Taxonomy and Phylogeny of Meruliaceae with Descriptions of Two New Species from China

Abstract

Two new wood-inhabiting fungi Hermanssonia fimbriata sp. nov. and Phlebia austroasiana sp. nov. in the Meruliaceae family are described and illustrated from southwestern China based on molecular and morphological evidence. The characteristics of H. fimbriata include annual, resupinate basidiomata, the absence of cystidia and cystidioles, oblong ellipsoid basidiospores of 5–6 × 2.4–3 μm, and growth on rotten gymnosperm wood in the east Himalayas. Its basidiomata change drastically upon drying, from being a light-coloured, juicy, papillose-to-wrinkled hymenophore, to a dark-coloured, corky-to-gelatinous, and more or less smooth hymenophore. The characteristics of Ph. austroasiana include annual, resupinate basidiomata, a hydnoid hymenophore, 2–3 spines per mm, the presence of tubular cystidia of 20–25 × 3–3.5 µm, oblong ellipsoid basidiospores of 4.4–5.2 × 2.1–3 μm, and growth on angiosperm wood in tropical forests in the southern Yunnan Province. The phylogenetic analyses based on the combined 2-locus dataset (ITS1-5.8S-ITS2 (ITS) + nuclear large subunit RNA (nLSU)) confirm the placement of two new species, respectively, in Hermanssonia and Phlebia s. lato. Phylogenetically, the closely-related species to these two new species are discussed.

1. Introduction

The phlebioid clade within Polyporales includes three lineages at a family level, namely Phanerochaetaceae, Irpicaceae, and Meruliaceae [1,2]. The taxonomy of many of the genera belonging to these families is not currently settled, and a case in point example is the genus Phlebia. In a recent study, Chen et al. [3] concluded that Phlebia s.l. is still polyphyletic, with members addressed in all families of the phlebioid clade. Based on their multigene phylogenetic analysis, the core Phlebia clade belongs to the Meruliaceae with three additional clades: the Hydnophlebia clade, the Mycoacia clade, and the Sarcodontia clade. The core Phlebia clade included the genera Aurantiopileus Ginns et al., Aurantiporus Murrill, Pappia Zmitr., and Phlebia s.s., as well as some species of Ceriporiopsis Domański s.l. and Mycoacia s.l. [3].

Phlebia Fr. was erected by Fries [4] and typified by Phlebia radiata Fr. As the delimitation of the genus Phlebia s. str. is not yet clarified, in the present paper, we treat Phlebia sensu in the same way as Chen et al. [3]. The genus is characterized by white-rot, resupinate or rarely pileate basidiocarps with a tuberculate, merulioid, folded, odontioid or hydnoid hymenophore, a monomitic hyphal system, generative hyphae with clamp connections, neither amyloid nor dextrinoid, and allantoid to ellipsoid, hyaline, thin-walled, smooth, neither amyloid nor dextrinoid, acyanophilous basidiospores [3,5]. Formerly, several genera have been proposed to accommodate different lineages of Phlebia s. lato, but still many of the species has no modern interpretation, e.g., [3,6]. The monotypic genus Hermanssonia Zmitr. (Meruliaceae, Polyporales) was erected by Zmitrovich [7], based on H. centrifuga (P. Karst.) Zmitr. (=Phlebia centrifuga P. Karst.). The genus is characterized by white-rot, resupinate to effuse-reflexed, ceraceous to cartilaginous basidiomata, a phlebioid (radially-costate) or tuberculate hymenophore, a monomitic hyphal system, generative hyphae with clamp connections, and cylindrical, hyaline, thin-walled, smooth, neither amyloid nor dextrinoid basidiospores [7].

Four resupinate phlebioid specimens were collected from southwestern China (Tibet and Yunnan Province) during studies on wood-inhabiting fungi, and their morphology corresponded to concepts of Hermanssonia and Phlebia. Phylogenetic analyses based on the ITS1-5.8S-ITS2 (ITS) and nuclear large subunit RNA (nLSU) rDNA sequences were conducted to confirm their affinity. Both morphological and molecular evidence demonstrated that these four specimens represent two undescribed species of Meruliaceae. Thus, they are described in this paper.

2. Materials and Methods

2.1. Morphological Studies

Macro-morphological descriptions were based on voucher specimens and field notes. Microscopic structures were prepared from slide preparations of dried tissues stained with Cotton Blue and Melzer’s reagent as described by Wu et al. [8]. The following abbreviations are used in the description: CB = Cotton Blue; CB– = acyanophilous in Cotton Blue; IKI = Melzer’s reagent; IKI– = neither amyloid nor dextrinoid in Melzer’s reagent; KOH = 5% potassium hydroxide; L = mean spore length (arithmetic average of basidiospores); W = mean spore width (arithmetic average of basidiospores); and Q = variation in the L/W ratios between the specimens studied, (n = a/b) = number of spores (a) measured from given number of specimens (b). When the variation in spore size is shown, 5% of the measurements were excluded from each end of the range, and these values are shown in parentheses. Special colour terms follow Petersen [9] and herbarium abbreviations follow Thiers [10]. The voucher specimens for the present study are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), Beijing, China.

2.2. DNA Extraction, PCR, and Sequencing

Total genomic DNA was extracted from dried specimens using a CTAB Rapid Plant Genome Extraction Kit (Aidlab Biotechnologies Company, Ltd., Beijing, China) according to the manufacturer’s instructions with some modifications [11]. The ITS regions were amplified with primers ITS4 and ITS5 [12]. The nLSU regions were amplified with primers LR0R and LR7 [13].

The polymerase chain reaction (PCR) procedure for the ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 54 °C for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for the nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 30 s, 48 °C for 1 min, and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min [14]. The purification and sequencing of the PCR products was conducted by the Beijing Genomics Institute, Beijing, China, with the same primers used in the PCR reactions. Species were identified by sequence comparison with accessions in the NCBI databases using the BLAST program.

2.3. Phylogenetic Analyses

Phylogenetic trees were constructed using ITS + nLSU rDNA sequences, and phylogenetic analyses were performed with the Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) methods. Sequences of the species and strains were primarily adopted from ITS-based and 28S-based tree topology, as described by Huang et al. [5] and Chen et al. [3]. New sequences generated in this study, along with reference sequences retrieved from GenBank (Table 1), were aligned by MAFFT 7 (Katoh et al. [15]; http://mafft.cbrc.jp/alignment/server/, accessed on 18 April 2022) using the “G-INS-i” strategy and manually adjusted in BioEdit v. 7.2.5 [16]. Unreliably aligned sections were removed before the analyses, and efforts were made to manually inspect and improve the alignment. The data matrix was edited in Mesquite v3.70 (https://www.mesquiteproject.org/ (accessed on 18 April 2022). [17]. The sequence alignment was deposited at TreeBase. Sequences of Hyphoderma mutatum (Peck) Donk and H. setigerum (Fr.) Donk obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 18 April 2022) were used as outgroups to root the trees in the ITS + nLSU analysis.

Table 1. Taxa information and GenBank accession numbers of the sequences used in this study.

Species	Sample	GenBank Accession No.		References
		ITS	nLSU
Aurantiopileus mayaensi	JV 1504/128	KT156706	—	—
A. mayaensi	TJB10228	HM772140	HM772139	[18]
Aurantiporus croceus	Miettinen-16483	KY948745	KY948901	[2]
A. roseus	Dai 13573	KJ698635	KJ698639	[19]
Ceriporiopsis alboaurantia	Cui 4136	KF845955	KF845948	[20]
C. alboaurantia	Cui 2877	KF845954	KF845947	[20]
C. fimbriata	Cui 1671	KJ698634	KJ698638	[19]
C. fimbriata	Dai 11672	KJ698633	KJ698637	[19]
C. gilvescens	BRNM 710166	FJ496684	FJ496684	[21]
C. gilvescens	BRNM 667882	FJ496685	FJ496719	[21]
C. guidella	HUBO 7659	FJ496687	FJ496722	[21]
C. kunmingensis	CLZhao 152	KX081072	KX081074	[22]
C. kunmingensis	CLZhao 153	KX081073	KX081075	[22]
C. lagerheimii	58240	KX008365	KX081077	[23]
C. pseudoplacenta	PRM 899297	JN592497	JN592504	[24]
C. pseudoplacenta	PRM 899300	JN592498	JN592505	[24]
C. semisupina	Cui 10222	KF845956	KF845949	[20]
C. semisupina	Cui 7971	KF845957	KF845950	[20]
Climacodon septentrionalis	AFTOL-767	AY854082	AY684165	[25]
C. septentrionalis	RLG-6890-Sp	KP135344	—	[26]
Crustodontia chrysocreas	HHB-3946	KP135357	—	[26]
C. chrysocreas	HHB-6333-Sp	KP135358	KP135263	[26]
C. nigrodontea	CLZhao 2758	MT896824	—	[5]
C. nigrodontea	CLZhao 2445	MT896821	MT896818	[27]
C. sp.	KUC20121123-24	KJ668482	—	[28]
C. tongxiniana	CLZhao 2255	MT020773	MT020751	[27]
C. tongxiniana	CLZhao 2316	MT020774	MT020752	[27]
Geesterania carneola	MCW 388/12	KY174999	KY174999	[29]
G. davidii	MCW 396/12	KY174998	KY174998	[29]
Hermanssonia centrifuga	CBS 125890	MH864088	MH875547	[30]
H. centrifuga	HHB-9239-Sp	KP135380	KP135262	[26]
H. fimbriata	Dai 23266	ON135436	ON135440	Present study
H. fimbriata	Dai 23305	ON135437	ON135441	Present study
H. fimbriata	Dai 23306	ON135438	ON135442	Present study
Hydnophanerochaete odontoidea	CLZhao 3882	MH784919	MH784929	[31]
H. odontoidea	CLZhao 4036	MH784927	MH784937	[31]
Hydnophlebia chrysorhiza	FD-282	KP135338	KP135217	[26]
H. chrysorhiza	HHB-18767	KP135337	—	[26]
Hyphoderma mutatum	HHB-15479-Sp	KP135296	KP135221	[26]
H. setigerum	FD-312	KP135297	KP135222	[26]
Lilaceophlebia livida	FCUG 2189	AF141624	AF141624	[21]
L. livida	FCUG 1290	HQ153414	—	[32]
L. subserialis	FCUG 1434	AF141631	AF141631	—
Luteochaete subglobosa	CLZhao 3639	MK881898	MK881788	[33]
L. subglobosa	CLZhao 3475	MK881897	MK881787	[33]
Luteoporia albomarginata	Dai 15229	KU598873	KU598878	[34]
L. albomarginata	GC 1702-1	LC379003	LC379155	[35]
L. citriniporia	Dai 19507	MT872218	MT872216	[36]
L. citriniporia	Dai 19622	MT872219	MT872217	[36]
L. lutea	CHWC 1506-68	MZ636997	MZ637157	[3]
L. lutea	GC 1409-1	MZ636998	MZ637158	[3]
Mycoacia aurea	DLL 2011263	KJ140747	—	[1]
M. aurea	RLG-5075-Sp	KY948759	MZ637161	[2,3]
M. aurea	DLL2011_100	KJ140614	—	[37]
M. fuscoatra	HHB 15354T	KP135367	—	[26]
M. cf. kurilensis	WEI 18-312	MZ637001	MZ637162	[3]
M. cf. kurilensis	WEI 18-324	MZ637002	MZ637163	[3]
M. fuscoatra	KHL 13275	JN649352	JN649352	[21]
M. nothofagi	HHB 12067	KP135370	—	[26]
M. nothofagi	KHL 13750	GU480000	GU480000	[21]
Mycoaciella bispora	EL13_99	—	AY586692	[38]
M. efibulata	WEI 19-057	MZ637012	MZ637172	[3]
M. efibulata	WEI 16-172	MZ637011	MZ637171	[3]
Odoria alborubescens	BP106943	MG097864	MG097867	[39]
O. alborubescens	BRNU 627479	JQ821319	JQ821318	[40]
Pappia fissilis	814	HQ728291	HQ729001	[41]
P. fissilis	BRNM 699803	HQ728292	HQ729002	[41]
Phlebia acanthocystis	KUC20131001-33	KJ668484	KJ668337	[26]
P. acanthocystis	FP150571	KY948767	KY948844	[2]
P. acerina	FD 301	KP135378	—	[2]
P. acerina	HHB 11146	KP135372	—	[26]
P. austroasiana	Dai 17556	ON135439	ON135443	Present study
P. austroasiana	E8898A	KJ654590	—	[42]
P. brevispora	HHB 7030	KP135387	—	[26]
P. brevispora	FBCC1463	LN611135	LN611135	[43]
P. floridensis	HHB 7175	KP135384	—	[26]
P. floridensis	HHB-9905-Sp	KP135383	KP135264	[26]
P. fuscotuberculata	CLZhao 10227	MT020759	MT020737	[27]
P. fuscotuberculata	CLZhao 10239	MT020760	MT020738	[27]
P. hydnoidea	HHB-1993-Sp	KY948778	KY948853	[2]
P. lindtneri	GB-1027	AB210076	—	[44]
P. lindtneri	GB-501	KY948772	KY948847	[2]
P. ludoviciana	HHB-8715-Sp	KY948770	KY948846	[2]
P. ludoviciana	FD-427	KP135342	—	[26]
P. nantahaliensis	HHB-2816-Sp	KY948777	KY948852	[2]
P. radiata	CBS 285.56	MH857642	MH869187	[30]
P. radiata	AFTOL-484	AY854087	AF287885	[25]
P. radiata	UBC: F19726	HQ604797	HQ604797	[1]
P. rufa	FBCC297	LN611092	LN611092	[43]
P. rufa	HHB-14924	KP135374	—	[26]
P. serialis	FCUG 2868	HQ153429	—	[32]
P. serialis	UC2023146	KP814195	—	[33]
P. setulosa	PH 11749	GU461312	—	[1]
P. setulosa	HHB-6891-Sp	KP135382	KP135267	[26]
P. setulosa	AH31879	GQ259417	GQ259417	[45]
P. subochracea I	KGN 162/95	EU118656	EU118656	[46]
P. subochracea II	FBCC295	LN611116	LN611116	[43]
P. subochracea II	HHB-8494-Sp	KY948768	KY948845	[2]
P. tomentopileata	CLZhao 9563	MT020765	MT020743	[27]
P. tomentopileata	CLZhao 9515	MT020764	MT020742	[27]
P. tremellosa	ES 20082	JX109859	JX109859	[1]
P. tremellosa	CBS 217.56	MH857589	MH869138	[30]
Phlebiporia bubalina	Dai 13168	KC782526	KC782528	[47]
P. bubalina	Dai 15179	KY131843	KY131902	[48]
Sarcodontia uda	FP-101544-Sp	KP135361	KP135232	[26]
Sarcodontia uda	USDA Kropp 1	KY948764	—	[2]
Scopuloides hydnoides	FP-150473	KP135355	KP135284	[26]
S. hydnoides	WEI 17-569	MZ637085	MZ637283	[3]
Stereophlebia tuberculata	FCUG 3157	HQ153427	—	[32]
S. tuberculata	Wu 1708-107	MZ637089	MZ637286	[3]

New sequences are in bold.

Table 1. Taxa information and GenBank accession numbers of the sequences used in this study.

Species	Sample	GenBank Accession No.		References
		ITS	nLSU
Aurantiopileus mayaensi	JV 1504/128	KT156706	—	—
A. mayaensi	TJB10228	HM772140	HM772139	[18]
Aurantiporus croceus	Miettinen-16483	KY948745	KY948901	[2]
A. roseus	Dai 13573	KJ698635	KJ698639	[19]
Ceriporiopsis alboaurantia	Cui 4136	KF845955	KF845948	[20]
C. alboaurantia	Cui 2877	KF845954	KF845947	[20]
C. fimbriata	Cui 1671	KJ698634	KJ698638	[19]
C. fimbriata	Dai 11672	KJ698633	KJ698637	[19]
C. gilvescens	BRNM 710166	FJ496684	FJ496684	[21]
C. gilvescens	BRNM 667882	FJ496685	FJ496719	[21]
C. guidella	HUBO 7659	FJ496687	FJ496722	[21]
C. kunmingensis	CLZhao 152	KX081072	KX081074	[22]
C. kunmingensis	CLZhao 153	KX081073	KX081075	[22]
C. lagerheimii	58240	KX008365	KX081077	[23]
C. pseudoplacenta	PRM 899297	JN592497	JN592504	[24]
C. pseudoplacenta	PRM 899300	JN592498	JN592505	[24]
C. semisupina	Cui 10222	KF845956	KF845949	[20]
C. semisupina	Cui 7971	KF845957	KF845950	[20]
Climacodon septentrionalis	AFTOL-767	AY854082	AY684165	[25]
C. septentrionalis	RLG-6890-Sp	KP135344	—	[26]
Crustodontia chrysocreas	HHB-3946	KP135357	—	[26]
C. chrysocreas	HHB-6333-Sp	KP135358	KP135263	[26]
C. nigrodontea	CLZhao 2758	MT896824	—	[5]
C. nigrodontea	CLZhao 2445	MT896821	MT896818	[27]
C. sp.	KUC20121123-24	KJ668482	—	[28]
C. tongxiniana	CLZhao 2255	MT020773	MT020751	[27]
C. tongxiniana	CLZhao 2316	MT020774	MT020752	[27]
Geesterania carneola	MCW 388/12	KY174999	KY174999	[29]
G. davidii	MCW 396/12	KY174998	KY174998	[29]
Hermanssonia centrifuga	CBS 125890	MH864088	MH875547	[30]
H. centrifuga	HHB-9239-Sp	KP135380	KP135262	[26]
H. fimbriata	Dai 23266	ON135436	ON135440	Present study
H. fimbriata	Dai 23305	ON135437	ON135441	Present study
H. fimbriata	Dai 23306	ON135438	ON135442	Present study
Hydnophanerochaete odontoidea	CLZhao 3882	MH784919	MH784929	[31]
H. odontoidea	CLZhao 4036	MH784927	MH784937	[31]
Hydnophlebia chrysorhiza	FD-282	KP135338	KP135217	[26]
H. chrysorhiza	HHB-18767	KP135337	—	[26]
Hyphoderma mutatum	HHB-15479-Sp	KP135296	KP135221	[26]
H. setigerum	FD-312	KP135297	KP135222	[26]
Lilaceophlebia livida	FCUG 2189	AF141624	AF141624	[21]
L. livida	FCUG 1290	HQ153414	—	[32]
L. subserialis	FCUG 1434	AF141631	AF141631	—
Luteochaete subglobosa	CLZhao 3639	MK881898	MK881788	[33]
L. subglobosa	CLZhao 3475	MK881897	MK881787	[33]
Luteoporia albomarginata	Dai 15229	KU598873	KU598878	[34]
L. albomarginata	GC 1702-1	LC379003	LC379155	[35]
L. citriniporia	Dai 19507	MT872218	MT872216	[36]
L. citriniporia	Dai 19622	MT872219	MT872217	[36]
L. lutea	CHWC 1506-68	MZ636997	MZ637157	[3]
L. lutea	GC 1409-1	MZ636998	MZ637158	[3]
Mycoacia aurea	DLL 2011263	KJ140747	—	[1]
M. aurea	RLG-5075-Sp	KY948759	MZ637161	[2,3]
M. aurea	DLL2011_100	KJ140614	—	[37]
M. fuscoatra	HHB 15354T	KP135367	—	[26]
M. cf. kurilensis	WEI 18-312	MZ637001	MZ637162	[3]
M. cf. kurilensis	WEI 18-324	MZ637002	MZ637163	[3]
M. fuscoatra	KHL 13275	JN649352	JN649352	[21]
M. nothofagi	HHB 12067	KP135370	—	[26]
M. nothofagi	KHL 13750	GU480000	GU480000	[21]
Mycoaciella bispora	EL13_99	—	AY586692	[38]
M. efibulata	WEI 19-057	MZ637012	MZ637172	[3]
M. efibulata	WEI 16-172	MZ637011	MZ637171	[3]
Odoria alborubescens	BP106943	MG097864	MG097867	[39]
O. alborubescens	BRNU 627479	JQ821319	JQ821318	[40]
Pappia fissilis	814	HQ728291	HQ729001	[41]
P. fissilis	BRNM 699803	HQ728292	HQ729002	[41]
Phlebia acanthocystis	KUC20131001-33	KJ668484	KJ668337	[26]
P. acanthocystis	FP150571	KY948767	KY948844	[2]
P. acerina	FD 301	KP135378	—	[2]
P. acerina	HHB 11146	KP135372	—	[26]
P. austroasiana	Dai 17556	ON135439	ON135443	Present study
P. austroasiana	E8898A	KJ654590	—	[42]
P. brevispora	HHB 7030	KP135387	—	[26]
P. brevispora	FBCC1463	LN611135	LN611135	[43]
P. floridensis	HHB 7175	KP135384	—	[26]
P. floridensis	HHB-9905-Sp	KP135383	KP135264	[26]
P. fuscotuberculata	CLZhao 10227	MT020759	MT020737	[27]
P. fuscotuberculata	CLZhao 10239	MT020760	MT020738	[27]
P. hydnoidea	HHB-1993-Sp	KY948778	KY948853	[2]
P. lindtneri	GB-1027	AB210076	—	[44]
P. lindtneri	GB-501	KY948772	KY948847	[2]
P. ludoviciana	HHB-8715-Sp	KY948770	KY948846	[2]
P. ludoviciana	FD-427	KP135342	—	[26]
P. nantahaliensis	HHB-2816-Sp	KY948777	KY948852	[2]
P. radiata	CBS 285.56	MH857642	MH869187	[30]
P. radiata	AFTOL-484	AY854087	AF287885	[25]
P. radiata	UBC: F19726	HQ604797	HQ604797	[1]
P. rufa	FBCC297	LN611092	LN611092	[43]
P. rufa	HHB-14924	KP135374	—	[26]
P. serialis	FCUG 2868	HQ153429	—	[32]
P. serialis	UC2023146	KP814195	—	[33]
P. setulosa	PH 11749	GU461312	—	[1]
P. setulosa	HHB-6891-Sp	KP135382	KP135267	[26]
P. setulosa	AH31879	GQ259417	GQ259417	[45]
P. subochracea I	KGN 162/95	EU118656	EU118656	[46]
P. subochracea II	FBCC295	LN611116	LN611116	[43]
P. subochracea II	HHB-8494-Sp	KY948768	KY948845	[2]
P. tomentopileata	CLZhao 9563	MT020765	MT020743	[27]
P. tomentopileata	CLZhao 9515	MT020764	MT020742	[27]
P. tremellosa	ES 20082	JX109859	JX109859	[1]
P. tremellosa	CBS 217.56	MH857589	MH869138	[30]
Phlebiporia bubalina	Dai 13168	KC782526	KC782528	[47]
P. bubalina	Dai 15179	KY131843	KY131902	[48]
Sarcodontia uda	FP-101544-Sp	KP135361	KP135232	[26]
Sarcodontia uda	USDA Kropp 1	KY948764	—	[2]
Scopuloides hydnoides	FP-150473	KP135355	KP135284	[26]
S. hydnoides	WEI 17-569	MZ637085	MZ637283	[3]
Stereophlebia tuberculata	FCUG 3157	HQ153427	—	[32]
S. tuberculata	Wu 1708-107	MZ637089	MZ637286	[3]

New sequences are in bold.

Maximum Parsimony analysis was applied to the ITS + nLSU dataset sequences. The approaches to phylogenetic analysis utilized those conducted by Chen and Cui [47], and the tree was constructed using PAUP* version 4.0 beta 10 [49]. All the characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with tree bisection and reconnection (TBR) branch swapping, and 1000 random sequence addition maxtrees were set to 5000. Branches of zero length were collapsed, and all the parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [50]. Descriptive tree statistics, including the Consistency Index (CI), Homoplasy Index (HI), Rescaled Consistency index (RC), Retention Index (RI), and tree length (TL), were calculated for each Maximum Parsimonious Tree (MPT) generated.

The research using ML was conducted using RAxML-HPC v. 8.2.3 [51] and RAxML-HPC through the CIPRES Science Gateway ([52]; http://www.phylo.org, accessed on 18 April 2022). Statistical support values (BS) were obtained using nonparametric bootstrapping with 1000 replicates. The BI analysis was performed with MrBayes 3.2.7a [53]. Four Markov chains were run for two runs from random starting trees for 3 million generations until the split deviation frequency value < 0.01, and the trees were sampled at every 1000 generation. The first 25% of the sampled trees were discarded as burn-in, and the remaining ones were used to reconstruct a majority rule consensus tree and calculate the Bayesian Posterior Probabilities (BPP) of the clades.

A total of 24 models of evolution were scored using PAUP* version 4.0 beta 10 [49]. Optimal substitution models for the combined dataset were then determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.3 [54,55]. The model GTR + I + G was selected for use in the Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.

Branches that received bootstrap support for Maximum Likelihood (BS), Maximum Parsimony (BP), and Bayesian Posterior Probabilities (BPP) > 75% (BS), 50% (BP), and 0.9 (BPP) were considered to be significantly supported. In addition, the ML analysis resulted in the best tree, and only the ML tree is shown along with the support values from the MP and BI analyses. FigTree v1.4.4 [56] was used to visualize the resulting tree.

3. Results

3.1. Phylogenetic Analyses

The combined ITS + nLSU dataset included sequences from 110 specimens representing 61 taxa (Table 1). The dataset had an aligned length of 2349 characters, of which 1503 were constant, 195 were variable but parsimony-uninformative, and 651 were parsimony-informative. MP analysis yielded nine equally parsimonious trees (TL = 3586, CI = 0.377, RI = 0.752, RC = 0.283, HI = 0.623). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR + I + G. Bayesian analysis and MP analysis resulted in a similar topology to the ML analysis, with an average standard deviation of split frequencies of 0.006112 (BI).

The phylogeny (Figure 1) inferred from the ITS and nLSU sequences demonstrated that the new species, Hermanssonia fimbriata and Phlebia austroasiana, clustered into the genera Hermanssonia and Phlebia, respectively. Hermanssonia fimbriata grouped with H. centrifuga with strong support (100% BS, 100% BP, and 1.00 BPP, Figure 1) and Phlebia austroasiana grouped with Ph. brevispora Nakasone with strong support (92% BP, 97% BS, 1.00 BPP, Figure 1).

3.2. Taxonomy

1.

Hermanssoniafimbriata Z.B. Liu & Y.C. Dai, sp. Nov. (Figure 2A,B and Figure 3)

MycoBank number: MB 844038.

Diagnosis—Hermanssonia fimbriata is characterized by annual, resupinate basidiomata, a monomitic hyphal system with clamp connections, the absence of cystidia and cystidioles, and basidiospores which are oblong ellipsoid, hyaline, thin-walled, smooth, IKI–, CB–, and 5–6 × 2.4–3 μm. Its basidiomata change drastically upon drying, from being a light-coloured, juicy, papillose-to-wrinkled hymenophore, to a dark-coloured, corky-to-gelatinous, and more or less smooth hymenophore.

Etymology—Fimbriata (Lat.): refer to the species having fimbriate margin.

Type—China. Tibet, Linzhi, Milin County, Nanyi Valley, ca. 94°22′E, 29°37′N, elev. 3000 m, on rotten wood of Picea, 22 October 2021, Dai 23266 (BJFC 037837).

Basidiomata—Annual, resupinate, adnate, when fresh ceraceous and salmon (6A4) when juvenile, gelatinous, darkening to pale mouse grey (7C2) to light vinaceous grey (13B2/3) when mature, becoming corky, salmon (6A4) and reddish brown (8/9E7) upon drying, first as small colonies, later confluent up to 10 cm or more in the longest dimension, 4 cm in the widest dimension, and less than 0.1 mm thick at center when dry; hymenial surface irregularly papillose and partly radially or unevenly wrinkled; margin white and fimbriate; subiculum very thin to almost absent.

Hyphal structure—Hyphal system monomitic; generative hyphae with clamp con-nections, IKI–, CB–; tissue unchanged in KOH.

Subiculum—Generative hyphae hyaline, thin- to thick-walled, smooth, rarely branched, loosely interwoven, 2–4 μm in diam.

Hymenium—Generative hyphae in subhymenium hyaline, thin-walled, smooth, oc-casionally branched, loosely interwoven, 1.5–3 μm in diam; cystidia and cystidioles ab-sent; basidia clavate, hyaline, bearing four sterigmata and a basal clamp connection, 25–30 × 5–6 μm; basidioles in shape similar to basidia, but slightly shorter.

Basidiospores—Ellipsoid to oblong ellipsoid, hyaline, thin-walled, smooth, IKI–, CB–, (4.5–) 5–6 × (2.2–) 2.4–3 μm, L = 5.51 μm, W = 2.78 μm, Q = 1.88–2.04 (n = 60/2).

Additional specimens (paratypes) examined—China. Tibet, Linzhi, Milin County, Nanyi Valley, ca. 94°22′E, 29°37′N, elev. 3000 m, on rotten wood of Picea, 22 October 2021, Dai 23305 (BJFC 037876), Dai 23306 (BJFC 037877).

2.

Phlebia austroasiana Z.B. Liu & Y.C. Dai, sp. Nov. Figure 2C and Figure 4

MycoBank number: MB 844039.

Diagnosis—Phlebia austroasiana is characterized by annual, resupinate basidiomata, a hymenophore with spines, 2–3 spines per mm, a monomitic hyphal system with clamp connections, the presence of tubular cystidia of 20–25 × 3–3.5 µm, and basidiospores which are oblong ellipsoid, hyaline, thin-walled, smooth, IKI–, CB–, 4.4–5.2 × 2.1–3 μm.

Etymology—Austroasiana (Lat.): refer to the species which is distributed in southeast Asia.

Type—China. Yunnan Province, Jinghong, Primeval Forest Park, ca. 100°52′E, 22°01′N, elev. 763 m, on angiosperm stump, 17 June 2017, Dai 17556 (BJFC 025088).

Basidiomata—Annual, resupinate, tightly adnate, gelatinous when dry, up to 5 cm long, 4 cm wide; hymenophore hydnoid, clay buff (6D4) when dry, not cracked; margin indistinct; spines crowded, clay buff (6D4), subulate, mostly separated, rarely fused, up to 2 mm long, 2–3 per mm at the base. Subiculum white, very thin to almost absent.

Hyphal structure—Hyphal system monomitic; generative hyphae with clamp con-nections, IKI–, CB–; tissue unchanged in KOH.Spines—Generative hyphae in spine trama hyaline, thin-walled, smooth, frequently branched, loosely interwoven, 2–3.5 μm in diam; cystidia tubular, thin-walled, with a basal clamp connection, 20–25 × 3–3.5 µm; cystidioles absent; basidia clavate, hyaline, bearing four sterigmata and a basal clamp connection, 18–26 × 4–5 μm; basidioles in shape similar to basidia, but slightly shorter.

Basidiospores—Ellipsoid to oblong ellipsoid, hyaline, thin-walled, smooth, IKI–, CB–, (4.1–)4.4–5.2 × (2–)2.1–3 μm, L = 4.86 μm, W = 2.53 μm, Q = 1.92 (n = 60/1).

4. Discussion

Chen et al. [3] divided the taxa of Meruliaceae into four clades: the core Phlebia clade, the Hydnophlebia clade, the Mycoacia clade, and the Sarcodontia clade. Two new species, Hermanssonia fimbriata and Phlebia austroasiana, are described in this study, based on morphological characters and phylogenetic analyses. Phylogenetically, they are nested in the core Phlebia clade, based on the ITS + nLSU sequence data (Figure 1).

Phylogenetically, three specimens of Hermanssonia fimbriata formed a lineage with strong support (100% BS, 100% BP, and 1.00 BPP, Figure 1) and grouped with H. centrifuga with strong support (100% BS, 100% BP, and 1.00 BPP). Both species share annual, resupinate basidiomata, a monomitic hyphal system, generative hyphae with clamp connections, thin-walled, IKI–, CB– basidiospores, and growth on rotten gymnosperm wood [57]. Hermanssonia fimbriata can be distinguished from H. centrifuga by its shorter basidiospores (5–6 × 2.4–3 µm vs. 6.5–9 × 2.5–3 µm, [57]). Hermanssonia centrifuga was described as Phlebia centrifuga P. Karst. from Finland [58], and an Asian taxon, Phlebia macra Litsch., was described from Siberia [59]. The latter was treated as a synonym of Ph. centrifuga [60]. Phlebia macra differs from Hermanssonia fimbriata by larger basidiospores (6–7.5 × 3–3.2 µm vs. 5–6 × 2.4–3 µm, [59]). Morphologically, H. fimbriata is similar to Phlebia coccineofulva Schwein., Ph. femsjoeensis (Litsch. & S. Lundell) J. Erikss. & Hjortstam, and Ph. radiata. These four species share the phlebioid hymenophore, but the last three species have cystidia, while cystidia are absent in Hermanssonia fimbriata. Above all, basidiospores of H. fimbriata are larger than that of Phlebia femsjoeensis (4–5 × 2–2.5 µm, [61]) and Ph. radiata (4–5 × 1.8–2 µm, [61]), but thinner than that of Ph. coccineofulva (2.8–3.5 µm in width, [61]). Hermanssonia fimbriata also resembles Phlebia subserialis (Bourdot & Galzin) Donk and Luteochaete subglobosa (Sheng H. Wu) C.C. Chen & Sheng H. Wu (=Phlebia wuliangshanensis C.L. Zhao) by the resupinate and ceraceous basidiomata when fresh, a monomitic hyphal system, and generative hyphae with clamp connections; however, cystidia are abundant in L. subglobosa and Phlebia subserialis, while cystidia are absent in Hermanssonia fimbriata. In addition, basidiospores of H. fimbriata are wider than that of Phlebia subserialis (2.4–3 µm vs. 2–2.5 µm, [61]), but thinner than that of Luteochaete subglobosa (2.4–3 µm vs. 3–3.7 µm, [5]). Hermanssonia remained a monotypic genus until the present paper which contributes the second species in the genus.

An ITS sequence KJ654590 of sample E8898A, named Phlebia sp. from GenBank, is almost identical to Dai 17556 in the ITS regions and the similarity between them is up to 99.65%. Hence, we believe the sample E8898A collected from Indonesia [42] represents the same species as our specimen (Dai 17556) collected from the Yunnan Province, China. Both samples were collected in tropical Asia, and formed a lineage with strong support (100% BS, 100% BP, and 1.00 BPP, Figure 1) in our phylogeny. Hence, Phlebia austroasiana is described based on these two samples. Ph. austroasiana is closely related to Ph. brevispora (92% BP, 97% BS, 1.00 BPP, Figure 1), however, morphologically, Ph. brevispora differs from Ph. austroasiana by its tuberculate hymenophore [62], while Ph. austroasiana has a hydnoid hymenophore. In addition, Ph. austroasiana is distinguished from Ph. brevispora by its larger basidiospores (4.4–5.2 × 2.1–3 µm vs. 4–4.5 × 2–2.5 µm, [62]). Morphologically, Ph. austroasiana is similar to Ph. capitata Bernicchia & Gorjón. in macromorphology, but the cystidia in Ph. capitata are capitate [61], while the cystidia in Ph. austroasiana are tubular. In addition, Ph. austroasiana is distinguished from Ph. capitata by its smaller basidiospores (4.4–5.2 × 2.1–3 µm vs. 5–5.5 × 2.5–3 μm, [61]).