka, Uno, and Kakishima (2012). Internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) of ribosomal RNA gene (rDNA) were amplified using the primer pairs ITS1/ITS4 (White, Bruns, Lee, & Taylor, 1990) and LR0R/LR5 (Vilgalys & Hester, 1990), respectively.

A total of four ITS and five LSU sequences from seven specimens of the present hydropoid fungus were newly generated and used for the phylogenetic analyses (Table 1). These sequences were deposited to the International Nucleotide Sequence Databases (INSD; accession nos.: OQ676551-OQ676559). Additionally, 22 ITS and 22 LSU sequences of Porotheleaceae fungi were retrieved from the NCBI GenBank databases (https://www.ncbi.nlm.nih.gov/) and included in the analyses (Table 1). DNA sequences were initially aligned using Muscle v.3.6 (Edgar, 2004a, 2004b), followed by manual alignment in the data editor of BioEdit ver. 7.0.1 (Hall, 1999). A total of 171 ITS and 714 LSU nucleotide positions were respectively excluded from the analyses because of the presence of ambiguously aligned regions. The final alignments were deposited in TreeBASE (https://treebase.org) under the accession number S30066. Phylogenetic analyses were performed for the combined dataset of ITS and LSU sequences under maximum likelihood (ML) and maximum parsimony (MP) criteria. ML analysis was performed using MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) after testing the best models according to the methods previously introduced by Kasuya, Uzawa, and Hosaka (2022). According to the lowest Bayesian information criterion scores, Tamura-Nei (Tamura & Nei, 1993) with gamma distributed rate heterogenetic and a proportion of invariant sites (TN93+G+I) was chosen as the optimal substitution model for the analysis of the combined ITS and LSU dataset. MP analysis was conducted under the equally weighted parsimony criterion using PAUP version 4.0b10 (Swofford, 2002) by the same method reported by Kasuya et al. (2012, 2022). Sequences of Trogia delicata Corner were selected for outgroup (Table 1), which species was strongly supported as the sister of the major clade containing the family Porotheleaceae (Consiglio et al., 2021).

Table 1. Specimen identification, voucher specimen and/or isolate numbers, origin and INSD accession numbers for ITS and LSU sequences used for the present phylogenetic analyses.

Species names	Herbarium voucher; isolate	Origin	INSD accession numbers
			ITS	LSU
Hydropodia silvae-nipponicae a	TNS-F-82592 (Holotype); Kasuya B426	Japan, Ibaraki, Miho	OQ676556	OQ676551
H. silvae-nipponicae	TNS-F-82593; Kasuya B427	Japan, Ibaraki, Miho	n/a b	OQ676552
H. silvae-nipponicae	INM-2-83331; Kasuya B953	Japan, Ibaraki, Miho	n/a	OQ676553
H. silvae-nipponicae	INM-2-83332; Kasuya B954	Japan, Ibaraki, Miho	n/a	OQ676554
H. silvae-nipponicae	TNS-F-82594; Kasuya B1696	Japan, Ibaraki, Miho	OQ676557	OQ676555
H. silvae-nipponicae	TNS-F-82597; Kasuya B4668	Japan, Kanagawa, Yokohama	OQ676558	n/a
H. silvae-nipponicae	TMI 12139; Kasuya B1652	Japan, Tottori	OQ676559	n/a
H. subalpina	OKA-TR-K364; Fagus orientalis forest	Turkey	MN701620	MN700170
H. subalpina	OKA-TR-K380; Fagus orientalis forest	Turkey	MN701621	MN700171
H. subalpina	OKA-TR-B400; Fagus orientalis forest	Turkey	MN701622	MN700172
H. subalpina	AMB 18782; n/a b	Italy	OM422763	OM423641
H. subalpina	AMB 18783; n/a	Italy	OM422764	OM423642
H. subalpina	AMB 18784; n/a	Italy	OM422761	OM423638
H. subalpina	AMB 18785; n/a	Italy	OM422762	OM423639
Porotheleum domingense	JBSD131801a; n/a	Dominican Republic	OM422768	OM423646
P. domingense	JBSD131801b; n/a	Dominican Republic	OM422769	OM423647
P. fimbriatum	n/a; AFTOL-ID 1725	Belgium	DQ490626	DQ457673
P. fimbriatum	KUC20131022-23; n/a	Korea	KJ668472	KJ668324
P. nigripes	JBSD131803; n/a	Dominican Republic	OM422771	OM423648
P. omphaliiforme	AMB 18843; n/a	Italy	OM422774	OM423651
P. omphaliiforme	AMB 18842; n/a	Italy	OM422773	OM423650
P. omphaliiforme	MCVE 25757; n/a	Spain	OM422772	OM423649
P. parvulum	JBSD131802; n/a	Dominican Republic	OM422783	OM423657
Pseudohydropus floccipes	BRNM 751633; n/a	Czech Republic	OM422759	OM423635
P. floccipes	BRNM 816173; n/a	Czech Republic	OM422758	OM423634
P. floccipes	BRNM 825631; n/a	Czech Republic	OM422760	OM423636
P. globosporus	BAP 661 (Holotype, SFSU); n/a	São Tomé	MH414566	MH385340
Pulverulina ulmicola	TENN:029208; n/a	USA	HQ179667	HQ179668
P. ulmicola	n/a; KUBOT-KRMK-2020-13	India	MW425325	MW425344
Trogia delicata	DED 8235 (SFSU); n/a	São Tomé	MH414567	MH385341

^a Informations on newly generated sequences in the present study were shown in bold.

^b “n/a” means information not available.

The combined dataset of ITS and LSU consisted of 29 ingroups and one outgroup taxa. It had an aligned in length of 1685 characters including gaps, of which 1267 variable and phylogenetically uninformative, and 398 phylogenetically informative. The highest log likelihood of the resulting ML tree of the combined ITS and LSU dataset was -6956.51. The MP analysis of the combined ITS and LSU dataset yielded 10,000 most parsimonious trees, of which 236 trees were found in the first step of the heuristic search. Consistency index, retention index and rescaled consistency index of the most parsimonious trees were 0.6404, 0.8379 and 0.5365, respectively. The ML and MP analyses resulted in trees that were almost identical in topology. Hence, only the ML tree topology of the combined ITS and LSU dataset is shown in Fig. 1.

Fig. 1 - A phylogenetic tree generated from maximum likelihood (ML) analysis based on the combined dataset of the nuclear rDNA ITS region and LSU sequences of selected Porotheleaceae species. Taxon names indicated by bold are newly generated sequences from the present study. Bootstrap support values (BS) of ML and maximum parsimony (MP) greater than 60% are shown for each node (ML/MP), and BS less than 60% are indicated by an asterisk (*). Scale bar indicates the number of substitutions per site.

By ML and MP, ITS and LSU sequences generated from specimens of the present hydropoid fungus were placed within a strongly supported clade by bootstrap (BS) values [ML BS (%) /MP BS (%) =98/95; Fig. 1], and this clade was distinct from those of the other species of Porotheleaceae. One ITS sequence successfully generated from a specimen housed at TMI which was preliminary reported as Hydropus subalpinus by Nagasawa (1990) was placed in the clade consisting by the other specimens from Japan (Fig. 1). Moreover, this clade was resolved as a sister to the clade containing H. subalpina (Fig. 1).

Morphologically, Japanese specimens including materials examined by Nagasawa (1990) were distinct from H. subalpina in its nodulose cystidium-like terminal elements of pileipellis, cylindrical to clavate pileocystidia with irregular excrescences, and dimorphic pleurocystidia. The present hydropoid fungus is morphologically different from H. subalpina and it was placed in a distinct position in the phylogenetic tree (Fig. 1). We therefore treat the present hydropoid fungus as a new member of the genus Hydropodia. Diagnosis, detailed description and illustrations of the salient features of the present new species are given below.

Taxonomy

Hydropodia silvae-nipponicae T. Kasuya, Reiich. Kaneko, Takehashi & K. Hosaka, sp. nov.

MycoBank no.: MB 848052.

Figs. 2, 3.

Fig. 2 - Basidiomata of Hydropodia silvae-nipponicae. A: Basidiomata in the natural habitat (TNS-F-82592). B: Whole basidiomata (TNS-F-82592). C: Basidiomata with pseudorhiza (TNS-F-82591). D: Surface of a pileus (TNS-F-82594). E: Lamellae with the upper part of a stipe (TNS-F-82594). F: Basal part of a stipe (INM-2-83332). Bars: A, B 30 mm; C, D, E 20 mm; F 5 mm.

Fig. 3 - Microscopic features of Hydropodia silvae-nipponicae (TNS-F-82592). A: Terminal elements of pileipellis with pileocystidia. B: Broadly lageniform to subfusiform type of pleurocystidia. C: Cylindrical to narrowly subfusiform type of pleurocystidia. D: Cheilocystidia. E: Basidia. F: Basidiospores. G: Hyphae of stipitipellis. Bars: A, C, D 10 µm; B 15 µm; E, G 5 μm; F 8 μm.

Diagnosis: This species is morphologically similar to H. subalpina, but different in its thin-walled, nodulose, cystidium-like terminal elements of pileipellis, cylindrical to clavate pileocystidia with irregular excrescences, and dimorphic pleurocystidia which are broadly lageniform to subfusiform and cylindrical to narrowly subfusiform. ITS and LSU sequences of this species are also distinct from those of H. subalpina.

Holotype: JAPAN, Ibaraki, Inashiki, Miho, Tsuchiura (approx. 36º0'59.35 N, 140º20'52.89 E, alt. 25 m), scattered, caespitose or gregarious and rooting to buried wood or debris, or on fallen branches, on the ground among plant debris and litter in an evergreen broad-leaved forest dominated by Machilus thunbergii Siebold & Zucc., Neolitsea sericea (Blume) Koidz. and Aucuba japonica Thunb., 21 May 2012, T. Kasuya, TNS-F-82592.

DNA sequence ex-Holotype: INSD accession no. OQ676556 for ITS and OQ676551 for LSU.

Etymology: The epithet refers to Japanese forest (“silvae” = forest in Latin and “nippon” = Nippon, Japan), the habitat and origin of the new species.

Description: Basidiomata mycenoid or collybioid. Pileus 10-50 mm broad, hemispherical, conical, conico-convex then plano-convex, usually with a low, broadly conical umbo, surface hygrophanous, subviscid when moist, radially fibrillose, often shiny, slightly pale greyish (2.5Y 7.5/2), ochraceous (10YR 6/7.5), pale ochraceous brown (8YR 7/6) to ochraceous brown (6YR 3/7), often in the center somewhat brighter and at margin somewhat paler; margin slightly translucently striate, involute at first, becoming straight. Lamellae crowded, L = 30-45, 1 = 1-4, deeply emarginate, adnexed, narrowly adnate or almost free, slightly ventricose, 3-6 mm wide, white (N9.5), with an irregularly eroded, whitish edge. Stipe 25-80(-130) × 2-5 mm, cylindrical, hollow, cartilaginous, often slightly longitudinally striate and pruinose, white (N9.5) to ivory (2.5Y 8.5/1.5) when young, then becoming pale greyish (2.5Y 7.5/2) to pale brown (8YR 6.5/5), often with hairy, white mycelial strands at base, attached with a short to rather long (up to 50 mm long) pseudorhiza rooting to buried wood or debris when growing on the ground. Context white, elastic. Odor indistinct. Taste mild. Spore print white.

Pileipellis a thin ixocutis consisting of radially arranged, cylindrical, thin-walled, 3-4 μm wide hyphae, with pale brown to brown intracellular pigment. Pileocystidia and pileocystidioid terminal elements 10-30 × 5-10 μm, variable in shape, nodulose, cylindrical to clavate with irregular excrescences, thin-walled, with pale brown to brown intracellular pigment. Subcutis consisting of ventricose to fusiform, thin-walled, 10-22 µm wide hyphae, with pale brown to brown intracellular pigment. Stipitipellis consisting of parallelly arranged, cylindrical, thin-walled, 2-5 μm wide hyphae with irregularly nodulose excrescences and crystalline deposits. Hymenophoral trama regular, consisting of parallel, cylindrical, thin-walled, hyaline, 5-12 µm wide hyphae. Pleurocystidia scattered, hyaline, thin-walled, dimorphic: (1) broadly lageniform to subfusiform, 51-70 × 12-18 μm, often with a pedicel and obtuse apex, with numerous refractive-granulose contents, usually with apical crystalline or mucous deposits; (2) cylindrical to narrowly subfusiform, 68-72 × 8-9 µm. Cheilocystidia abundant, 30-65 × 11-16 μm, narrowly to broadly lageniform, fusiform or clavate, often with a pedicel and obtuse apex, hyaline, thin-walled but slightly thick-walled at the apex, sometimes with refractive-granulose contents, sometimes with apical crystalline or mucous deposits. Basidia 19-25 × 6-7 μm, clavate, 4-spored, with granular content, with a basal clamp-connection. Basidiospores 6-8(-8.5) μm × 2.5-3.5(-4) μm, mean = 7.3 × 2.9 μm (708/14/11), Q = 2.13-2.67, Q_m = 2.43, subcylindrical, narrowly cylindrical to allantoid, smooth, hyaline, thin-walled, with small hilar appendage, with granulose content or mono-guttulate, inamyloid, slightly congophilic. Clamp-connections present.

Habit and habitat: Solitary, scattered, caespitose or gregarious and rooting to buried wood or debris, or on fallen branches, on the ground among plant debris and litter in the evergreen broad-leaved forest which mainly dominated by M. thunbergii, N. sericea, Quercus acuta Thunb. and A. japonica, in May to Jun.

Additional specimens examined: JAPAN, Ibaraki, Inashiki, Miho, Tsuchiura: 23 May 2009, T. Kasuya and Y. Kitadate, INM-2-67145; 4 Jun 2011, T. Kasuya, TNS-F-82591; 21 May 2012, T. Kasuya, TNS-F-82593; 19 May 2013, T. Kasuya, INM-2-83331, INM-2-83332, INM-2-83333; 22 May 2014, T. Kasuya and M. Omori, TNS-F-82594. JAPAN, Kanagawa, Yokohama, Aoba, Jike: 3 Jun 2019, M. Nakajima, TNS-F-82596. JAPAN, Kanagawa, Yokohama, Kohoku, Hiyoshi Campus of Keio University: 18 May 2022, R. Kaneko, TNS-F-82597. JAPAN, Kanagawa, Miura, Hayama, Nagae: 29 May 2019, M. Nakajima, TNS-F-82595. JAPAN, Tottori, Tottori, Kokoge: 1 Jun 1985, E. Nagasawa, TMI 12139; 10 Jun 1988, E. Nagasawa, TMI 13822; 5 Jun 1986, E. Nagasawa, TMI 14656; 30 May 1992, E. Nagasawa, TMI 16002.

Japanese name: Kicha-mori-no-kasa [proposed by Nagasawa (1990) for specimens preliminary reported as Hydropus subalpinus].

Notes: Macroscopically, H. silvae-nipponicae is very similar to H. subalpina. However, several microscopic characteristics between them are quite different. Pileocystidia of H. subalpina are thick-walled, versiform, sublageniform, fusiform, subcylindrical, clavate or capitate (Hausknecht et al., 1997; Malysheva & Svetasheva, 2011; Fernández-Vicente, 2012; Kaygusuz et al., 2020; Consiglio et al., 2021) while those of H. silvae-nipponicae are thin-walled, cylindrical to clavate, with irregular excrescences. Although pleurocystidia of H. silvae-nipponicae are dimorphic, those of H. subalpina are monomorphic (lageniform to subfusiform; Consiglio et al., 2021). Basidiospores of H. silvae-nipponicae are smaller than those of H. subalpina (8.1 × 3.58 μm; Consiglio et al., 2021). Ecologically, H. subalpina has been collected mainly from Fagus spp. forests in Europe, Caucasus and Turkey (Hausknecht et al., 1997; Malysheva & Svetasheva, 2011; Fernández-Vicente, 2012; Kaygusuz et al., 2020; Consiglio et al., 2021). Hydropodia silvae-nipponicae is also distinguishable from H. subalpina in their habitat because the former species usually forms its basidiomata in evergreen broad-leaved forests dominated by Machilus, Neolitsea, Quercus and Aucuba spp. trees. The present new species is probably widespread to the south of the warm-temperate region of Honshu, Japan.

Disclosure

The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.

Acknowledgments

We are very much obliged to Ms. K.-O. Nam and Ms. Megumi Otsuka of the National Museum of Nature and Science for help with assisting molecular experiments. Special thanks also go to Mr. Yusuke Kitadate, Mr. Minoru Nakajima and Ms. Maya Omori for facilitating the fieldwork. We also deeply thank Mr. Eiji Nagasawa, Tottori Mycological Institute, Japan, for his valuable helps to loan of Hydropodia specimens and suggestions to this study. This work was supported in part by JSPS KAKENHI Grant Numbers JP20K06805 and JP23K05895.

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