
==== Front
Mycoscience
Mycoscience
mycosci
Mycoscience
1340-3540
1618-2545
The Mycological Society of Japan Tokyo, JAPAN

mycosci.2023.09.002
10.47371/mycosci.2023.09.002
Full Paper
Taxonomic and ecological significance of synnema-like structures/acanthophyses produced by Physisporinus (Meripilaceae, Polyporales) species
Shino Ryotaro rshino756@gmail.com

a *
Sotome Kozue b
Endo Naoki b
Maekawa Nitaro b
Nakagiri Akira b
a The United Graduate School of Agricultural Sciences, Tottori University
b Fungus/Mushroom Resource and Research Center (FMRC), Faculty of Agriculture, Tottori University
* Corresponding author. The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori, Tottori 680-8553, Japan. E-mail address:rshino756@gmail.com (R. Shino).
20 11 2023
2024
64 6 136149
5 7 2023
14 9 2023
18 9 2023
2024, by The Mycological Society of Japan
https://creativecommons.org/licenses/by-nc-nd/4.0/ 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/).
Physisporinus, a genus in Polyporales, Basidiomycota, is a versatile fungus that lives as a wood decomposer, a potential pathogen of standing trees, and an orchid mycobiont. We previously reported that some Physisporinus species inhabiting wet wood in aquatic environments such as streams and waterfalls form synnema-like structures (SSs) bearing acanthophyses at their apices, and that they produce acanthophyses on vegetative hyphae when cultured on agar media. In this study, we investigated the acanthophysis-forming ability in Physisporinus and allied genera, and experimentally demonstrated the function of SSs. Phylogenetic analyses and observations of Meripilus, Physisporinus and Rigidoporus cultures showed that all of the strains forming acanthophyses belonged to Physisporinus, whereas strains of Meripilus and Rigidoporus did not produce acanthophyses. These findings suggest that SS/acanthophysis formation is a useful taxonomic character for members of Physisporinus. When Physisporinus strains were cultured under oxygen (O2) concentrations of 5, 10, 20 and 40%, most of those cultured under 20% O2 formed the most acanthophyses. According to these experimental data, the SSs/acanthophyses in Physisporinus were considered to have a respiratory function. Physisporinus probably acquired the SS/acanthophysis-forming ability to adapt to moist and/or aquatic habitats and to decay wet wood in which the O2 concentration is often low.

freshwater
Meripilus
oxygen concentration
phylogenetic analysis
Rigidoporus
==== Body
pmc1. Introduction

Physisporinus P. Karst. is a poroid fungus in Meripilaceae, Polyporales, Agaricomycetes, Basidiomycota (Justo et al., 2017). This cosmopolitan genus mainly decays dead broad-leaved and coniferous trees (Breitenbach & Kränzlin, 1986; Dai, 2012; Gilbertson & Ryvarden, 1987; Núñez & Ryvarden, 2001; Ryvarden et al., 2022; Ryvarden & Gilbertson, 1994; Ryvarden & Melo, 2017). Some species may cause butt rot in living Japanese cedars [Noguchi et al., 2007 (as Basidiomycete-B)], or establish mycorrhizal relationships with mycoheterotrophic orchids (Yamashita et al., 2020). Based on morphological and phylogenetic studies of aquatic fungi inhabiting wet wood in streams, we previously reported that five clades of fungi in the genus Physisporinus, i.e., two new species (P. microacanthophysis Shino, Sotome & Nakagiri and P. rhizomorphae Shino, Sotome & Nakagiri) and three unidentified groups (P. cf. 1 eminens, P. cf. 2 eminens, and P. cf. furcatus) form synnema-like structures (SSs) and produce numerous acanthophyses at their apices (Shino et al., 2022). Since cultures isolated from SSs or basidiocarps produced several types of acanthophyses on agar media, we considered that these characters might be useful as a taxonomic trait of Physisporinus. However, our previous study did not examine whether genera that are closely related to Physisporinus also produce SSs/acanthophyses. Phylogenetically, Physisporinus is closely related to both Meripilus P. Karst. (Chen & Dai, 2021; Shino et al., 2022; Tomšovský et al., 2010), which is the type genus of the Meripilaceae (Binder et al., 2013; Jülich, 1981; Justo et al., 2017), and Spongipellis Pat. (Kotiranta et al., 2017; Spirin et al., 2022; Wang & Dai, 2022). Spongipellis forms basidiocarps composed of a duplex context and generative hyphae with clamp connections (Ryvarden, 1991; Spirin et al., 2022) and can be distinguished from Physisporinus having a simplex context and generative hyphae without clamp connections in the basidiocarps (Gilbertson & Ryvarden, 1987). Meripilus species produce pileate basidiocarps with single to numerous brownish pilei arising from a short stipe or a base. This type of basidiocarp differs from the whitish resupinate basidiocarps found in Physisporinus, but the micromorphological features in basidiocarps of Meripilus (monomitic hyphal system, generative hyphae without clamp connections, and smooth and broadly ellipsoid to subglobose basidiospores with inamyloid reaction in Melzer's reagent) are similar to those found in Physisporinus (Gilbertson & Ryvarden, 1987). These characteristics are also observed in Leucophellinus Bondartsev & Singer, Oxyporus (Bourdot & Galzin) Donk, and Rigidoporus Murrill, which have been phylogenetically assigned to Hymenochaetales (Wu et al., 2017). Leucophellinus, which produces clavate and occasionally septate cystidia and has distinctly thick-walled basidiospores (Núñez & Ryvarden, 2001), is distinguishable from Physisporinus and related genera. Many species of Oxyporus and Rigidoporus resemble each other in that they form cystidia in their basidiocarps, but these taxa differ from Physisporinus, which has no cystidia (Ryvarden & Gilbertson, 1994). Regarding the former two genera, Pouzar (1966) treated Oxyporus as a subgenus of Rigidoporus because of their morphological similarities including the above features. However, several mycologists (Corner, 1987; Donk, 1967; Ryvarden & Johansen, 1980) proposed that these genera should remain separate because Rigidoporus typically produces basidiocarps with bright colors and forms cystidia in tramae while Oxyporus has pale-colored basidiocarps with cystidia in hymenia (Ryvarden, 1991). Recently, Wu et al. (2017) integrated Oxyporus into Rigidoporus since their phylogenetic analysis showed that the type species of the two genera grouped in the same clade in Hymenochaetales. Moreover, they transferred part of the remaining species of Rigidoporus, which were found belonging to Polyporales, to Physisporinus. As a result of this and other studies, Physisporinus currently accommodates several species that form apically encrusted cystidia [e.g., P. eminens (Y.C. Dai) F. Wu, Jia J. Chen & Y.C. Dai, formerly treated as R. eminens Y.C. Dai (Dai, 1998); P. furcatus (Núñez & Ryvarden) F. Wu, Jia J. Chen & Y.C. Dai, formerly R. furcatus Núñez & Ryvarden (Núñez et al., 2001); P. lineatus (Pers.) F. Wu, Jia J. Chen & Y.C. Dai, formerly R. lineatus (Pers.) Ryvarden (Ryvarden, 1972; Ryvarden & Johansen, 1980); P. pouzarii (Vampola & Vlasák) F. Wu, Jia J. Chen & Y.C. Dai, formerly R. pouzarii Vampola & Vlasák (Vampola & Vlasák, 2012)] and have pore surfaces with vivid colors when fresh [e.g., P. lavendulus F. Wu, Jia J. Chen & Y.C. Dai (Wu et al., 2017); P. roseus Jia J. Chen & Y.C. Dai (Chen & Dai, 2021); P. sulphureus Y.C. Dai (Dai & Dai, 2018)]. Thus, Physisporinus and Rigidoporus have become difficult to clearly distinguish by the morphology of their basidiocarps. Hence, the first objective of this study is to evaluate the taxonomic significance of SS/acanthophysis formation among Physisporinus, phylogenetically related genus Meripilus and morphologically similar genus Rigidoporus, and to identify other taxonomically important characters found in their cultures, such as formation of clamp connections at hyphal septa, as well as the presence of conidia and plectenchymata in mycelia.

We previously reported that acanthophyses on the apices of SSs are not conidia because they neither easily detach from SSs nor germinate hyphae (Shino et al., 2022). Since SSs have been often found at the water-boundary part of wet wood in aquatic environments such as streams and waterfalls, we hypothesized that the SSs of Physisporinus may be associated with the respiration for mycelia creeping in the water-saturated wood tissue where oxygen (O2) levels tend to be lower than in the atmosphere. Therefore, as the second objective of this study, we aim to verify this hypothesis by experiments using cultures of Physisporinus and to discuss the ecological significance of SSs/acanthophyses.

2. Materials and methods

2.1. Samples

24 specimens and 41 strains were tested in this study (Figs. 1, 6; Table 1). Procedures for the establishment of dried specimens and living isolates followed Shino et al. (2022). We also used strains preserved in the Fungus/Mushroom Resource and Research Center (FMRC), Faculty of Agriculture, Tottori University, and strains obtained from the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (Table 1).

Fig. 1 - Basidiocarps (A-C), SSs (D) bearing acanthophyses (E), and rhizomorphs (F) of Physisporinus in nature. A: Whitish basidiocarps produced on wet wood nearby streams (P. cf. 2 eminens TUMH 65445). B: Basidiocarps (P. cf. 1 eminens TUMH 65440). C: Pore surface of basidiocarps (P. cf. 1 eminens TUMH 65442). D: SSs on the water-boundary part of wood in streams (the source for P. pouzarii TUFC 101965). Arrowheads show parts forming acanthophyses. E: Acanthophyses on the apex of SS (P. microacanthophysis TUMH 64311). F: Rhizomorphs on the submerged part of wood in streams (P. rhizomorphae TUMH 64298). Bars: C, D 1 mm; E 30 µm.

Table 1. Data of samples used in this study.

Species name	Strain No. a	Herbarium Specimen No.	Locality	Collection date	Isolation date	Source b	Habitat c	
Meripilus giganteus	CBS 421.48	-	Germany	-	-	-	-	
M. giganteus	TUFC 100564	(TUMH 60367)d	Tottori Pref., Japan	17 Aug 2012	17 Aug 2012	T	F	
Physisporinus cf. 1 eminens	TUFC 101880	TUMH 64307	Miyagi Pref., Japan	22 Sep 2014	22 Sep 2014	S	A	
P. cf. 1 eminens	TUFC 101957	TUMH 65440	Tottori Pref., Japan	05 Nov 2019	05 Nov 2019	B	A	
P. cf. 1 eminens	TUFC 101958	TUMH 65441	Tottori Pref., Japan	05 Nov 2019	05 Nov 2019	B	A	
P. cf. 1 eminens	TUFC 101959	TUMH 65442	Tottori Pref., Japan	05 Nov 2019	05 Nov 2019	B	A	
P. cf. 2 eminens	TUFC 101881	TUMH 64308	Tottori Pref., Japan	25 Jun 2014	25 Jun 2014	S	A	
P. cf. 2 eminens	TUFC 101960	TUMH 65443	Hokkaido Pref., Japan	26 Sep 2017	26 Sep 2017	B	F	
P. cf. 2 eminens	TUFC 101961	TUMH 65444	Tottori Pref., Japan	14 Oct 2017	14 Oct 2017	B	A	
P. cf. 2 eminens	TUFC 101962	TUMH 65445	Tottori Pref., Japan	30 Oct 2019	30 Oct 2019	B	A	
P. cf. furcatus	TUFC 101883	TUMH 64310	Gifu Pref., Japan	29 May 2012	29 May 2012	S	A	
P. cf. furcatus	TUFC 101884	TUMH 64310	Gifu Pref., Japan	29 May 2012	29 May 2012	S	A	
P. crocatus	CBS 107806	-	Canada	-	1982	-	-	
P. lineatus	CBS 167.65	-	USA	-	24 Aug 1960	-	-	
P. lineatus	CBS 700.94	-	Germany	-	-	-	-	
P. lineatus	CBS 109425	-	Taiwan	27 Aug 1996	-	-	-	
P. lineatus	TUFC 13809	TUMH 60931	Tokyo Metropolis, Japan	19 Nov 2010	-	T	F	
P. lineatus	TUFC 13812	TUMH 60932	Tokyo Metropolis, Japan	19 Nov 2010	-	T	F	
P. microacanthophysis	TUFC 101885	TUMH 64311	Tottori Pref., Japan	07 Jul 2011	07 Jul 2011	S	A	
P. microacanthophysis	TUFC 101888	TUMH 64312	Miyazaki Pref., Japan	24 Nov 2013	24 Nov 2013	S	A	
P. microacanthophysis	TUFC 101889	TUMH 64313 T e	Tottori Pref., Japan	23 Oct 2019	23 Oct 2019	B	A	
P. microacanthophysis	TUFC 101963	TUMH 65446	Tottori Pref., Japan	04 Apr 2020	16 Apr 2020	S	A	
P. microacanthophysis	TUFC 101964	TUMH 65447	Tottori Pref., Japan	19 May 2020	19 May 2020	B	A	
P. pouzarii	TUFC 101965	No specimen	Osaka Pref., Japan	01 Apr 2013	01 Apr 2013	S	A	
P. pouzarii	TUFC 101966	TUMH 65448	Tottori Pref., Japan	05 Nov 2019	05 Nov 2019	B	A	
P. rhizomorphae	TUFC 101870	TUMH 64297	Tottori Pref., Japan	24 Sep 2013	24 Sep 2013	S	A	
P. rhizomorphae	TUFC 101871	TUMH 64298	Tottori Pref., Japan	19 Oct 2014	19 Oct 2014	R	A	
P. rhizomorphae	TUFC 101876	TUMH 64303 T	Tottori Pref., Japan	14 Oct 2017	14 Oct 2017	B	A	
P. rhizomorphae	TUFC 101967	TUMH 65449	Tottori Pref., Japan	04 Apr 2020	08 May 2020	S	A	
“P. sanguinolentus”	CBS 139.76	-	Belgium	Sep 1975	-	-	-	
“P. sanguinolentus”	CBS 193.76	-	Netherlands	-	-	-	-	
“P. sanguinolentus”	CBS 679.70	-	USA	-	-	-	-	
“P. sanguinolentus”	CBS 107146	-	Denmark	-	1980	-	-	
Physisporinus sp.	TUFC 101892	TUMH 64316	Kagoshima Pref., Japan	04 Sep 2018	04 Sep 2018	B	A	
Physisporinus sp.	TUFC 101968	TUMH 65450	Tottori Pref., Japan	05 Sep 2020	05 Sep 2020	T	F	
Rigidoporus ulmarius	CBS 186.60	-	USA	05 Nov 1952	-	T f	-	
“R. vinctus”	CBS 153.84	-	New Zealand	-	16 Oct 1973	-	-	
“R. vinctus”	CBS 174.71	-	Costa Rica	20 Jun 1963	-	-	-	
“R. vinctus”	TUFC 11175	(TUMH 60851)	Kagoshima Pref., Japan	19 Sep 2007	19 Sep 2007	B	F	
“R. vinctus”	TUFC 13815	(TUMH 63562)	Tokyo Metropolis, Japan	20 Nov 2010	-	B	F	
“R. vinctus”	TUFC 35082	(TUMH 60896)	Okinawa Pref., Japan	13 Jul 2003	13 Jul 2003	B	A	
a Strains in bold were used for the experiments incubating cultures under different O2 concentrations.

b “B”, “R”, “S”, and “T” mean basidiospores, a rhizomorph, a SS, and tissue of the basidiocarp as the source of isolation, respectively.

c “A” means that the sample was collected in or nearby an aquatic area. “F” means the sample from a forest area, not aquatic.

d Parenthesis means the specimen that we did not examine in the present study.

e T means the type specimen.

f FP 103737, other strain number of CBS 186.60, was isolated from tissue of the basidiocarp (Lombard et al., 1960).

2.2. Molecular phylogeny

2.2.1. DNA extraction, amplification and sequencing

DNA extraction from mycelia cultured on agar media was performed using a modified cetyltrimethylammonium bromide (CTAB) method (Shino et al., 2022). From the obtained genomic DNA, the internal transcribed spacer (ITS) region and D1/D2 domains of the large subunit (LSU) of nuclear ribosomal DNA (nrDNA) were amplified by the polymerase chain reaction (PCR) using a thermal cycler (PC-812 or PC-818; ASTEC Co., Ltd., Fukuoka, Japan). As primers, we used ITS5 and ITS4 for the ITS region (White et al., 1990), and LR0R and LR5 for the LSU region (Rehner & Samuels, 1994; Vilgalys & Hester, 1990). PCRs were conducted using the protocol described in Shino et al. (2022). Amplicons were purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc., Shiga, Japan), and Fasmac Co., Ltd. (Kanagawa, Japan) was commissioned to perform the DNA sequencing. All of the sequences except the ITS regions of CBS 186.60 and TUFC 101965 were readable by direct sequencing. Of the above two samples showing partial heterogeneity between the gene copies, we performed a cloning for TUFC 101965 using pGEM-T Easy Vector Systems (Promega K.K., Tokyo, Japan) and competent bacterial cells (Escherichia coli (Migula) Castellani & Chalmers JM109). Sequence data were deposited at the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/index-e.html).

2.2.2. Sequence alignment and phylogenetic analyses

Alignment of the data sets and creation of phylogenetic trees were performed online using MAFFT v. 7 (Katoh & Standley, 2013; https://mafft.cbrc.jp/alignment/server/; Jun 2023) and MEGA7 (Kumar et al., 2016). DNA sequences of the ITS and/or LSU regions of nrDNA retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) were included in phylogenetic analyses which were performed using the maximum likelihood (ML) method. Based on the results of the best-fitting model test in MEGA7, the GTR+G+I model was adopted as a model of molecular evolution in the ML analyses using a combined data set of nrDNA ITS and LSU sequences for the Meripilaceae group (Meripilus, Physisporinus, and Spongipellis) and only nrDNA ITS for the Cerrenaceae group [Cerrena Gray, Irpiciporus Murrill, Pseudolagarobasidium J.C. Jang & T. Chen, Pseudospongipellis Y.C. Dai & Chao G. Wang, Radulodon Ryvarden, “Rigidoporus hypobrunneus” (Petch) Corner, and “R. vinctus” (Berk.) Ryvarden] in Polyporales, whereas the TN93+G model was applied to only nrDNA LSU for Rigidoporus in Hymenochaetales. The confidence coefficient of each node in the phylogenetic trees was confirmed by bootstrap (BS) analysis with 1,000 replicates (Felsenstein, 1985). Outgroups for the phylogenetic analyses of each data set for the above three clusters were selected as follows; Abortiporus biennis (Bull.) Singer (FD-319), Hyphoderma setigerum (Fr.) Donk (FD-312), and Hypochnicium sp. (FP-110227-sp) (Floudas & Hibbett, 2015; Justo et al., 2017) for the data set consisting of the nrDNA ITS and LSU sequences of the Meripilaceae, Polyporales group, following Yamashita et al. (2020); Cymatoderma sp. (OMC-1427), Panus conchatus (Bull.) Fr. (Miettinen 13966), and P. fragilis O.K. Mill. (HHB-11042-Sp) (Floudas & Hibbett, 2015; Justo et al., 2017; Miettinen et al., 2012) for the data set consisting of nrDNA ITS sequences of the Cerrenaceae, Polyporales group, referring to Justo et al. (2017); Bridgeoporus sinensis (X.L. Zeng) F. Wu, Jia J. Chen & Y.C. Dai (Cui 10013), Leucophellinus hobsonii (Berk. ex Cooke) Ryvarden (Cui 6468), and L. irpicoides (Bondartsev ex Pilát) Bondartsev & Singer (Yuan 2690) (Wu et al., 2017) for the data set consisting of nrDNA LSU sequences of Rigidoporus, Hymenochaetales, referring to Wu et al., 2017. The DNA sequences analyzed in this study and retrieved from GenBank to infer phylogenetic relationships among the Meripilaceae, Polyporales group are listed in Table 2. The lists of DNA sequences for the Cerrenaceae, Polyporales group and Rigidoporus, Hymenochaetales are shown in Supplementary Table S1 and S2. Sequence alignment data are added as Supplementary alignment S1 for the Meripilaceae, Polyporales group, S2 for the Cerrenaceae, Polyporales group, and S3 for Rigidoporus, Hymenochaetales.

Table 2. DNA sequence data newly obtained in this study (bold-face type) and employed from GenBank for the phylogenetic analysis of Meripilaceae in Polyporales.

Species name	Sample No.	Locality	GenBank accession No.	
nrDNA ITS	nrDNA LSU	
Abortiporus biennis	FD-319	USA	KP135300	KP135195	
Hyphoderma setigerum	FD-312	USA	KP135297	KP135222	
Hypochnicium sp.	FP-110227-sp	USA	KY948804	KY948862	
Meripilus giganteus	CBS 421.48	Germany	MH856418 a	LC770099	
M. giganteus	FP-135344-Sp	UK	KP135307	KP135228	
M. giganteus	TUFC 100564	Japan	LC643683	LC643708	
Physisporinus castanopsidis	Dai 20396 T b	China	MT309485	MT309470	
P. castanopsidis	Dai 20397	China	MT309486	MT309472	
P. castanopsidis	Dai 20398	China	MT840113	MT840131	
P. cf. 1 eminens	TUFC 101880	Japan	LC643670	LC643695	
P. cf. 1 eminens	TUFC 101957	Japan	LC770057	LC770082	
P. cf. 1 eminens	TUFC 101958	Japan	LC770058	LC770083	
P. cf. 1 eminens	TUFC 101959	Japan	LC770059	LC770084	
P. cf. 2 eminens	TUFC 101881	Japan	LC643671	LC643696	
P. cf. 2 eminens	TUFC 101960	Japan	LC770060	LC770085	
P. cf. 2 eminens	TUFC 101961	Japan	LC770061	LC770086	
P. cf. 2 eminens	TUFC 101962	Japan	LC770062	LC770087	
P. cf. furcatus	TUFC 101883	Japan	LC643673	LC643698	
P. cf. furcatus	TUFC 101884	Japan	LC643674	LC643699	
P. cinereus	Cui 3266	China	KY131844	KY131903	
P. crataegi	Dai 15497 T	China	KY131845	KY131904	
P. crataegi	Dai 15499	China	KY131846	KY131905	
P. crocatus	CBS 107806	Canada	LC770074	LC770100	
P. crocatus	Dirks 374051	USA	ON364084	ON369536	
P. crocatus	MJ 19/09	Slovakia	JQ409466	OM669978	
P. eminens	Cui 9520	China	KY131847	KY131906	
P. eminens	Cui 10341	China	KY131849	KY131907	
P. eminens	Cui 10344	China	KY131850	KY131908	
P. eminens	Cui 10475	China	MT840114	MT840132	
P. eminens	Dai 11400	China	KY131852	KY131909	
P. eminens	Dai 12685	Czechia	MT840115	MT840133	
P. eminens	Dai 17200	Unknown	MT279690	MT279911	
P. eminens	Dai 19861	China	MT840116	MT840134	
P. eminens	Dai 20832	China	MT279689	MT279910	
P. eminens	Dai 20868	China	MT840117	MT840135	
P. eminens	Dai 22472	China	OM669900	OM669983	
P. furcatus	Dai 2105	China	KY131854	KY131911	
P. furcatus	Dai 2544	China	KY131855	KY131912	
P. furcatus	Dai 11313	China	KY131856	KY131913	
P. furcatus	Dai 12938	China	KY131857	KY131914	
P. furcatus	Dai 20976	Belarus	MT840118	MT840136	
P. furcatus	Dai 20977	Belarus	MT840119	MT840137	
P. furcatus	TAA 15097 T	Russia	KY131853	KY131910	
P. lavendulus	Dai 9925	China	KY131858	KY131915	
P. lavendulus	Dai 13587A T	China	KY131859	KY131916	
P. lineatus	CBS 167.65	USA	LC770075	LC770101	
P. lineatus	CBS 700.94	Germany	LC770076	LC770102	
P. lineatus	CBS 109425	Taiwan	LC770077	LC770103	
P. lineatus	Dai 17986	China	MT840121	MT840139	
P. lineatus	Dai 18280	Vietnam	MT840122	MT840140	
P. lineatus	JV 1407/37	Costa Rica	OM669903	OM669986	
P. lineatus	TUFC 13809	Japan	LC770063	LC770088	
P. lineatus	TUFC 13812	Japan	LC770064	LC770089	
P. longicystidius	Cui 16630	Australia	ON417177	ON417227	
P. longicystidius	Cui 16725	Australia	ON417178	ON417228	
P. microacanthophysis	TUFC 101885	Japan	LC643675	LC643700	
P. microacanthophysis	TUFC 101888	Japan	LC643678	LC643703	
P. microacanthophysis	TUFC 101889 T	Japan	LC643679	LC643704	
P. microacanthophysis	TUFC 101963	Japan	LC770065	LC770090	
P. microacanthophysis	TUFC 101964	Japan	LC770066	LC770091	
P. pouzarii	Dai 15005	China	KP420014	KP420017	
P. pouzarii	Dai 21043	Belarus	MT840124	MT840142	
P. pouzarii	JV 0511/23	Czechia	JQ409465	KY131921	
P. pouzarii	TUFC 101965	Japan	LC770067	LC770092	
P. pouzarii	TUFC 101966	Japan	LC770068	LC770093	
P. rhizomorphae	TUFC 101870	Japan	LC643660	LC643685	
P. rhizomorphae	TUFC 101871	Japan	LC643661	LC643686	
P. rhizomorphae	TUFC 101876 T	Japan	LC643666	LC643691	
P. rhizomorphae	TUFC 101967	Japan	LC770069	LC770094	
P. roseus	Dai 19877 T	China	MT840126	MT840144	
“P. sanguinolentus”	CBS 139.76	Belgium	MH860969 c	MH872738 c	
“ P. sanguinolentus ”	CBS 193.76	Netherlands	LC770078	LC770104	
“P. sanguinolentus”	CBS 679.70	USA	MH859899 c	MH871689 c	
“ P. sanguinolentus ”	CBS 107146	Denmark	LC770079	LC770105	
“P. sanguinolentus”	Dai 20995	Belarus	MT309483	MT309480	
“P. sanguinolentus”	DM1068	Denmark	MT644902	MT644902	
“P. sanguinolentus”	JV 1610/2	Czechia	OM669921	OM669999	
“P. sanguinolentus”	KHL 11913	Sweden	JX109843	JX109843	
Physisporinus sp. 1	Dai 11693	China	KY131865	KY131922	
Physisporinus sp. 2	Dai 6720	China	KY131867	KY131923	
Physisporinus sp. 4	Dai 15184	Unknown	KY131868	KY131924	
Physisporinus sp.	Cui 16852	Puerto Rico	ON417179	ON417229	
Physisporinus sp.	CWU 3874	Ukraine	OM971903	OM971889	
Physisporinus sp.	JV 0308/58	USA	OM669909	OM669991	
Physisporinus sp.	JV 0509/47	USA	OM669906	OM669988	
Physisporinus sp.	JV 0709/188	USA	OM971904	OM971890	
Physisporinus sp.	JV 0909/3	Czechia	OM669939	OM670011	
Physisporinus sp.	JV 1407/36	Costa Rica	OM669933	OM670008	
Physisporinus sp.	Miettinen 15239	Indonesia	KY948732	KY948867	
Physisporinus sp.	Miettinen 16699	USA	KY948733	KY948863	
Physisporinus sp.	TUFC 101892	Japan	LC643682	LC643707	
Physisporinus sp.	TUFC 101968	Japan	LC770070	LC770095	
P. subcrocatus	Dai 12800	USA	KY131869	KY131925	
P. subcrocatus	Dai 15917 T	China	KY131870	KY131926	
P. sulphureus	Dai 17839 T	Singapore	MG132179	MG132181	
P. sulphureus	Dai 17841	Singapore	MG132180	MG132182	
P. tibeticus	Cui 9381 T	China	KY131871	KY131927	
P. tibeticus	Cui 9588	China	KY131873	KY131929	
P. tibeticus	Cui 10478	China	MT840128	MT840146	
P. undatus	JV 0110/48	Czechia	OM669931	OM670005	
P. undatus	Miettinen 13591	Finland	KY948731	KY948870	
P. undatus	MJ 129/04	Czechia	OM669932	OM670006	
“P. vinctus”	Cui 16903	China	MT840129	MT840147	
P. vitreus	Dai 21060	Belarus	MT840130	MT840148	
P. vitreus	KHL 11959	Norway	JQ031129	JQ031129	
P. yunnanensis	CLZhao 21583	China	OP852341	OP852343	
P. yunnanensis	CLZhao 21647 T	China	OP852340	OP852342	
Polyporales sp.	422b	Japan	AB470242	AB470242	
Polyporales sp.	TK-10	Japan	AB716748	AB762089	
Uncultured mycorrhizal fungus	Polyporales218	Taiwan	KP238183	KP238182	
Uncultured mycorrhizal fungus	Polyporales859	Taiwan	KP238186	KP238184	
Uncultured mycorrhizal fungus	Physisporinus222	Taiwan	KP238185	KP238181	
Spongipellis ambiens	Niemelä 6407	China	ON979313	ON979313	
S. ambiens	Spirin 5389	Russia	ON979322	ON979322	
S. profissilis	Dai 3934	China	ON979321	ON979321	
S. profissilis	Kotiranta 26990	Russia	OP104014	OP104014	
S. spumea	JV 1511/6	Czechia	ON979318	ON979318	
S. spumea	Kotiranta 26889	Finland	ON979311	ON979311	
S. spumea	Spirin 6741	Russia	ON979326	ON979326	
S. variispora	Niemelä 6423	China	ON979320	ON979320	
S. variispora	Spirin 3737 T	Russia	ON979312	ON979312	
a The nrDNA ITS sequence of M. giganteus CBS 421.48 had been already registered with this accession number in GenBank before our study. We used it for the analysis of the Meripilaceae group because the sequence of this strain obtained in the present study corresponded to the above sequence with high homology (99%) by the Standard Nucleotide BLAST (Basic Local Alignment Search Tool) of the GenBank database.

b T means the type specimen or ex-type culture.

c The nrDNA ITS and LSU sequences of “P. sanguinolentus” CBS 139.76 and CBS 679.70 had been already registered with these accession numbers in GenBank before our study. We used them for the analysis of the Meripilaceae group because the sequences of the two strains obtained in the present study corresponded to the above sequences with high homology (Both were 100% in the ITS and 99% in the LSU region) by the BLAST.

2.3. Observation of cultures and specimens

Strains including new isolates were precultured on an antibiotics-added corn meal agar medium (Shino et al., 2022) at room temperature (20-25 °C) for 1-2 mo. After agar discs containing mycelia were cut out or stamped out from the precultured plates using a flame sterilized scalpel or autoclaved sterilized plastic straws (6 mm diam), they were inoculated on the following four media; a corn meal agar medium [CMA; Corn Meal Agar “Nissui” (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan; containing 2 g/L cornmeal extract and 15 g/L agar)], a malt extract agar medium [MA; 15 g/L malt extract (Oriental Yeast Co., Ltd., Tokyo, Japan) and 15 g/L agar (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)], a potato dextrose agar medium [PDA; Potato Dextrose Agar “Nissui” (Nissui Pharmaceutical Co., Ltd.; containing 3.9 g/L potato extract, 21 g/L glucose and 14.1 g/L agar)], and a starch agar medium [SA; 10 g/L starch, soluble (FUJIFILM Wako Pure Chemical Corporation), 5 g/L malt extract, and 20 g/L agar]. These plates were incubated at 25 °C for 7-31 d and the following cultural properties were examined using a Nikon ECLIPSE 80i differential interference contrast microscope (DICM) (Nikon Corporation, Tokyo, Japan); presence/absence and morphology of acanthophyses, clamp connections, conidia, and plectenchymata. Samples were mounted in 3% potassium hydroxide (KOH) on a slide glass, and the size was measured by PhotoRuler ver. 1.1.3 software (http://inocybe.info). Specimens of SSs and basidiocarps from which strains were isolated were observed by the above method to confirm the consistency to the results of phylogenetic analyses. Acanthophyses were also observed using a scanning electron microscope (SEM) (SU1510; Hitachi High-Tech Corporation, Tokyo, Japan). Preparation and observation of SEM samples followed Shino et al. (2022).

2.4. Investigation of acanthophysis production on agar media during incubation under different O 2 concentrations

A schematic illustration of the following experiment is shown in Fig. 2. Selected strains (Physisporinus cf. 1 eminens TUFC 101880, P. cf. 2 eminens TUFC 101881, P. cf. furcatus TUFC 101883, P. lineatus TUFC 13809, P. microacanthophysis TUFC 101885, and P. pouzarii TUFC 101965; see Table 1) were preincubated at room temperature (20-25 °C). After mycelia covered the entire surface of the medium, they were stamped out as discs with agars using autoclaved sterilized plastic straws (6 mm diam) and the agar discs were used to inoculate CMA plates: six discs were inoculated per a plate (three discs were placed facing up and the other three discs were placed facing down). After checking the number of acanthophyses on the surface of the discs under the DICM, as they were already formed during preincubation before experiment, the CMA plates with their inoculated agar discs were placed in four desiccators [Vacuum Polycarbonate Desiccator 240G (or 240GA) or 300G (or 300GA) (AS ONE Corporation, Osaka, Japan)]. Then, the air in each desiccator was exhausted using a diaphragm type dry vacuum pump (DA-20D; ULVAC KIKO, Inc., Miyazaki, Japan) and each desiccator was filled with one of the following standard gas mixtures or air to prepare four different O2 conditions: 5% O2 (O2 4.95%, CO2 402 ppm, and N2 as a base gas), 10% O2 (O2 10.0%, CO2 405 ppm, and N2), 20% O2 (the atmospheric air: O2 21%, CO2 400 ppm, and N2 78%; all the values of concentrations are approximate), and 40% O2 (O2 39.5%, CO2 395 ppm, and N2). To prepare the 5, 10 and 40% O2 conditions, we used calibration gas cylinders that were prepared by Taiyo Nippon Sanso JFP Corporation (Kanagawa, Japan) and a gas regulator (GHN-3; CHIYODA SEIKI Co., Ltd., Hyogo, Japan). The gas in each desiccator was exchanged daily: the process of exhausting and filling of each gas was repeated twice per exchange. Uncovered plates in desiccators were incubated for 2-5 d at room temperature. After finishing the incubation, the number of acanthophyses that formed on the upper surface and side of each disc was counted under the DICM. The number of acanthophyses produced during preincubation was excluded from the data.

Fig. 2 - The outline of experiment for acanthophysis production under different O2 concentrations (For details, see section 2.4. in materials and methods).

3. Results

3.1. Relationships between molecular phylogeny and cultural characteristics with emphasis on acanthophysis formation

The phylogenetic analysis of Meripilaceae in Polyporales that was based on the combined sequences of the ITS and LSU regions of nrDNA showed that Meripilus, Physisporinus, and Spongipellis form a clade, and Meripilus was included within the Physisporinus clade (BS = 99%; Fig. 3). The topology of this phylogenetic tree was almost correspondent with trees estimated in several recent studies (Chen & Dai, 2021; Shino et al., 2022; Spirin et al., 2022; Wang & Dai, 2022). Acanthophysis-forming strains on agar media in the present study belonged only to the Physisporinus clade (Figs. 3, 4, 5). In this clade, a monophyletic cluster including P. castanopsidis Jia J. Chen & Y.C. Dai, P. crocatus (Pat.) F. Wu, Jia J. Chen & Y.C. Dai, P. microacanthophysis, P. pouzarii, “P. sanguinolentus” (Alb. & Schwein.) Pilát, P. subcrocatus F. Wu, Jia J. Chen & Y.C. Dai, P. tibeticus F. Wu, Jia J. Chen & Y.C. Dai, and P. vitreus (Pers.) P. Karst. (BS = 100%) formed shorter acanthophyses (10-30 µm long: the ornamented part with warts or spines, but not including spines) than the rest of species in this genus which are known to produce SSs/acanthophyses (20-70 µm long), except for P. cf. 1 eminens (12-29 µm long). Moreover, the strains in this cluster had sparse clamp connections at the septa of vegetative hyphae (Figs. 3, 4, 6E), whereas strains in other clades of Physisporinus lacked clamp connections on the hyphae. Meripilus strains did not produce acanthophyses on the agar media employed in this study. Both Meripilus and Physisporinus formed plectenchymata in cultures (Fig. 6B, F). Formation of the plectenchymata in both genera have been described previously, e.g., Larsen and Lombard (1988) and Lombard and Chamuris (1990).

Fig. 3 - Phylogenetic tree of Meripilaceae in Polyporales inferred from connected sequences of the ITS and LSU regions of nrDNA by ML method. A total of 1,171 sites in the final data set were used for this analysis. The values at nodes indicate BS in ML method (≥ 70%), and bold branches mean BS ≥ 90% in the above method. The species names and numbers of strains used in this study are shown in bold, and the strain number followed by “S” or “R” indicates the isolate from a SS or rhizomorph. T on the sample number means the sequence obtained from the type specimen or ex-type culture. Filled circles show the strains forming acanthophyses in culture. Open squares show the strains having clamp connections at the septa of vegetative hyphae on agar media (white arrow indicates a monophyletic clade characterized by this feature). The strains without sufficient cultural investigations in the present study are unmarked.

Fig. 4 - The relationship between molecular phylogeny and acanthophysis formation in Physisporinus. A figure at the upper left is the reduced Fig. 3, and a box in it shows a magnified part for this figure. As with Fig. 3, filled circles show the strains forming acanthophyses in culture, and open squares show the strains having clamp connections at the septa of vegetative hyphae on agar media (white arrow indicates a monophyletic clade characterized by this feature). The strains without sufficient cultural investigations in the present study are unmarked. The sizes of acanthophyses of Physisporinus species investigated in this study are described under the species name. The appearance of a typical acanthophysis of each species is exhibited by photographs using SEM at the right of this figure. Bars: 10 µm.

Fig. 5 - The relationship between molecular phylogeny and acanthophysis formation in Physisporinus. A figure at the upper left is the reduced Fig. 3, and a box in it shows a magnified part for this figure. As with Fig. 3, filled circles show the strains forming acanthophyses in culture. The strains without sufficient cultural investigations in the present study are unmarked. The sizes of acanthophyses of Physisporinus species investigated in this study are described under the species name. The appearance of a typical acanthophysis of each species is exhibited by photographs using SEM at the right of this figure. Bars: 10 µm.

Fig. 6 - Cultural characteristics of the strains of Meripilus giganteus (A-C) and Physisporinus species (D-F) in Meripilaceae. A: Colony on 1.5% MA (TUFC 100564). B: Plectenchymata in mycelia (CBS 421.48). C: Vegetative hyphae (CBS 421.48). Arrow heads show clampless septa. D: Colony on 1.5% MA (P. pouzarii TUFC 101965). E: Clamp connection on a septum of vegetative hyphae (P. pouzarii TUFC 101965). F: Plectenchymata and vegetative hyphae (P. lineatus CBS 167.65). Arrow heads show clampless septa. Bars: B, C, E, F 10 µm.

The phylogenetic analysis based on the nrDNA ITS or LSU region showed that “Rigidoporus” species in the traditional usage separated in two different lineages, Polyporales and Hymenochaetales (Supplementary Figs. S1, S2) as reported by Wu et al. (2017). The phylogenetic tree of the Cerrenaceae group in Polyporales (Supplementary Fig. S1) showed that this family clusters with Cerrena, Irpiciporus, Pseudolagarobasidium, Pseudospongipellis, Radulodon, and “Rigidoporus” (BS = 100%), as reported in previous studies (Justo et al., 2017; Wang & Dai, 2022; Westphalen & Motato-Vásquez, 2022), and a highly supported clade (BS = 99%) of “R. hypobrunneus”/“R. vinctus” accommodates five strains of “R. vinctus” examined in this study. Arthroconidia production in “R. vinctus” strains was observed (Supplementary Fig. S1) as reported previously [Setliff, 1972 (as oidia); Stalpers, 1978], but they did not produce any acanthophyses. The topology of the tree estimated for Rigidoporus in Hymenochaetales was similar to that in Wu et al. (2017) and Yuan et al. (2020). Rigidoporus ulmarius (Sowerby) Imazeki CBS 186.60 formed a clade together with nine sequences, including four sequences of R. microporus (Sw.) Overeem (BS = 98%; Supplementary Fig. S2) in Hymenochaetales. This strain did not form acanthophyses, but it did produce vesicular cells on vegetative hyphae laterally and terminally in culture (Supplementary Fig. S2), as described previously in Lombard et al. (1960) and Stalpers (1978; as terminal vesicles).

3.2. Acanthophysis production on agar media under different O 2 concentrations

The results of the experiments using six strains (Physisporinus cf. 1 eminens TUFC 101880, P. cf. 2 eminens TUFC 101881, P. cf. furcatus TUFC 101883, P. lineatus TUFC 13809, P. microacanthophysis TUFC 101885, and P. pouzarii TUFC 101965) cultured under four different O2 conditions (5, 10, 20, and 40% O2) are shown in Fig. 7. In the case of the agar discs placed facing up, the three strains (TUFC 101880, TUFC 101881, and TUFC 101885) formed acanthophyses most abundantly under the atmospheric condition, i.e., 20% O2, whereas the two strains (TUFC 101883 and TUFC 101965) formed most acanthophyses under 40% O2. The four strains other than TUFC 13809 and TUFC 101880 tended to form more acanthophyses on the upper surface than on the sides of the agar discs in the situation placed facing up, probably because aerial vegetative hyphae on the discs are easy to contact to the air.

Fig. 7 - Differences in the number of acanthophyses produced on agar discs under four different O2 concentrations (5, 10, 20, and 40%) by six Physisporinus strains. The vertical axis of the bar graph shows the number of acanthophyses. “T” and “B” under the horizontal axis mean the agar discs inoculated on a CMA plate as top face up and back face up, respectively. “T” is shown by a blue bar and “B” by a white bar. “U” and “S” indicate the upper surface and side face of the agar discs.

In the case of the agar discs placed facing down, the four strains (TUFC 101880, TUFC 101881, TUFC 101885, and TUFC 101965) produced more acanthophyses under the 20% O2 condition than under the 40% O2 condition. TUFC 101883 produced acanthophyses only under the 40% O2 condition. When the agar discs were set on the plates with facing down, the aerial vegetative hyphae at the upper surface were facing the CMA plates, which resulted in most of the hyphae existing inside the discs. Hence, this latter situation in which the hyphae spreading inside the agar disc is somewhat analogous to that of hyphae growing within water-saturated wood tissue in the natural wet habitats of SS-forming Physisporinus species.

The number of acanthophyses formed by the five strains (TUFC 101880, TUFC 101881, TUFC 101883, TUFC 101885, and TUFC 101965) on the discs placed both facing up and down under the 5% and 10% O2 conditions was lower than 20% or 40% O2 conditions, but TUFC 13809 produced acanthophyses most abundantly under 10% O2 in the case of discs being placed facing up and under 5% O2 in the case of discs being placed facing down. The above five strains tended to mainly produce acanthophyses from the upper surface and/or side face of the agar discs placed facing up or down on CMA plates, while TUFC 13809 formed acanthophyses abundantly on the aerial vegetative hyphae that spread on the plates as well as on the entire surface of the agar discs.

4. Discussion

Some basidiomycetes have been known to produce acanthophyses on vegetative hyphae in culture, and most of these taxa are now placed in Physisporinus; for example, P. crocatus, which was formerly treated as Poria nigrescens Bres. (Nobles, 1958); P. lineatus, formerly treated as Polyporus zonalis Berk. [Bakshi et al., 1963; Davidson et al., 1942 (acanthophyses were described as hyphal ends covered with short knobs or definite spines); Nobles, 1958], as Rigidoporus zonalis (Berk.) Imazeki (Kobayashi, 1972), and as R. lineatus [Hood et al., 1997 (as acanthohyphidia); Motato-Vásquez et al., 2016 (as the spiny and clavate cystidia); Stalpers, 1978 (as acanthohyphidia)]; P. undatus (Pers.) Pilát, formerly treated as R. undatus (Pers.) Donk (Motato-Vásquez et al., 2016); P. vitreus, formerly treated as R. vitreus (Pers.) Donk (Lombard & Chamuris, 1990; Schmidt et al., 1996, 1997). In these previous studies, species identification was based mainly on the morphological characteristics of basidiocarps. Our phylogenetic studies showed that the above acanthophysis-forming species are accommodated in Physisporinus. Except for Physisporinus, some species of Xylobolus P. Karst. have also been reported to form acanthophyses on agar media; for example, X. frustulatus (Pers.) Boidin [Lombard & Chamuris, 1990; Nakasone, 1990 (termed as acanthohyphidia); Stalpers, 1978 (as acanthohyphidia)]; X. subpileatus (Berk. & M.A. Curtis) Boidin (Stalpers, 1978), although the two species scarcely produce acanthophyses (Stalpers, 1978). In addition to Xylobolus, Aleurodiscus Rabenh. ex J. Schröt. sensu lato (Wu et al., 2001), Megalocystidium Jülich [only M. diffissum (Sacc.) K.H. Larss. & Spirin (Spirin et al., 2021)], and Stereum Hill ex Pers. are also known to produce acanthophyses (or termed as acanthocystidia or acanthohyphidia) in their basidiocarps (e.g., Bernicchia & Gorjón, 2010; Larsson & Ryvarden, 2021). These genera belong to Stereaceae, Russulales (Miller et al., 2006; Wu et al., 2022), and acanthophysis formation on vegetative hyphae in culture has been known only in the above Xylobolus species. On the other hand, Physisporinus seldom or never produce acanthophyses in their basidiocarps. Among Physisporinus, the closely related Meripilus and morphologically similar Rigidoporus, only Physisporinus species produce SSs in nature and/or acanthophyses in culture. Previous studies on the culture of Meripilus and Rigidoporus species, except for the species currently transferred to Physisporinus, did not observe SS/acanthophysis formation (Campbell, 1937; Davidson et al., 1942; Go et al., 2021; Kaewchai et al., 2010; Larsen & Lombard, 1988; Lombard et al., 1960; Nobles, 1948, 1965; Setliff, 1972; Stalpers, 1978). These results support our previous suggestion that SS/acanthophysis formation could be a taxonomic character for defining the genus Physisporinus, which is currently difficult to distinguish from Rigidoporus based on the morphology of the basidiocarp (Shino et al., 2022). All the Physisporinus samples used in our previous study were collected from aquatic environments (Shino et al., 2022). However, in addition to samples from streams, this study includes new samples from terrestrial environments (Table 1). Therefore, the Physisporinus species that inhabit forest areas may also form acanthophyses on their vegetative hyphae. Physisporinus species that have not been proven to produce SSs or acanthophyses, especially those that form perennial and/or brightly colored basidiocarps, should be investigated for their SS/acanthophysis-forming ability on wet wood or on media. Moreover, we found that the species group producing small acanthophyses (10-30 µm long) and rare clamp connections on septa of vegetative hyphae formed a highly supported clade that harbored at least eight Physisporinus species (P. castanopsidis, P. crocatus, P. microacanthophysis, P. pouzarii, “P. sanguinolentus”, P. subcrocatus, P. tibeticus, and P. vitreus), whereas other acanthophysis-forming clades in this genus produce larger acanthophyses (20-70 µm long, except for P. cf. 1 eminens which forms acanthophyses of 12-29 µm long) and no clamp connections (Figs. 3, 4, 5). These characteristics suggest that the size of acanthophyses is related to the phylogeny of Physisporinus. In this study, we were unable to find any clearly distinctive characteristics in basidiocarps of the species group having short acanthophyses and clamp connections in culture in comparison with other Physisporinus species contained in different clades. Further studies focusing on both the basidiocarps and isolates are therefore needed.

We currently face a raft of challenges related to the taxonomy of Physisporinus and the allied genera, Meripilus and Rigidoporus. Physisporinus still contains taxonomically confused species probably composed of plural species [e.g., P. sanguinolentus (Runnel et al., 2021, refer to Additional file 5; Wu et al., 2017); P. furcatus group and P. undatus group (Chen & Dai, 2021); P. vitreus (Runnel et al., 2021)]. The question remains about the validity of P. subcrocatus from the perspective of the very close similarity to P. crocatus in terms of morphology of basidiocarps and phylogeny. The phylogenetic position of Meripilus (i.e., whether this genus is truly nested in the Physisporinus clade or not) has not been confirmed by multi-gene phylogenetic analyses with sufficient sequences yet, although this study showed that, unlike Physisporinus, Meripilus species do not produce acanthophyses. In the Cerrenaceae, Polyporales group, a taxonomic problem regarding the “R. hypobrunneus”/“R. vinctus” clade, which was also pointed out by Nakasone and Ortiz-Santana (2022), was more clearly highlighted by our phylogenetic analysis (Supplementary Fig. S1). This clade should be treated as a new or another genus, but we do not treat it as such in this study because we could not investigate the type specimens of these species. In addition, it is important to reexamine specimens that are currently treated as P. vinctus (Berk.) Murrill, a synonym of R. vinctus, in the phylogenetic trees by Chen and Dai (2021), Shino et al. (2022), Wu et al. (2017), and this study (Fig. 3). To solve these taxonomic issues, investigations of currently overlooked or underestimated characters, such as the cultural properties of asexual states and vegetative hyphae, as well as the ecological characteristics of these species should be conducted in addition to the currently dominant studies focusing on the morphology of sexual states (basidiocarps) and molecular phylogeny.

When we find SSs of Physisporinus species in freshwater areas, they are often formed on the water-boundary part of dead and wet wood of broad-leaved or coniferous trees. The wood substrate is carried by water flow as drift and caught between rocks, then exposed to the flow and splash for extended periods, mostly resulting in barkless and sometimes partly getting mossy. The insides of wet wood at the water-boundary and submerged parts are saturated with water and the dissolved O2 concentrations within the wood tissue most likely decrease due to the low level of gas exchange. The results of the present experiments that exposed cultures of Physisporinus to gas mixtures with different O2 concentrations suggested that acanthophyses were produced in response to higher O2 concentrations and that they probably play a role in obtaining O2. Our experiments clearly showed that the number of acanthophyses produced on agar discs was markedly increased when incubated under O2 concentrations of 20-40% compared to when incubated under O2 concentrations of 5-10% O2 (Fig. 7). The numerous spines of the acanthophyses probably function to increase the surface area of acanthophysis cells for gas exchange. This thought is supported by the fact that acanthophyses are formed on aerial vegetative hyphae, not on submerged hyphae in agar medium. Thus, it is possible that SSs furnished with numerous acanthophyses are formed at the water-boundary part that is exposed to the air and that they play a role in respiration at the closest site to the submerged part. The synnematous morphology of the SSs possibly serves to maintain the distance from the water surface and the water-saturated part of the wood substrate, so that the acanthophyses are exposed to the atmosphere, and also to withstand the force of the water flow. When the agar discs containing mycelia were placed on media facing down, four strains (P. cf. 1 eminens TUFC 101880, P. cf. 2 eminens TUFC 101881, P. microacanthophysis TUFC 101885 and P. pouzarii TUFC 101965) formed acanthophyses more abundantly under 20% O2 condition than under 40% O2 condition (Fig. 7). This finding might be explained as follows; under the 40% O2 condition, high levels of O2 permeate the agar media, so only a fewer number of acanthophyses need to be produced in order to obtain sufficient O2 for extending hyphae into the media. However, P. cf. furcatus TUFC 101883 responded differently, as producing more acanthophyses under the 40% O2 condition than under the 20% O2 condition (Fig. 7). Therefore, the sensitivity to O2 may differ among species and/or strains. In the present experiments, most strains formed less acanthophyses under the low O2 conditions, 5% or 10%. This is assumed that there was insufficient difference in oxygen concentration between inside and outside the culture medium to induce acanthophysis formation. Further detailed physiological study is required to verify this hypothesis. Interestingly, P. lineatus TUFC 13809 exceptionally formed numerous acanthophyses, even under 5% O2 condition (Fig. 7). This species is known to show a high level of mycelial growth rate even in low O2 concentrations (Hood et al., 1997) and to cause the decay in heartwood, especially root and butt rot of living trees in Asia and North and South America [Dai et al., 2007 (as Rigidoporus lineatus); Kobayashi, 1972 (as R. zonalis); Overholts, 1953 (as Polyporus zonalis); Rajchenberg & Robledo, 2013 (as R. lineatus)]. The internal part of trees is not normally exposed to the atmosphere, so such heart rot fungi must be adapted to the low oxygen condition. Though SS formation by P. lineatus has not been reported yet, the ability to produce large numbers of acanthophyses even under low O2 concentrations is likely to contribute to the high rate of hyphal growth and the heart rot in trees. Additional investigations on the ecology of this species are needed. Hyde and Goh (1998) reported an unidentified fungus that formed tufts of acanthophyses on the apices of root-like hyphal strands on wet wood collected in several tropical streams. Based on the habits and observations of the characteristics of the fungus, they guessed that the function of acanthophyses was to take up O2 in water. However, their discussion was speculative at the time. Because the fungus was not identified or observed in its sexual state, the taxonomic assignment of their fungus should be clarified by further study.

Basidiomycetous fungi have been considered to prefer terrestrial environments to aquatic environments; this is suggested by the fewer number of aquatic species compared to terrestrial species (Jones et al., 2014; Shearer et al., 2007). However, our findings showed that Physisporinus species have adapted to humid environments such as streams and waterfalls by acquiring the ability to form SSs/acanthophyses, which appear to function as respiratory organs. This may be a strategy for terrestrial fungi in origin to adapt to aquatic habitats and decay water-saturated wood with low O2 concentrations. Further research of the basidiomycetous fungi inhabiting wet habitats should be undertaken to better clarify their biodiversity and ecology.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

This study was supported, in part, by a Grant-in-Aid from the Institute for Fermentation, Osaka (IFO). We appreciate staff of Westerdijk Fungal Biodiversity Institute for providing living cultures of Meripilus, Physisporinus and Rigidoporus species. We also great thank Ms. Sachiko Ueta and Kaori Shimizu, FMRC, for taking on the cryopreservation and maintenance of strains investigated in the present study. Deposition and utilization of Tottori University Fungal Culture Collection (TUFC) strains were supported by FMRC through the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (http://nbrp.jp).
==== Refs
Bakshi, B.K. , Singh, S. , &Singh, B. (1963) .A re-examination of Fomes lignosus and Polyporus zonalis. Transactions of the British Mycological Society, 46 , 426–430. https://doi.org/10.1016/S0007-1536(63)80036-4
Bernicchia, A. , &Gorjón, S.P. (2010) .Corticiaceae s.l., Fungi Europaei(Vol.12 ). Edizioni Candusso.
Binder, M. , Justo, A. , Riley, R. , Salamov, A. , Lopez-Giraldez, F. , Sjökvist, E. , Copeland, A. , Foster, B. , Sun, H. , Larsson, E. , Larsson, K.-H. , Townsend, J. , Grigoriev, I.V. , & Hibbett, D.S. . (2013). Phylogenetic and phylogenomic overview of the Polyporales. Mycologia, 105 , 1350-1373. https://doi.org/10.3852/13-003 23935031
Breitenbach, J. , &Kränzlin, F. (1986). Fungi of Switzerland . 2 . Verlag Mykologia, Lucerne, Switzerland.
Campbell, W.A. (1937). The cultural characteristics of Fomes connatus. Mycologia, 29 , 567-571. https://doi.org/10.1080/00275514.1937.12017225
Chen, J.J. , & Dai, Y.C. (2021). Two new species of Physisporinus (Polyporales, Basidiomycota) from Yunnan, Southwest China. Mycological Progress, 20 , 1-10. https://doi.org/10.1007/s11557-020-01647-8
Corner, E.J.H. (1987). Ad Polyporaceas IV. Beihefte zur Nova Hedwigia, 86 , 1-265.
Dai, S.J. , &Dai, Y.C. (2018). Morphological characters and molecular data reveal a new species of Physisporinus (Basidiomycota) from Southeast Asia. Mycosystema, 37 , 145-150. https://manu40.magtech.com.cn/Jwxb/EN/10.13346/j.mycosystema.170210
Dai, Y.C. (1998). Changbai wood-rotting fungi 9. Three new species and other species in Rigidoporus, Skeletocutis and Wolfiporia (Basidiomycota, Aphyllophorales). Annales Botanici Fennici, 35 , 143-154. https://www.jstor.org/stable/23726542
Dai, Y.C. (2012). Polypore diversity in China with an annotated checklist of Chinese polypores. Mycoscience, 53 , 49-80. https://doi.org/10.1007/s10267-011-0134-3
Dai, Y.C. ,Cui, B.K. ,Yuan, H.S. ,&Li, B.D. (2007). Pathogenic wood-decaying fungi in China. Forest Pathology, 37 , 105-120. https://doi.org/10.1111/j.1439-0329.2007.00485.x
Davidson, R.W. ,Campbell, W.A. ,&Vaughn, D.B. (1942). Fungi causing decay of living oaks in the Eastern United States and their cultural identification. Technical Bulletin, 785 , 1-68.
Donk, M.A. (1967). Notes on European polypores-II. Persoonia, 5 , 47-130.
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39 , 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x 28561359
Floudas, D. ,&Hibbett, D.S. (2015). Revisiting the taxonomy of Phanerochaete (Polyporales, Basidiomycota) using a four gene dataset and extensive ITS sampling. Fungal Biology, 119 , 679-719. https://doi.org/10.1016/j.funbio.2015.04.003 26228559
Gilbertson, R.L. ,Ryvarden, L. (1987). North American polypores ,2 .Fungiflora, Oslo, Norway.
Go, W.Z. , Chin, K.L. , H’ng, P.S. , Wong, M.Y. , Luqman, C.A. , Surendran, A. , Tan, G.H. , Lee, C.L. , Khoo, P.S. , &Kong, W.J. (2021). Virulence of Rigidoporus microporus isolates causing white root rot disease on rubber trees (Hevea brasiliensis) in Malaysia. Plants, 10 , 2123. https://doi.org/10.3390/plants10102123 34685932
Hood, I. , Ramsden, M. , Dot, T.D. , &Self, M. (1997). Rigidoporus lineatus (Pers.) Ryvarden in fire salvaged logs stored under water sprinklers in south east Queensland. Material und Organismen, 31 , 123-143.
Hyde, K.D. , &Goh, T.K. (1998). Acanthophysis-like structures from wood submerged in freshwater streams in the tropics. Mycoscience, 39 , 199-203. https://link.springer.com/article/10.1007/BF02464060
Jones, E.B.G. , Hyde, K.D. , &Pang, K.L. (2014). Freshwater fungi: and fungal-like organisms. Walter de Gruyter GmbH, Berlin, Germany.
Jülich, W. (1981). Higher taxa of Basidiomycetes. Bibliotheca Mycologica, 85 , 1-485.
Justo, A. , Miettinen, O. , Floudas, D. , Ortiz-Santana, B. , Sjökvist, E. , Lindner, D. , Nakasone, K. , Niemelä, T. , Larsson, K.-H. , Ryvarden, L. , &Hibbett, D.S. (2017). A revised family-level classification of the Polyporales (Basidiomycota). Fungal Biology, 121 , 798-824. https://doi.org/10.1016/j.funbio.2017.05.010 28800851
Kaewchai, S. , Lin, F.C. , Wang, H.K. , &Soytong, K. (2010). Characterization of Rigidoporus microporus isolated from rubber trees based on morphology and ITS sequencing. Journal of Agricultural Technology, 6 , 289-298.
Katoh, K. , &Standley, D.M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30 , 772-780. https://doi.org/10.1093/molbev/mst010 23329690
Kobayashi, T. (1972). Rigidoporus zonalis (Berk.) Imazeki causing root and butt rot of Paulownia trees [In Japanese]. Bulletin of the Government Forest Experiment Station, 246 , 69-73. https://www.ffpri.affrc.go.jp/pubs/bulletin/201/documents/246-6.pdf
Kotiranta, H. , Kulju, M. , &Miettinen, O. (2017). Caudicicola gracilis (Polyporales, Basidiomycota), a new polypore species and genus from Finland. Annales Botanici Fennici, 54 , 159-167. https://doi.org/10.5735/085.054.0325
Kumar, S. , Stecher, G. , &Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33 , 1870-1874. https://doi.org/10.1093/molbev/msw054 27004904
Larsen, M.J. , &Lombard, F.F. (1988). The status of Meripilus giganteus (Aphyllophorales, Polyporaceae) in North America. Mycologia, 80 , 612-621. https://doi.org/10.1080/00275514.1988.12025591
Larsson, K.-H. , &Ryvarden, L. (2021). Corticioid fungi of Europe, Vol. 1. Synopsis Fungorum, 43 , 1-266.
Lombard, F.F. , &Chamuris, G.P. (1990). Basidiomycetes. In: C.J.K. Wang, & R.A. Zabel (Eds.), Identification manual for fungi from utility poles in the Eastern United States (pp. 21-104). American Type Culture Collection.
Lombard, F.F. , Davidson, R.W. , &Lowe, J.L. (1960). Cultural characteristics of Fomes ulmarius and Poria ambigua. Mycologia, 52 , 280-294. https://doi.org/10.1080/00275514.1960.12024901
Miettinen, O. , Larsson, E. , Sjökvist, E. , &Larsson, K.-H. (2012). Comprehensive taxon sampling reveals unaccounted diversity and morphological plasticity in a group of dimitic polypores (Polyporales, Basidiomycota). Cladistics, 28 , 251-270. https://doi.org/10.1111/j.1096-0031.2011.00380.x 34872189
Miller, S.L. , Larsson, E. , Larsson, K.-H. , Verbeken, A. , &Nuytinck, J. (2006). Perspectives in the new Russulales. Mycologia, 98 , 960-970. https://doi.org/10.1080/15572536.2006.11832625 17486972
Motato-Vásquez, V. , Pires, R.M. , Vitali, V.M.V. , &Gugliotta, A.M. (2016). Cultural and ligninolytic activity studies of some polypores (Basidiomycota) from brazilian Atlantic Forest, São Paulo State, Brazil. Hoehnea, 43 , 289-300. https://doi.org/10.1590/2236-8906-81/2015
Nakasone, K.K. (1990). Cultural studies and identification of wood-inhabiting Corticiaceae and selected Hymenomycetes from North America. Mycologia Memoir, 15 , 1-412.
Nakasone, K.K. , &Ortiz-Santana, B. (2022). New species and combinations in the Cerrenaceae (Polyporales, Basidiomycota). Lilloa, 59 , 89-113. https://doi.org/10.30550/j.lil/2022.59.S/2022.08.16
Nobles, M.K. (1948). Studies in forest pathology: VI. Identification of cultures of wood-rotting fungi. Canadian Journal of Research, 26 , 281-431. https://doi.org/10.1139/cjr48c-026 18873270
Nobles, M.K. (1958). Cultural characters as a guide to the taxonomy and phylogeny of the Polyporaceae. Canadian Journal of Botany, 36 , 883-926. https://doi.org/10.1139/b58-071
Nobles, M.K. (1965). Identification of cultures of wood-inhabiting Hymenomycetes. Canadian Journal of Botany, 43 , 1097-1139. https://doi.org/10.1139/b65-126
Noguchi, T. , Ohtani, Y. , Hattori, T. , Abe, Y. , &Sahashi, N. (2007). Basidiomycetous fungi associated with butt rot disease of Cryptomeria japonica in the Aso District, Kumamoto Prefecture, Japan [In Japanese]. Journal of the Japanese Forest Society, 89 , 225-229. https://doi.org/10.4005/jjfs.89.225
Núñez, M. , Parmasto, E. , &Ryvarden, L. (2001). New and interesting polypores from East Russia. Fungal Diversity, 6 , 107-114.
Núñez, M. , &Ryvarden, L. (2001). East Asian polypores, Vol. 2. Synopsis Fungorum, 14 , 170-522.
Overholts, L.O. (1953). The Polyporaceae of the United States, Alaska, and Canada. University of Michigan Press.
Pouzar, Z. (1966). Studies in the taxonomy of the Polypores II. Folia Geobotanica et Phytotaxonomica, 1 , 356-375. https://link.springer.com/article/10.1007/BF02854587
Rajchenberg, M. , &Robledo, G. (2013). Pathogenic polypores in Argentina. Forest Pathology, 43 , 171-184. https://doi.org/10.1111/efp.12032
Rehner, S.A. , &Samuels, G.J. (1994). Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Research, 98 , 625-634. https://doi.org/10.1016/S0953-7562(09)80409-7
Runnel, K. , Miettinen, O. , &Lõhmus, A. (2021). Polypore fungi as a flagship group to indicate changes in biodiversity - a test case from Estonia. IMA Fungus, 12 , 2. https://doi.org/10.1186/s43008-020-00050-y 33461627
Ryvarden, L. (1972). A critical checklist of the Polyporaceae in tropical East Africa. Norwegian Journal of Botany, 19 , 229-238.
Ryvarden, L. (1991). Genera of polypores: Nomenclature and taxonomy. Synopsis Fungorum, 5 , 1-363.
Ryvarden, L. , Decock, C. , Mossebo, D. , &Masuka, A. (2022). Poroid fungi of Africa. Synopsis Fungorum, 45 , 1-271.
Ryvarden, L. , &Gilbertson, R.L. (1994). European polypores, Part 2. Synopsis Fungorum, 7 , 394-743.
Ryvarden, L. , &Johansen, I. (1980). A preliminary polypore flora of East Africa. Fungiflora, Oslo, Norway.
Ryvarden, L. , &Melo, I. (2017). Poroid fungi of Europe, 2nd ed. Synopsis Fungorum, 37 , 1-431.
Schmidt, O. , &Liese, W. , Moreth, U. (1996). Decay of timber in a water cooling tower by the basidiomycete Physisporinus vitreus. Material und Organismen, 30 , 161-177.
Schmidt, O. , Schmitt, U. , Moreth, U. , &Potsch, T. (1997). Wood decay by the white-rotting basidiomycete Physisporinus vitreus from a cooling tower. Holzforschung, 51 , 193-200. https://doi.org/10.1515/hfsg.1997.51.3.193
Setliff, E.C. (1972). Cultural characteristics of Poria vincta. Mycologia, 64 , 641-646. https://doi.org/10.1080/00275514.1972.12019310
Shearer, C.A. , Descals, E. , Kohlmeyer, B. , Kohlmeyer, J. , Marvanová, L. , Padgett, D. , Porter, D. , Raja, H.A. , Schmit, J.P. , Thorton, H.A. , &Voglymayr, H. (2007). Fungal biodiversity in aquatic habitats. Biodiversity and Conservation, 16 , 49-67. https://doi.org/10.1007/s10531-006-9120-z
Shino, R. , Shibata, S. , Sotome, K. , Endo, N. , Maekawa, N. , &Nakagiri, A. (2022). Taxonomy and ecology of Physisporinus forming synnema-like structures in freshwater environments. Mycologia, 114 , 587-606. https://doi.org/10.1080/00275514.2022.2050127 35605127
Spirin, V. , Vlasák, J. , Niemelä, T. , &Miettinen, O. (2022). Studies in Spongipellis sensu stricto (Polyporales, Basidiomycota). Lilloa, 59 , 341-358. https://doi.org/10.30550/j.lil/2022.59.S/2022.09.27
Spirin, V. , Volobuev, S. , Malysheva, V. , Miettinen, O. , Kotiranta, H. , &Larsson, K.-H. (2021). Identity of the subalpine-subarctic corticioid fungus Megalocystidium leucoxanthum (Russulales, Basidiomycota) and six related species. Plant Ecology and Evolution, 154 , 231-244. https://doi.org/10.5091/plecevo.2021.1857
Stalpers, J.A. (1978). Identification of wood-inhabiting Aphyllophorales in pure culture. Studies in Mycology, 16 , 1-248.
Tomšovský, M. , Menkis, A. , &Vasaitis, R. (2010). Phylogenetic relationships in European Ceriporiopsis species inferred from nuclear and mitochondrial ribosomal DNA sequences. Fungal Biology, 114 , 350-358. https://doi.org/10.1016/j.funbio.2010.02.004 20943145
Vampola, P. , &Vlasák, J. (2012). Rigidoporus pouzarii, a new polypore species related to Rigidoporus crocatus. Czech Mycology, 64 , 3-11. http://www.czechmycology.org/_cmo/CM64102.pdf
Vilgalys, R. , &Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 172 , 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 2376561
Wang, C.G. , &Dai, Y.C. (2022). Phylogeny and taxonomy of Spongipellis (Polyporales, Basidiomycota) and its micromorphological similar genera. Mycological Progress, 21 , 73. https://doi.org/10.1007/s11557-022-01817-w
Westphalen, M.C. , &Motato-Vásquez, V. (2022). A new species of Pseudolagarobasidium from Brazil and new insights on Cerrena (Basidiomycota, Cerrenaceae). Phytotaxa, 555 , 159-168. https://doi.org/10.11646/phytotaxa.555.2.4
White, T.J. , Bruns, T. , Lee, S. , &Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M.A. Innis, D.H. Gelfand, J.J. Sninsky, & T.J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315-322). Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Wu, F. , Chen, J.J. , Ji, X.H. , Vlasák, J. , &Dai, Y.C. (2017). Phylogeny and diversity of the morphologically similar polypore genera Rigidoporus, Physisporinus, Oxyporus, and Leucophellinus. Mycologia, 109 , 749-765. https://doi.org/10.1080/00275514.2017.1405215 29336678
Wu, S.H. , Hibbett, D.S. , &Binder, M. (2001). Phylogenetic analyses of Aleurodiscus s.l. and allied genera. Mycologia, 93 , 720-731. https://doi.org/10.1080/00275514.2001.12063203
Wu, S.H. , Wei, C.L. , &Chang, C.C. (2022). Aleurodiscus bicornis and A. formosanus spp. nov. (Basidiomycota) with smooth basidiospores, and redescription of A. parvisporus. Mycological Progress, 21 , 147-157. https://doi.org/10.1007/s11557-021-01733-5
Yamashita, Y. , Kinoshita, A. , Yagame, T. , Ogura-Tsujita, Y. , Yokoyama, J. , &Yukawa, T. (2020). Physisporinus is an important mycorrhizal partner for mycoheterotrophic plants: Identification of mycorrhizal fungi of three Yoania species. Mycoscience, 61 , 219-225. https://doi.org/10.1016/j.myc.2020.05.003
Yuan, H.S. , Lu, X. , Dai, Y.C. , Hyde, K.D. , Kan, Y.H. , Kušan, I. , He, S.H. , Liu, N.G. , Sarma, V.V. , Zhao, C.L. , Cui, B.K. , Yousaf, N. , Sun, G. , Liu, S.Y. , Wu, F. , Lin, C.G. , Dayarathne, M.C. , Gibertoni, T.B. , Conceição, L.B. , …&Zhou, L.W. (2020). Fungal diversity notes 1277-1386: taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity, 104 , 1-266. https://doi.org/10.1007/s13225-020-00461-7
