Two new species of Fuscoporia (Hymenochaetales, Basidiomycota) from southern China based on morphological characters and molecular evidence.

Fuscoporia (Hymenochaetaceae) is characterized by annual to perennial, resupinate to pileate basidiocarps, a dimitic hyphal system, presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Phylogenetic analyses based on the nLSU and a combined ITS, nLSU and RPB2 datasets of 18 species of Fuscoporia revealed two new lineages that are equated to two new species; Fuscoporia ramulicola sp. nov. grouped together with F. ferrea, F. punctatiformis, F. subferrea and F. yunnanensis with a strong support; Fuscoporia acutimarginata sp. nov. formed a strongly supported lineage distinct from other species. The individual morphological characters of the new species and their related species are discussed. A key to Chinese species of Fuscoporia is provided. Qian Chen, Yu-Cheng Dai.

Introduction

Murrill (1907) was established based on (Schrad.) Murrill. However, the genus has been unconsidered for a long time, reduced as a synonym of Quél. (e.g., Overholts 1953; Ryvarden and Johansen 1980; Larsen and Cobb-Poulle 1990). Fiasson and Niemelä (1984) firstly used morphological features to segregate some members of into distinct taxonomic entities, including Murrill, Murrill, Murrill, Murrill and .

Fiasson and Niemelä (1984) defined by annual to perennial, resupinate to pileate basidiocarps, a dimitic hyphal system with generative hyphae in the dissepiment edge and the tube trama often encrusted with crystals, presence of hymenial setae and hyaline, thin-walled, smooth basidiospores. Later on, phylogenetic studies based on nuclear large subunit (nLSU) ribosomal RNA-based phylogeny confirmed that formed a lineage distinct from s. s. (Wagner and Fischer 2001, 2002). Previous studies on were mostly based on morphological characteristics (Groposo et al. 2007; Baltazar et al. 2009; Baltazar and Gibertoni 2010; Raymundo et al. 2013) but, recently, more new taxa were described based on both molecular analyses and morphology (Niemelä et al. 2001; Jang et al. 2012; Pires et al. 2015; Chen and Yuan 2017; Chen et al. 2019).

In our study, phylogenetic analyses were carried out based on the nLSU and combined ITS, nLSU and RPB2 datasets including 99 (60 newly generated) sequences representing 18 species of . From the analyses, two new species of were found and described. In addition, a key to Chinese species in the genus was provided.

Materials and methods

Morphological studies

The studied specimens are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions are based on field observations and notes and dry herbarium specimens. The microscopic analyses followed that described by Cui et al. (2019). Sections were studied at ultimate magnification ×1000 applying Nikon Eclipse 80i microscopy and phase contrast illumination. Drawings were made with the aid of a drawing tube. The measurements and drawings were made from slide preparations stained with Cotton Blue. In recording spore size variation, 5% of measurements were excluded from each end of the range and given in parentheses. The following abbreviations are used in the article: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB- = acyanophilous, IKI = Melzer’s reagent, IKI- = neither amyloid nor dextrinoid, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between specimens studied, and n (a/b) = number of spores (a) measured from given number of specimens (b). Special color terms are cited from Petersen (1996).

DNA extraction and sequencing

Extract total genomic DNA was extracted from dried specimens by CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer’s instructions with some modifications (Chen et al. 2016; Han et al. 2016). To generate PCR amplicons, the following primer pairs were used: ITS4 and ITS5 (White et al. 1990) for the internal transcribed spacer (ITS), LR0R and LR7 (Vilgalys and Hester 1990) for nuclear large submit (nLSU) and bRPB2-6F and bRPB2-7.1R (Matheny 2005) for partial RNA polymerase II, second largest submit (RPB2). The PCR procedures followed Song and Cui (2017) and Zhu et al. (2019). DNA sequencing was performed at Beijing Genomics Institute and the sequences are deposited in GenBank and listed in Table 1.

Table 1.

Taxa and GenBank accession numbers for ITS, nLSU and RPB2 sequences used in the phylogenetic analyses (Fig. 2).

Species	Sample no.	Locality	GenBank accession no.
			ITS	nLSU	RPB2
Fuscoporia acutimarginata	Dai 15137	China	MH050751	MH050765	MN159384
	Dai 16892	China	MH050752	MH050766	MH079393
	Dai 15115	China	MN121764	MN121823	MN159385
F. atlantica	SP 445618	Brazil	KP058515	KP058517	–
	SP 465829	Brazil	KP058514	KP058516	–
F. callimorpha	Dai 17388	Brazil	MN121765	MN121824	–
	Dai 17389	Brazil	MN121766	MN121825	–
F. contigua	Dai 16025	USA	MG008401	MG008454	MH079406
	JV 1204/22.6-J	USA	MG008403	MG008456	MH079407
	Dai 13567A	Romania	MG008402	MG008455	MN159386
F. ferrea	MUCL 45984	France	KX961112	KY189112	–
	Cui 11801	China	KX961101	KY189101	MN159387
	JV 1105/3-J	USA	MH050760	MH050770	MH079392
F. ferruginosa	JV 1309/4	Slovakia	KX961102	KY189102	MH079405
	JV 1507/11-CN	Europe	MG008400	MG008453	MH079404
F. gilva	Cui 11209	China	MN121767	MN121826	MN159388
	Dai 15681	China	MN121768	MN121827	MN159389
F. insolita	SP 5251	Russia	KJ677113	–	–
	SP 5208	Russia	MN121769	MN121828	–
F. punctatiformis	Doll#872	USA	MH050753	–	–
	Dai 17443	Brazil	MH050755	MH050764	–
F. ramulicola	Dai 15723	China	MH050749	MH050762	MH079398
	Dai 16155	China	MH050750	MH050763	MH079399
F. rufitincta	JV 1008/25	USA	KJ940029	KX058575	–
	JV 0904/142	USA	KJ940030	KX058574	–
F. senex	KUC 20110922-13	Korea	JX463658	JX463652	–
	MEL:2382630	Australia	KP012992	KP012992	–
F. setifer	Dai 15710	China	MH050758	MH050767	MN159390
	Dai 15706	China	MH050759	MH050769	MN159391
F. subferrea	Dai 16326	China	KX961097	KY053472	MH079400
	Dai 16327	China	KX961098	KY053473	MH079401
F. torulosa	JV 1405/2	Czech	KX961106	KY189106	MN159392
	JV 1312/19-Kout	Spain	KX961107	KY189107	MN159393
F. viticola	JV 0911/6	Czech	KX961110	KY189110	–
	He 2081	USA	MN121770	MN121829	–
F. wahlbergii	Dai 15636	China	MG008397	MG008450	MH079402
	Dai 15659	China	MG008398	MG008451	MH079403
F. yunnanensis	Cui 8182	China	MH050756	–	MN159394
	Dai 15637	China	MH050757	MH050768	MN159395
Coniferiporia sulphurascens	Cui 10429	China	KR350565	KR350555	–
C. weirii	CFS 504	Canada	AY829341	AY829345	–

Note: New sequences produced by this work are in bold.

Figure 2.

Phylogeny of inferred from ITS+nLSU+RPB2 sequences. Topology is from MP tree and statistical values (MP/BI/ML) are indicated for each node that simultaneously received BS from ML and MP not below 75%, and BPP from BI not below 0.9. Names of new species are in bold.

Phylogenetic analyses

Sixty new sequences (nineteen ITS, seventeen nLSU and twenty-four RPB2) of species were newly generated (Table 1). All sequences of ITS+nLSU+RPB2 analysis (Fig. 2) were shown in Table 1. Additional sequences of representatives genera of included in nLSU analysis (Fig. 1) were downloaded from GenBank to explore the phylogenetic relationships of , which were used in the previous phylogenetic study (Zhou et al. 2016; Chen et al. 2019), thus not shown in Table 1. (Fr.) Ryvarden, (Schumach.) Donk, and (Bres.) J. Erikss. were included as outgroups in nLSU analysis based on previous studies (Zhou et al. 2016; Chen et al. 2019). The outgroups selected for ITS+nLSU+RPB2 analysis were (Murrill) L.W. Zhou & Y.C. Dai and (Pilát) L.W. Zhou & Y.C. Dai because resulted as a sister group of in previous studies (Fig. 1; Zhou et al. 2016; Chen and Yuan 2017; Chen et al. 2019).

Figure 1.

Phylogenetic positions of and the new species within the inferred from the nLSU sequences. Topology is from MP tree and statistical values (MP/BI/ML) are indicated for each node that simultaneously received BS from ML and MP not below 75%, and BPP from BI not below 0.9. Names of new species are in bold.

Sequences were aligned with BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps were manually adjusted to optimize the alignment. Sequence alignment was deposited at TreeBase (http://purl.org/phylo/treebase; submission ID 22620). Phylogenetic analysis was carried out according to previous studies (Zhou 2015; Shen et al. 2019; Zhu et al. 2019). Maximum parsimony (MP), bayesian inference (BI) and maximum likelihood (ML) methods were employed to perform phylogenetic analysis of the two aligned datasets. MP analysis were performed using PAUP* 4.0b10 (Swofford 2002); BI was calculated with MrBayes3.1.2 (Ronquist and Huelsenbeck 2003); RAxML v.7.2.6 (Stamatakis 2006) was used for ML analysis. The three phylogenetic methods resulted in similar topologies for each dataset, and, thus, only the topology from the MP tree is presented along with statistical values from the ML/BI/MP algorithms (simultaneous MP/BI not less than 75 % and BPP not less than 0.9) at the nodes.

Results

The nLSU datasets included 23 representatives genera of and the combined ITS+nLSU+RPB2 datasets included 41 fungal specimens representing 20 species. In addition to sequences of new species, 14 new sequences of three species without published DNA sequences were uploaded – (Murrill) Zmitr., Malysheva & Spirin, (T. Hatt.) Y.C. Dai and Y.C. Dai.

The nLSU dataset had an aligned length of 1386 characters, of which 996 were constant, 96 were variable but parsimony-uninformative, and 294 were parsimony-informative. Maximum Parsimony (MP) analysis yielded four equally most parsimonious trees (TL = 1639, CI = 0.350, RI = 0.733, RC = 0.256, HI = 0.650). Bayesian (BI) resulted in a similar consensus tree to that of the Maximum Parsimony (MP) and Maximum Likelihood (ML) analysis, with 1 million generations and an average standard deviation of split frequencies = 0.009570.

The three-gene dataset had an aligned length of 2950 characters, of which 1990 were constant, 90 were variable but parsimony-uninformative, and 870 were parsimony-informative. Maximum Parsimony (MP) analysis yielded 4 most parsimonious trees with near-identical topologies (TL = 2631, CI = 0.569, RI = 0.807, RC = 0.459, HI = 0.431). Bayesian (BI) resulted in a similar consensus tree to that of the Maximum Parsimony (MP) and Maximum Likelihood (ML) analysis, with 1 million generations and an average standard deviation of split frequencies = 0.005640.

Eighteen species of formed a well-supported clade (94/1/96 in Fig. 1) within the . is a sister genus to . Two samples from southern China are clustered into a new highly supported lineage (100/1/100 in Fig. 2) and in a clade with (Pers.) G. Cunn., , Q. Chen & Y. Yuan, and with high support (99/1/98 in Fig. 1; 100/1/100 in Fig. 2); it is described as sp. nov. Another three specimens formed a distinct lineage with strong support (100/1/100 in Fig. 2) in . This clade is interpreted as a new species, sp. nov.

Taxonomy

Y.C. Dai & Q. Chen sp. nov.

4298BC90-BB1A-58F6-84B0-C050C6D59056

824732

Figs 3A , 4

Figure 3.

Basidiocarps of . A (Dai 15137) B (Dai 16155). Scale bars: 10 mm.

Figure 4.

Microscopic structures of (Holotype, Dai 16892) A basidiospores B basidia and basidioles C cystidioles D hymenial setae E generative hyphae at dissepiment edge F hyphae from trama G hyphae from subiculum.

Type.

China. Yunnan Province: Kunming, Wild Duck Park, 2 August 2016, on fallen angiosperm branch, Dai 16892 (holotypes: BJFC 022998).

Etymology.

“Acutimarginata” (Latin): referring to the species with a sharp margin of fruiting body.

Description.

Basidiocarps annual, effused-reflexed to pileate, broadly attached, without taste or odor and soft corky when fresh. Pilei conchate, laterally fused, convex towards margin, projecting up to 1.5 cm, 7 cm wide and 6 mm thick at base. Pileal surface yellowish brown to dark brown, velutinate, concentrically sulcate with zoned; margin acute, yellowish brown. Pore surface yellowish brown when dry, glancing; margin distinct, yellowish, up to 2 mm wide; pores circular to angular, 5–7 per mm; dissepiments thin, entire. Context yellowish brown to dull brown, corky, up to 3 mm thick. Tubes yellowish brown, paler than context, corky, up to 3 mm long.

Hyphal structure.

Hyphal system dimitic; generative hyphae simple septate; tissue darkening but otherwise unchanged in KOH.

Subiculum.

Generative hyphae rare, hyaline to pale yellowish, thin- to slightly thick-walled, occasionally branched, 2–3.5 μm in diam; skeletal hyphae dominant, yellowish brown, thick-walled with a wide lumen, unbranched, aseptate, interwoven, 2–4.3 μm in diam.

Tubes.

Generative hyphae hyaline, thin-walled, occasionally branched, 2–3 μm in diam, occasionally encrusted at dissepiment edges; skeletal hyphae dominant, yellowish brown, thick-walled with a wide lumen, unbranched, aseptate, straight, subparallel along the tubes, 2–4 μm in diam. Irregular crystals occasionally present among trama and hymenia.

Hymenium.

Hymenial setae rare, mostly originating from tramal hyphae, subulate, dark brown, thick-walled, 20–40 × 3–7 μm; cystidioles frequent, fusoid, sometimes covered with crystals, hyaline, thin-walled, 16.5–26 × 4–6.5 μm; basidia broadly clavate, with four sterigmata and a simple septum at the base, 14–17 × 4.8–6.5 μm; basidioles similar in shape to basidia, but slightly smaller. Basidiospores cylindrical, hyaline, thin-walled, smooth, IKI–, CB–, (7–)7.5–9(–9.8) × (2.2–)2.5–3.2 μm, L = 8.12 μm, W = 2.87μm, Q = 2.73–2.95 (n = 60/2).

Additional specimens examined (paratypes).

China. Hunan Province: Yizhang County, Mangshan Nature Reserve, Guizizhai, 16 Aug 2014, on fallen angiosperm trunk, Dai 15115 (BJFC 018227), Dai 15137 (BJFC 018253).

Y.C. Dai & Q. Chen sp. nov.

A45B5EAD-2319-5EAD-A1B3-1EA8E6AA1096

824734

Figs 3B , 5

Figure 5.

Microscopic structures of (Holotype, Dai 15723) A basidiospores B basidia and basidioles C cystidioles D hymenial setae E generative hyphae at dissepiment edge F hyphae from trama G hyphae from subiculum.

China. Yunnan Province: Binchuan County, Jizushan Park, 30 August 2015, on fallen angiosperm branch, Dai 15723 (holotypes: BJFC 019827).

“” (Latin) referring to the species growing on branches.

Basidiocarps annual, resupinate, effused, inseparable, without taste or odor and corky when fresh, light-weight and hard corky when dry, up to 10 cm long, 2.2 cm wide and 1 mm thick at center. Pore surface grayish brown, fawn, cracked with age; sterile margin yellowish brown to olivaceous buff, distinctly paler than tubes, up to 1 mm wide; pores more or less angular, 6–7 per mm; dissepiments thin, sometimes irregular to slightly lacerate; abundant setae seen in tube cavities (under lens). Subiculum reddish brown, corky, very thin, about 0.1 mm thick. Tubes olivaceous buff, paler contrasting with pores and subiculum, hard corky, up to 0.9 mm long.

Hyphal system dimitic; generative hyphae simple septate; tissue darkening but otherwise unchanged in KOH.

Generative hyphae rare, hyaline, thin-walled, occasionally branched and simple septate, 2.5–3 μm in diam; skeletal hyphae dominant, rust-brown, thick-walled with a wide lumen, unbranched, aseptate, flexuous, loosely interwoven, 3–3.8 μm in diam.

Generative hyphae rare, mostly present at dissepiment edges and subhymenium, hyaline, thin-walled, occasionally branched and frequently simple septate, 1.8–2.8 μm in diam, some of them at dissepiment edges and in the hymenium encrusted with small crystals; skeletal hyphae dominant, yellowish brown, thick-walled with a wide lumen, unbranched, aseptate, flexuous to more or less straight, subparallel along the tubes, 2.5–3.8 μm in diam. Irregular crystals usually present among trama and hymenia.

Hymenial setae frequent, mostly originating from hymenium, subulate, dark brown, thick-walled, 35–60 × 4.5–7 μm; cystidioles fusoid, sometimes covered with crystals, hyaline and thin-walled, 15–22 × 3–5 μm; basidia barrel-shaped, with four sterigmata and a simple septum at the base, 9–11 × 4.5–5.5 μm; basidioles frequently in hymenium, similar in shape to the basidia, but slightly smaller. Basidiospores cylindrical, hyaline, thin-walled, smooth, usually glued in tetrads, IKI–, CB–, with some small guttules, (5.2–)5.8–7(–7.2) × (1.8–)2–2.5(–2.8) μm, L = 6.37 μm, W = 2.28 μm, Q = 2.57–2.88 (n = 60/2).

Additional specimen examined (paratype).

China. Hainan Province: Wuzhishan County, Wuzhishan Nature Reserve, 14 Nov 2015, on fallen angiosperm branch, Dai 16155 (BJFC 020252).

Discussion

In the study, sixteen previously accepted species of were referred to morphological examination and phylogenetic analyses. Two species of from China, and , are described as new on the basis of molecular data and morphology. is characterized by annual, effused-reflexed to pileate basidiocarps, small pores (5–7 per mm), a dimitic hyphal structure, hymenial setae rarely present, originating from tramal hyphae, the presence of cystidioles, and cylindrical basidiospores measuring 7.5–9 × 2.5–3.2 μm. Phylogenetically, samples of formed a well-supported monophyletic lineage distinct from other species (Fig. 2). is very similar to in having annual, effused-reflexed basidiocarps and the presence of cystidioles, but the latter has bigger pores (3–4 per mm) and smaller basidiospores (5.8–7 × 2–2.5 μm, Dai 2010). has a similar macromorphology to (Schwein.) T. Wagner & M. Fisch., but the latter has frequently septate skeletal hyphae and ellipsoid to ovoid basidiospores (4–5 × 3–3.5 μm, Ryvarden 2004).

is distributed in southern China and characterized by annual and resupinate basidiocarps, small pores (6–7 per mm), a dimitic hyphal system, subulate hymenial setae, the presence of cystidioles, and cylindrical basidiospores measuring 5.8–7 × 2–2.5 μm. , , and have overlapping distribution in China and clustered together with in a clade with strong support (99/1/98 in Fig. 1, 100/1/100 in Fig. 2). They are distinguishable according to their morphology and molecular data: is distinguished from by its perennial basidiocarps and widely distributed in northern China, Europe and North America (Ryvarden and Gilbertson 1994, Lowe 1966, Chen et al. 2019); has smaller pores (7–10 per mm) and shorter basidiospores (4.2–6.2 × 2–2.6 μm, Chen and Yuan 2017); has larger pores (2–4 per mm) and wider basidiospores (6–8.3 × 2.4–3 μm, Dai 2010); has shorter hymenial setae (18–25 μm vs. 35–60 μm), subcylindrical basidiospores with a pointed apex (4–6 × 1.5–2 μm, Ryvarden and Johansen 1980) and is reported from America, Brazil and USA (Ryvarden 2004; Groposo et al. 2007). is similar to and in having resupinate basidiocarps; however, and have mycelial, extra-hymenial setae (Chen et al. 2019); in addition, the three species are distantly related in our phylogenies (Figs 1, 2). Y.C. Dai & Niemelä and (Lév.) Baltazar & Gibertoni are also similar to in sharing resupinate basidiocarps, abundant subulate hymenial setae, absence of mycelial setae and distributed in southern China. differs from in having ovoid basidiospores 6.5–8.2 × 3.2–4.2 μm (Niemelä et al. 2001) whereas is different from by its smaller pores (9–10 per mm) and shorter basidiospores (3–4 × 2–2.5 μm, Dai 2010).

1	Basidiocarps usually laterally stipitate; hymenial setae absent	F. discipes
–	Basidiocarps sessile; hymenial setae present	2
2	Basidiocarps completely resupinate	3
–	Basidiocarps effuse-reflexed to pileate	11
3	Mycelial setae present in the decayed wood and margin of basidiocarps (by hand lens)	4
–	Mycelial setae absent from the decayed wood and margin of basidiocarps (by hand lens)	5
4	Pores 7–8 per mm	F. ferruginosa
–	Pores 2–3 per mm	F. contigua
5	Basidiocarps perennial	6
–	Basidiocarps annual	9
6	Pores 5–7 per mm	7
–	Pores 7–10 per mm	8
7	Basidiospores cylindrical, 6–7.8 × 2–2.5 μm	F. ferrea
–	Basidiospores subcylindrical, 4–6 × 1.5–2 μm	F. punctatiformis
8	Pores 9–10 per mm; basidiospores ellipsoid, < 5 μm long	F. chrysea
–	Pores 7–8 per mm; basidiospores narrowly ovoid, > 5 μm long	F. montana
9	Pores 2–4 per mm	F. yunnanensis
–	Pores 6–10 per mm	10
10	Pores 7–10 per mm; basidiospores 4.2–6.2 µm long	F. subferrea
–	Pores 6–7 per mm; basidiospores 6–7 µm long	F. ramulicola
11	Hymenial setae usually hooked	F. wahlbergii
–	Hymenial setae straight	12
12	Pores 3–4 per mm	F. setifera
–	Pores 5–11 per mm	13
13	Basidiocarps annual, margin acute	14
–	Basidiocarps perennial, margin obtuse	15
14	Skeletal hyphae septate, spores ellipsoid, 3.3–4.2 × 2.2–3 μm	F. gilva
–	Skeletal hyphae aseptate, spores cylindrical, 7.5–9 × 2.5–3.2 μm	F. acutimarginata
15	Basidiocarps subungulate; contextual skeletal hyphae aseptate	F. torulosa
–	Basidiocarps usually applanate; contextual skeletal hyphae septate	16
16	Basidiospores 3.3–4.1 × 2.1–2.4 μm, skeletal hyphae unchanged in KOH	F. rhabarbarina
–	Basidiospores 4–4.8 × 3.6–3.9 μm, skeletal hyphae swelling in KOH	F. senex