Taxonomy and Phylogeny of Cystostereaceae (Agaricales, Basidiomycota): A New Genus, Five New Species, and Three New Combinations

This paper aims to understand the species diversity, taxonomy, and phylogeny of Cystostereaceae (Agaricales), which is based primarily on material from East and Southeast Asia. Cystostereaceae is a small, understudied family of saprobes of woody plants with a worldwide distribution. Phylogenetic analyses of the LSU and ITS sequences revealed four distinct clades in the Cystostereaceae, representing the genera Crustomyces, Cystostereum, Effusomyces gen. nov., and Parvodontia. In addition, phylogenetic analyses showed that Cystidiodontia and Rigidotubus are synonyms of Crustomyces for their type of species nested within the Crustomyces clade. The new monotypic genus Effusomyces, based on specimens from Thailand, lacks any distinctive morphological features. Parvodontia, originally erected for a species from South America, is reported for the first time from Asia. The widely distributed and morphologically well-characterized Cystostereum is represented in East Asia by two new species: Cystostereum crassisporum and C. submurrayi. In addition, three new species, viz., Crustomyces albidus, Effusomyces thailandicus, and Parvodontia austrosinensis, are described and illustrated. Finally, three new combinations are proposed: Crustomyces isabellinus, C. laminiferus, and C. tephroleucus. A key to the genera and species of Cystostereaceae is provided. Our results proved that the species diversity of wood-decaying fungi in East and Southeast Asia is rich and suggested that more investigations and studies should be carried out in the future.


Introduction
Cystostereaceae Jülich is a small family in the Agaricales of wood-decaying fungi composed of seven genera [1,2]. Established in 1982, it included two genera -Cystostereum Pouzar and Crustomyces Jülich [1,3,4] Basidiomes in the Cystostereaceae are effused to effused-reflexed with smooth, odontoid, hydnoid, or poroid hymenophores with a monomitic or dimitic hyphal system, clamped generative hyphae, gloeocystidia or hyphidia, and smooth basidiospores that do Thin, freehand sections were made from dried basidiomes and mounted in 2% (weight/volume) aqueous potassium hydroxide (KOH) and 1% (w/v) aqueous phloxine. Amyloidity and dextrinoidity of hyphae and basidiospores were checked in Melzer's reagent (IKI). Cyanophily of hyphal and basidiospore walls were observed in 1% (w/v) cotton blue in 60% (w/v) lactic acid (CB). Microscopic examinations were carried out with a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) at magnifications up to 1000×. Drawings were made with the aid of a drawing tube. The following abbreviations are used: IKI-= neither amyloid nor dextrinoid, CB+ = cyanophilous, CB-= acyanophilous, L = mean spore length, W = mean spore width, Q = L/W ratio, and n (a/b) = number of spores (a) measured from the number of specimens (b). Color codes and names follow Kornerup and Wanscher [7].

DNA Extraction and Sequencing
A CTAB plant genomic DNA extraction kit, DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China), was used to extract total genomic DNA from dried specimens, amplified by the polymerase chain reaction (PCR), according to the manufacturer's instructions. The ITS1-5.8S-ITS2 region was amplified with the primer pair ITS5/ITS4 [8] using the following protocol: initial denaturation at 95 • C for 4 min, followed by 34 cycles at 94 • C for 40 s, 58 • C for 45 s and 72 • C for 1 min, and final extension at 72 • C for 10 min. The D1-D2 region of the nucleic ribosomal LSU was amplified with the primer pair LR0R/LR7 [http://www. biology.duke.edu/fungi/mycolab/primers.htm (accessed on 1 August 2022)] employing the following procedure: initial denaturation at 94 • C for 1 min, followed by 34 cycles at 94 • C for 30 s, 50 • C for 1 min and 72 • C for 1.5 min, and final extension at 72 • C for 10 min. DNA sequencing was performed at the Beijing Genomics Institute, and newly gener-ated sequences were deposited in GenBank [https://www.ncbi.nlm.nih.gov/(accessed on 1 August 2022)]. BioEdit v. 7.0. 5.3 [9] and Geneious Basic v. 11. 1.15 [10] were used to review the chromatograms and for contig assembly. Table 1 lists the taxa and sequences used in the phylogenetic analyses. Two datasets, the LSU sequences of representative taxa of Agaricales and the concatenated ITS-LSU sequences of species of the Cystostereaceae, were constructed. Coniophora olivacea (Fr.) P. Karst. and C. puteana (Schumach.) P. Karst. were selected as the outgroup for the LSU dataset, whereas Chondrostereum purpureum (Pers.) Pouzar was used in the ITS-LSU dataset [2,5]. For the latter dataset, sequences of ITS and LSU were aligned separately using MAFFT v.7 [http://mafft.cbrc.jp/alignment/server/(accessed on 1 August 2022)] [11] with the G-INS-I iterative refinement algorithm and optimized manually in BioEdit v. 7.0. 5. 3. The separate alignments were then concatenated using Mesquite v. 3. 5.1 [12].

Phylogenetic Analyses
Maximum parsimony (MP), maximum likelihood (ML) analyses, and Bayesian inference (BI) were carried out by using PAUP* v. 4.0b10 [13], RAxML v. 8.2.10 [14] and MrBayes 3.2.6 [15], respectively. In MP analysis, trees were generated using 100 replicates of random stepwise addition of sequence and the tree-bisection reconnection (TBR) branch-swapping algorithm with all characters given equal weight. Branch supports for all parsimony analyses were estimated by performing 1000 bootstrap replicates with a heuristic search of 10 random-addition replicates for each bootstrap replicate. In ML analysis, statistical support values were obtained using rapid bootstrapping with 1000 replicates, with default settings for other parameters. For BI, the best-fit substitution model was estimated with jModeltest v. 2.17 [16]. Four Markov chains were run for two million for the Agaricales LSU and Cystostereaceae ITS-LSU datasets; until the split deviation frequency value was lower than 0.01. Trees were sampled every 100th generation. The first quarter of the trees, which represented the burn-in phase of the analyses, were discarded. The remaining trees were used to calculate posterior probabilities (BPP) in the majority rule consensus tree.

Phylogenetic Analyses
The LSU dataset consisted of 45 samples representing 31 taxa in the Agaricales, representing seven families with crust or effused basidiomes and the outgroup (Table 1). In this dataset, the aligned length was 1374 characters, of which 276 were parsimony informative. MP analysis yielded eight equally parsimonious trees (TL = 1119, CI = 0.512, RI = 0.757, RC = 0.388, HI = 0.488). The concatenated ITS-LSU dataset comprised 28 ITS and 26 LSU sequences from 35 samples representing 13 taxa in the Cystostereaceae and the outgroup ( Table 1). This dataset had an aligned length of 1989 characters, and 277 characters were parsimony informative. MP analysis yielded two equally parsimonious tree (TL = 790, CI = 0.730, RI = 0.839, RC = 0.613, HI = 0.270). jModelTest suggested GTR + I+G was the best-fit model of nucleotide evolution for both datasets. The average standard deviation of the split frequencies of BI was 0.003928 (for the LSU dataset) and 0.003985 (for the ITS-LSU) at the end of the run. ML and BI analyses resulted in almost identical tree topologies compared to the MP analysis for both datasets. The ML trees of Agaricales and Cystostereaceae are shown in Figures 1 and 2, respectively, with the parsimony bootstrap values (≥50%, first value), likelihood bootstrap values (≥50%, second value) and Bayesian posterior probabilities (≥0.95, third value) labeled along the branches. 26 LSU sequences from 35 samples representing 13 taxa in the Cystostereaceae and the outgroup ( Table 1). This dataset had an aligned length of 1989 characters, and 277 characters were parsimony informative. MP analysis yielded two equally parsimonious tree (TL = 790, CI = 0.730, RI = 0.839, RC = 0.613, HI = 0.270). jModelTest suggested GTR + I+G was the best-fit model of nucleotide evolution for both datasets. The average standard deviation of the split frequencies of BI was 0.003928 (for the LSU dataset) and 0.003985 (for the ITS-LSU) at the end of the run. ML and BI analyses resulted in almost identical tree topologies compared to the MP analysis for both datasets. The ML trees of Agaricales and Cystostereaceae are shown in Figures 1 and 2, respectively, with the parsimony bootstrap values (≥50%, first value), likelihood bootstrap values (≥50%, second value) and Bayesian posterior probabilities (≥0.95, third value) labeled along the branches.  In Figure 1, seven families were recognized in the Agaricales, confirming the results presented by Song et al. [5], with the Cystostereaceae clade receiving strong support (97/94/1). Four subclades (lineages) corresponding to the genera Crustomyces, Cystostereum, Effusomyces gen. nov., and Parvodontia were strongly supported within the Cystostereaceae in both phylogenetic trees (Figures 1 and 2). Significantly, the type species of Cystidiodontia (represented by Crustomyces laminifera) and Rigidotubus, R. tephroleucus, nested within the Crustomyces clade. In addition, five strongly supported lineages were recovered, representing the new species Crustomyces albidus, Cystostereum submurrayi, C. crassisporum, Effusomyces thailandicus, and Parvodontia austrosinensis ( Figure 2). In Figure 1, seven families were recognized in the Agaricales, confirming the results presented by Song et al. [5], with the Cystostereaceae clade receiving strong support (97/94/1). Four subclades (lineages) corresponding to the genera Crustomyces, Cystostereum, Effusomyces gen. nov., and Parvodontia were strongly supported within the Cystostereaceae in both phylogenetic trees (Figures 1 and 2). Significantly, the type species of Cystidiodontia (represented by Crustomyces laminifera) and Rigidotubus, R. tephroleucus, nested within the Crustomyces clade. In addition, five strongly supported lineages were recovered, representing the new species Crustomyces albidus, Cystostereum submurrayi, C. crassisporum, Effusomyces thailandicus, and Parvodontia austrosinensis ( Figure 2). Basidiomes annual or perennial, resupinate, widely effused, adnate, ceraceous to crustaceous, rarely membranous, lacking hyphal cords or rhizomorphs. Hymenophore; smooth, papillate to spinose or poroid, white, cream, gray to grayish brown. Hyphal system; mono-or dimitic; generative hyphae clamped and skeletal hyphae, branched or not, sometimes dextrinoid. Gloeocystidia; usually present, empty, or containing dark yellow  Basidiomes annual or perennial, resupinate, widely effused, adnate, ceraceous to crustaceous, rarely membranous, lacking hyphal cords or rhizomorphs. Hymenophore; smooth, papillate to spinose or poroid, white, cream, gray to grayish brown. Hyphal system; mono-or dimitic; generative hyphae clamped and skeletal hyphae, branched or not, sometimes dextrinoid. Gloeocystidia; usually present, empty, or containing dark yellow material. Dendrohyphidia; present or absent. Basidia clavate; up to 25 µm long, thin-walled, colorless, with four sterigmata and a basal clamp connection. Basidiospores; subglobose, ellipsoid, short-cylindrical, thin-or thick-walled, colorless, smooth, IKI-, CB-, or CB+ if thick-walled.
As revised, we accept seven species in Crustomyces, the two original species as well as C. indecorus Hjortstam, the new taxon C. albidus described below, and three new combinations proposed below-C. isabellinus, C. laminiferum, and C. tephroleucus. Crustomyces expallens (Bres.) Hjortstam is excluded from Crustomyces because its narrowly clavate basidia, 30-40 × 5-7 µm, are decidedly different from the typical, shorter clavate basidia found in other species in the genus.  In Figures 1 and 2, C. albidus is sister to C. subabruptus, a north temperate species from North America and Europe that is characterized by a gray to pale ochraceous basidiome with densely crowded aculei, a dimitic hyphal system with skeletal hyphae, often with dendrohyphidia, and shorter basidiospores, 3.5-4.5 µ m long [24].  Etymology refers to the color of the basidiomes. Fruiting body-Basidiomes annual, resupinate, widely effused, adnate, inseparable from substrate, coriaceous, first as small patches, later confluent up to 10 cm long, 3.5 cm wide, up to 80 µm thick in section. Hymenophore; smooth, white (3A1) to yellowish white (3A2), turning black in KOH, not cracked; margin thinning out, adnate, indistinct, fimbriate, concolorous with hymenophore surface. Context white.
Notes-Crustomyces albidus is easily identified because of the moniliform gloeocystidia. It is found in the Yunnan Province, southwestern China, where it is locally common. In Figures 1 and 2, C. albidus is sister to C. subabruptus, a north temperate species from North America and Europe that is characterized by a gray to pale ochraceous basidiome with densely crowded aculei, a dimitic hyphal system with skeletal hyphae, often with dendrohyphidia, and shorter basidiospores, 3.5-4.5 µm long [24].     Descriptions and illustrations-Hallenberg and Ryvarden [25], as Cystostereum artocreas; Ryvarden [26], as H. grandinioides; Niemelä et al. [27]. Habitat and distribution-on woody angiosperms from Asia and Africa. Notes-Kneiffia isabellina was described from Sri Lanka and later reported from Taiwan and Africa [27,28]. Our specimens from mainland China and Vietnam have grandinioid to odontoid hymenophores and strongly dextrinoid tissues. The closely related Crustomyces laminiferus, discussed below, has a South American and Caribbean distribution [27,29]. The phylogenetic trees in Figures 1 and 2  Description and illustration-Hjortstam and Ryvarden [29], as Cystidiodontia atrocreas. Habitat and distribution-on dead trunks and branches of angiosperms, reported from the Caribbean Islands and Central and South America.
Notes-Hjortstam [30] created Cystidiodontia for Hydnum artocreas, but later, he determined it was a synonym of Hydnum laminiferum [31]. Thereafter, Cystidiodontia laminifera was adopted and reported from South and Central America [31], Taiwan [28], and mainland China [32]. A sample from Nigeria (TF10, ITS and LSU GenBank accession number MH625705) formed a lineage with one from Costa Rica (KHL13057, ITS and LSU GenBank accession number EU118622), thus extending the distribution of C. laminiferus into Africa. The reports of Crustomyces laminiferus from Asia [28,32] need to be confirmed, for they may be C. isabellinus (see discussion above). Habitat and distribution-on dead trunks and stumps of woody angiosperms from subtropical China.
Notes-Song et al. [5] erected the monotypic genus Rigidotubus based on molecular evidence and the poroid hymenophore configuration of R. tephroleucus. Figures 1 and 2 show that Crustomyces (Rigidotubus) tephroleucus nested within the Crustomyces clade and is sister to the C. isabellinus and C. laminiferus lineage. Despite its unusual poroid hymenophore configuration, Rigidotubus is placed in synonymy under Crustomyces based on phylogenetic evidence (Figures 1 and 2).

Cystostereum crassisporum Yue Li, Nakasone & S.H. He, sp. nov. Figures 5 and 6
Notes-Cystostereum crassisporum is characterized by thin basidiomes with a smooth to slightly tuberculate hymenophore, a dimitic hyphal system, abundant gloeocystidia, and thick-walled, cyanophilous basidiospores. The species is easily mistaken to be monomitic since microbinding hyphae are difficult to find. Cystostereum submurrayi, described below, is also from China but has a distinctly tuberculate hymenophore and thinwalled basidiospores. Cystostereum kenyense Hjortstam has thick-walled basidiospores as in C. crassisporum but is characterized by a tuberculate to odontoid hymenophore, a monomitic hyphal system, and produces two kinds of cystidia [33]. Cystostereum sirmaurense R. Kaur, Avn. P. Singh & Dhingra from India is distinguished from C. crassisporum by its smooth hymenophore with minute, scattered tubercules, a dimitic hyphal system with skeletal hyphae, and smaller, thin-walled basidiospores [34]. In the phylogenetic trees, C. crassisporum formed a distinct lineage sister to the C. murrayi (Berk. & M.A. Curtis) Pouzar and C. submurrayi clade.  Etymology-refers to the morphological similarity and close phylogenetic relationship to C. murrayi. Etymology-refers to the thick-walled basidiospores.
Microscopic structures-Hyphal system dimitic; generative hyphae bearing clamp connections and microbinding hyphae in a substrate, scarce to locally abundant. Subiculum not observed. Subhymenium composing most of context, thickening, up to 235 µm thick, a densely agglutinated tissue of hyphae and cystidia with two distinct layers-next to substrate with collapsed gloeocystidia often containing dark yellow, resinous-like material and an upper layer with elongate gloeocystidia enclosed by a thin, agglutinated matrix of hyphae, often stratose; hyphae thin-walled, colorless, smooth, interwoven, moderately septate, 1.5-2.5 µm in diam; gloeocystidia embedded, vesicular to subfusiform, sometimes with a distinct, short stalk, usually with a constriction in the upper part, empty or filled with dark yellow, solid, resinous-like material, slightly thick-walled, colorless, smooth, 17-53 × 6-19 µm. Basidia clavate, sometimes with slight constriction, thin-walled, colorless, smooth, with four sterigmata and a basal clamp connection, 14-24 × 4-6 µm. Basidiospores ellipsoid to broadly ellipsoid, with a distinct apiculus, thick-walled, colorless, smooth, usually with oil-drops, often 2-4 in groups, IKI-, CB+, Habitat and distribution-on Ficus or other angiosperm trees from subtropical China. Notes-Cystostereum crassisporum is characterized by thin basidiomes with a smooth to slightly tuberculate hymenophore, a dimitic hyphal system, abundant gloeocystidia, and thick-walled, cyanophilous basidiospores. The species is easily mistaken to be monomitic since microbinding hyphae are difficult to find. Cystostereum submurrayi, described below, is also from China but has a distinctly tuberculate hymenophore and thinwalled basidiospores. Cystostereum kenyense Hjortstam has thick-walled basidiospores as in C. crassisporum but is characterized by a tuberculate to odontoid hymenophore, a monomitic hyphal system, and produces two kinds of cystidia [33]. Cystostereum sirmaurense R. Kaur, Avn. P. Singh & Dhingra from India is distinguished from C. crassisporum by its smooth hymenophore with minute, scattered tubercules, a dimitic hyphal system with skeletal hyphae, and smaller, thin-walled basidiospores [34]. In the phylogenetic trees,  Figures 7 and 8 ture is like that in C. crassisporum, also from China, that differs by developing slightly broader, cyanophilous, thick-walled basidiospores and a smoother hymenophore. Cystostereum murrayi has significantly thicker basidiomes (up to 1 mm thick) and is reported from North America and Europe. The report of C. murrayi in China by Dai [32] needs to be confirmed. In our phylogenetic analyses, C. submurrayi formed a distinct lineage sister to C. murrayi, which appears to have two distinct lineages in Europe (Figures 1 and 2). Cystostereum sirmaurense also has thin-walled basidiospores as in C. submurrayi but develops skeletal hyphae, smaller gloeocystidia (26-44 × 7-8.5 µ m), and a mostly smooth hymenophore [34]).  Basidiomes annual, resupinate, effused, adnate, membranaceous. Hymenial surface smooth, pale yellow to greyish yellow, unchanged in KOH. Hyphal system monomitic; generative hyphae clamped. Subiculum thin; hyphae slightly thick-walled, colorless, smooth. Subhymenium indistinct. Hyphidia rare. Cystidia or gloeocystidia absent. Basidia clavate to subcylindrical, thin-walled, with four sterigmata and a basal clamp connection. Basidiospores ellipsoid, thin-walled, colorless, smooth, IKI-, CB-. Associated with a white rot of dead hardwood branches and bamboo.
Notes-Effusomyces are characterized by thin, membranous basidiomes, a smooth hymenophore, a monomitic hyphal system with clamped generative hyphae, and a lack of Etymology-refers to the morphological similarity and close phylogenetic relationship to C. murrayi.
Notes-Cystostereum submurrayi is characterized by a tuberculate hymenophore, a dimitic hyphal system, embedded gloeocystidia, and thin-walled basidiospores. Its structure is like that in C. crassisporum, also from China, that differs by developing slightly broader, cyanophilous, thick-walled basidiospores and a smoother hymenophore. Cystostereum murrayi has significantly thicker basidiomes (up to 1 mm thick) and is reported from North America and Europe. The report of C. murrayi in China by Dai [32] needs to be confirmed. In our phylogenetic analyses, C. submurrayi formed a distinct lineage sister to C. murrayi, which appears to have two distinct lineages in Europe (Figures 1 and 2). Cystostereum sirmaurense also has thin-walled basidiospores as in C. submurrayi but develops skeletal hyphae, smaller gloeocystidia (26-44 × 7-8.5 µm), and a mostly smooth hymenophore [34] Notes-Effusomyces are characterized by thin, membranous basidiomes, a smooth hymenophore, a monomitic hyphal system with clamped generative hyphae, and a lack of cystidia or gloeocystidia. Although lacking distinctive morphological features, Effusomyces are nested within the Cystostereaceae and are sister to Crustomyces (Figures 1 and 2). Crustomyces (Rigidotubus) tephroleucus also is monomitic and lacks cystidia and gloeocystidia but has a tough, thick (up to 0.5 mm) basidiome and poroid hymenophore. Within the family, monomitic hyphal systems with clamped generative hyphae are also reported in Parvobasidium, Parvodontia, and some species of Crustomyes and Cystostereum. Still, these taxa also have cystidia, gloeocystidia, or thick-walled basidiospores.   Etymology-refers to Thailand, the type locality.

Discussion
Cystostereaceae is a small, monophyletic family in the Agaricales, with affinities to Lyophyllum and allied taxa [2,3]. Phylogenetic analyses of LSU and ITS sequences confirm that Cystostereum, Crustomyces, Cystidiodontia, Parvodontia, and Rigidotubus belong in the Cystostereaceae. In contrast, the placement of Cericium and Parvobasidium needs to be confirmed by molecular evidence. Our phylogenetic analyses support the monophyly of the family and clarify generic and species relationships within the family (Figures 1 and 2). We demonstrate that Crustomyces has a wide range of hymenophore configurations from smooth, odontoid, and spinose to poroid for the types species of Crustomyces, Cystidiodontia, and Rigidotubus all clustered together in a monophyletic clade with high support values. The newly emended Crustomyces includes seven species-C. subabruptus, the type species, C. indecorus Hjortstam, C. pini-canadensis, the new taxon C. albidus described herein, and three new combinations proposed above, C. isabellinus, C. laminiferum, and C. tephroleucus. Excluded from the genus is Crustomyces expallens (Bres.) Hjortstam because its large, narrowly clavate basidia are unlike the typical, shorter clavate basidia of other species in the genus. Crustomyces heteromorphum (Hallenb.) Hjortstam from eastern Europe is a synonym of C. subabruptus, as posited by Larsson and Ryvarden [24]. There are additional species of Crustomyces from China and New Zealand that appear as distinct lineages represented by Cui 12283 and HHB 19125, respectively, in Figure 2.
In addition to Crustomyces and Cystostereum, two lineages, Parvodontia and Effusomyces, were revealed in the phylogenetic analyses (Figures 1 and 2). Parvodontia is similar to other species in the Cystostereaceae that it produces vesicles and gloeocystidia. Effusomyces is an unlikely member because it has a very simple basidiome structure and lacks vesicles and gloeocystidia.
Recently, many new macrofungal taxa have been described from Southern China [42][43][44][45][46][47][48], and the area is very rich in wood-inhabiting fungi [49][50][51]. In the present paper, five new species belonging to Cystostereaceae are described, including four species from Southern China. It seems more unknown macrofungal species exist in the area, and further investigations are needed to demonstrate the fungal diversity in Southern China.