Global Phylogeny and Taxonomy of the Wood-Decaying Fungal Genus Phlebiopsis (Polyporales, Basidiomycota)

An in-depth study of the phylogeny and taxonomy of the corticioid genus Phlebiopsis (Phanerochaetaceae) was conducted. Phylogenetic analyses of the ITS1-5.8S-ITS2 and nrLSU sequences demonstrated that Phlebiopsis is a strongly supported clade which is distinct from its sister clades of Phaeophlebiopsis, Hapalopilus, and Rhizochaete. Two genera, Australohydnum and Hjortstamia, are reduced to synonyms under Phlebiopsis as generic type species A. griseofuscescens and H. friesii, respectively, are embedded in the Phlebiopsis clade. Twenty-four lineages are resolved in the ITS phylogenetic tree of Phlebiopsis, including six new taxa, viz. P. albescens, P. brunnea, P. cylindrospora, P. magnicystidiata, P. membranacea and P. sinensis, from Sri Lanka and China. Five new combinations, viz. Phaeophlebiopsis mussooriensis, Phlebiopsis bambusicola, P. dregeana, P. griseofuscescens and P. novae-granatae, are proposed. Phlebiopsis crassa is a morphological species complex with three distinct lineages. Phlebiopsis lamprocystidiata is determined to be a later synonym of P. darjeelingensis. The new taxa are described, illustrated, and compared and contrasted to morphologically similar species. An emended description of Phlebiopsis is provided along with an identification key to 27 accepted species.

The generic circumscription of Phlebiopsis was expanded when molecular studies showed that Phanerochaete crassa (Lév.) Burds. and Phlebiopsis gigantea (Fr.) Jülich were closely related (de Koker et al., 2003;Greslebin et al., 2004;Wu et al., 2010;Floudas and Hibbett, 2015). With the inclusion of P. crassa, Phlebiopsis now also includes species with effusedreflexed, coriaceous basidiomata, a dimitic hyphal system, and lamprocystidia or skeletocystidia with light brown walls. In addition, based on both phylogenetic and morphological evidence, Floudas and Hibbett (2015) created Phaeophlebiopsis Floudas & Hibbett to accommodate Phlebiopsis peniophoroides Gilb. & Adask. and similar species with ceraceous, beige-brown basidiomata and subicula, lamprocystidia with brown walls, and small basidiospores. The limits of the Phlebiopsis clade were extended by Miettinen et al. (2016) who transferred six species into Phlebiopsis. The results of their phylogenetic study showed that the type species of Castanoporus Ryvarden, Merulius castaneus Lloyd, was nested in a clade with P. gigantea and, therefore, a synonym of Phlebiopsis. Similarly, Dentocorticium pilatii (Parmasto) Duehm & Michel, Lopharia papyrina (Mont.) Boidin, Phanerochaete brunneocystidiata Sheng H. Wu, and Phanerochaete laxa Sheng H. Wu clustered in the Phlebiopsis clade, and were all transferred to the genus. Based on the morphological similarity of Thelephora friesii Lév., the type of Hjortstamia Boidin and Gilles, 2003 to L. papyrina and P. crassa, they also transferred T. friesii to Phlebiopsis, thereby reducing Hjortstamia to a synonym of Phlebiopsis. Phlebiopsis pilatii (Parmasto) Spirin & Miettinen is unique in the genus for it has a dimitic hyphal system of simple-septate generative and microbinding (squelettoligatives) hyphae and finely branched hyphidia but lacks lamprocystidia or skeletal cystidia (Larsen and Gilberston, 1977;Duhem and Michel, 2009).
Among the 24 names of Phlebiopsis recovered in Index Fungorum 1 (accessed on 21 January2020), four species were transferred to Phaeophlebiopsis. Of the remaining 20 species, 11 were described originally from Asia (Dhingra, 1987;Wu, 2000Wu, , 2004Priyanka et al., 2011;Kaur et al., 2015;Zhao et al., 2018;Xu et al., 2020). More than 150 specimens of Phlebiopsis were collected by the corresponding author from China and Southeast Asia in recent years. Based on these specimens and sequences obtained from GenBank, the phylogenetic analyses and taxonomic study of Phlebiopsis and related taxa in the Phanerochaetaceae were undertaken. This study is a contribution to the understanding of the diversity and phylogenetic relationships of crust fungi in China.

Specimen Collection
Field trips for specimen collection in many kinds of Nature Reserves and Forest Parks in China and other countries were carried out by the authors. In situ photos of the fungi were taken with a Canon camera EOS 70D (Canon Corporation, Japan). Fresh specimens were dried with a portable drier (manufactured in Finland). Dried specimens were labeled and then stored in a refrigerator of minus 40 • C for 2 weeks to kill the insects and their eggs before they were ready for morphological and molecular studies.

Morphological Studies
Voucher specimens are deposited at the herbaria of Beijing Forestry University, Beijing, China (BJFC), Centre for Forest Mycology Research, U.S. Forest Service, Madison, WI, United States (CFMR), National Museum of Natural Science, Taichung, Taiwan, China (TNM) and Beijing Museum of Natural History, Beijing, China (BJM). The Sri Lankan voucher specimens are deposited in the Faculty of Agriculture, University of Ruhuna, Kamburupitiya, Sri Lanka and the herbarium of Beijing Forestry University, Beijing, China (BJFC), and were studied under the material transfer agreement signed by the two universities. Freehand sections were made from dried basidiomata and mounted in 2% (w/v) potassium hydroxide (KOH), 1% (w/v) phloxine, Melzer's reagent (IKI) or cotton blue (CB). Microscopic examinations were carried out with a Nikon Eclipse 80i microscope (Nikon Corporation, 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-, acyanophilous; L, mean spore length; W, mean spore width; Q, L/W ratio; n (a/b), number of spores (a) measured from number of specimens (b). Color codes and names follow Kornerup and Wanscher (1978).

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 then 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 (White et al., 1990) 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 nrLSU D1-D2 region was amplified with the primer pair LR0R/LR7 2 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 Beijing Genomics Institute, and the sequences were deposited in GenBank 3 ( Table 1). BioEdit v.7.0.5.3 (Hall, 1999) and Geneious Basic v.11.1.15 (Kearse et al., 2012) were used to review the chromatograms and for contig assembly.
Maximum parsimony (MP), maximum likelihood (ML) analyses and Bayesian inference (BI) were carried out by using PAUP * v.4.0b10 (Swofford, 2002), RAxML v.8.2.10 (Stamatakis, 2014, and MrBayes 3.2.6 (Ronquist et al., 2012), respectively. In MP analysis, trees were generated using 100 replicates of random stepwise addition of sequence and treebisection 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 used for other parameters. For BI, the best-fit substitution model was estimated with jModeltest v.2.17 (Darriba et al., 2012). Four Markov chains were run for five million and three million generations for the Phanerochaetaceae ITS-LSU and Phlebiopsis ITS datasets, respectively, 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, and the remaining trees were used to calculate posterior probabilities (BPP) in the majority rule consensus tree.

Phylogenetic Analyses
Forty-three ITS and 37 nrLSU sequences were generated for this study. The concatenated ITS-LSU dataset contained 101 ITS and 107 nrLSU sequences from 107 samples representing 86 Phanerochaetaceae taxa and the outgroup, while the ITS dataset contained 71 samples representing 21 Phlebiopsis s.s. taxa, a sample of Irpex vellereus and the outgroup ( Table 1). The concatenated dataset had an aligned length of 2339 characters, of which 554 were parsimony-informative. MP analysis yielded one equally parsimonious tree (TL = 3603, CI = 0.360, RI = 0.695, RC = 0.250, HI = 0.640). The ITS dataset had an aligned length of 726 characters, of which 178 were parsimony-informative. MP analysis yielded 92 equally parsimonious trees (TL = 658, CI = 0.579, RI = 0.870, RC = 0.504, HI = 0.421). jModelTest suggested GTR + I + G and HKY + G were the best-fit models of nucleotide evolution for the concatenated ITS-LSU and ITS datasets, respectively. The average standard deviation of split frequencies of BI was 0.009223 and 0.007710 at the end of the run. ML and BI analyses resulted in almost identical    (2015) New species are set in bold with type specimens indicated with an asterisk (*).
tree topologies compared to the MP analysis. The MP trees are shown in Figures 1, 2 with the parsimony bootstrap values (≥50%, first), Bayesian posterior probabilities (≥0.95, second) and likelihood bootstrap values (≥50%, third) labeled along the branches.
Notes -This is a poorly understood species that has been interpreted differently by various researchers. We take a narrow concept of P. dreageana based on studies of the type specimen and specimens restricted to Africa as described and illustrated by Massee (1891), Talbot (1951), Reid (1975), and Hjortstam and Ryvarden (1990). The ellipsoid basidiospores based on these studies are approximately 6.5-8 × 4-5 µm in size. Note that the cylindrical basidiospores illustrated by Reid (1975) are questionable for Hjortstam (1989) noted that basidia and spores were not observed in the type. Hjortstam and Ryvarden (1990) took a broad interpretation of A. dreageanum when they placed Hydnum griseofuscescens Reichardt from Australia and Irpex vellereus Berk. & Broome from Sri Lanka in synonymy; see below for further discussion of these two taxa. Although A. dregeanum has since been reported from India (De, 1998, as Oxyporus vellereus), South Korea (Lim, 2001;Lim and Jung, 2003), New Zealand (Buchanan and Ryvarden, 2000), Portugal (Melo and Hjortstam, 2002), Israel (Ţura et al., 2011), and Italy (Saitta et al., 2014), the basidiospore size, when given, is significantly smaller than the African collections.
Sequences from authentic specimens of the species are not available at present, but ITS sequences labeled "Australohydnum dregeanum" in GenBank, from United States, Korea and Sri Lanka, formed a strongly supported lineage within Phlebiopsis (Figure 2). The identity of the taxa in this lineage needs further study. Notes -Hydnum griseofuscescens was described from Australia and is the type of Australohydnum (Jülich, 1978). It is characterized by resupinate to effused-reflexed basidiomata with a hydnoid, purplish brown hymenophore, a pseudodimitic hyphal system with simple-septate, colorless, generative hyphae, 4-9 µm broad, encrusted hymenial cystidia with colorless walls, and small ellipsoid basidiospores, 4-6 × 2.5-3 µm (Reid, 1956as Irpex vellerus, Jülich, 1978. We follow Reid (1956Reid ( , 1963 who determined that H. griseofuscescens and I. vellereus, described from Sri Lanka, were synonyms after studying the types of both species. Reid (1967) also reported that T. venustum sensu Cunningham (1965) is H. griseofuscescens. Based on morphological studies and sequence analyses, we determined that P. lacerata described from southern China (Xu et al., 2020) is conspecific with P. griseofuscescens. Gilbertson and Adaskaveg (1993) described and illustrated I. griseofuscescens from Hawaii, but this species lacks encrusted hymenial cystidia and has small basidiospores, 4-4.5 × 2-2.5 µm. Similarly, De's (1998) description of O. vellereus from India appears to represent a different species with a monomitic hyphal system of colorless to pale brown hyphae and cylindrical basidiospores, 5.2-7 × 2-3 µm. One of the specimens cited, VBMN 80451, is also at CBS, CBS 515.92, and its ITS sequence is available from GenBank (AF479670) as "Irpex vellereus." This sequence was included in Lim and Jung (2003) and Figure 2, herein, where it is on a long branch, sister to P. griseofuscescens. Notes -Reported from Colombia on bamboo, this species is characterized by a pale brown hymenophore and smooth skeletocystidia but lacking lamprocystidia (Welden, 1975;Hjortstam and Ryvarden, 1990). Because of its morphological similarity to P. crassa, the transfer of P. novae-granatae is proposed.  Figure 9E).

Phlebiopsis crassa Species Complex
Phlebiopsis Notes -Our phylogenetic analyses showed that samples of P. crassa group A from Vietnam, Sri Lanka and southern China formed a distinct lineage and represent P. crassa s.s., for the type was described from Vietnam (Figures 1, 2). Collections from southern China and Japan, group B, and the United States, group C, clustered into two lineages in the ITS tree (Figure 2). All three lineages of P. crassa are morphologically similar, however. Unraveling this species complex is beyond the scope of this study, involving a number of presumed synonyms of P. crassa; see Lentz (1955) and Burdsall (1985). Dhingra, Nova Hedwigia 44: 222, 1987 Synonyms: Phanerochaete lamprocystidiata Sheng. H. Wu, Mycotaxon 90: 426, 2004. Phlebiopsis lamprocystidiata (Sheng H. Wu) Sheng H. Wu & Hallenb., Fungal Diversity 42: 116, 2010. Notes -Because P. darjeelingensis, from India, and P. lamprocystidiata, from Taiwan, are nearly identical in morphology -basidiomata ceraceous when fresh then corneous when dried, well-developed subiculum of compactly packed, colorless hyphae, and cystidia and basidiospores of similar shape and size (Dhingra, 1987;Wu, 2004), we consider P. lamprocystidiata to be a later synonym of P. darjeelingensis. Zmitrovich (2018) transferred Phlebiopsis lamprocystidiata to Phaeophlebiopsis based on morphology, our phylogenetic analyses show that it belongs to Phlebiopsis s.s., however.

DISCUSSION
The generic limits of Phlebiopsis has expanded over the last 40 years since its introduction in 1978 to include significant morphological range in basidiomata habit and texture and hymenophore configuration with the aid of molecular phylogenetic methods (Floudas and Hibbett, 2015;Miettinen et al., 2016;Zhao et al., 2018;Xavier de Lima et al., 2020;Xu et al., 2020). In this study, we emphasized sampling of Phlebiopsis taxa, and our overall results confirm those of Floudas and Hibbett (2015), Miettinen et al. (2016), and Chen et al. (2018b). In Figures 1, 2, Phlebiopsis, including the types of Australohydnum, P. griseofuscescens and Hjortstamia, P. friesii, formed a wellsupported clade in the Phanerochaetaceae and is closely related to Phaeophlebiopsis, Hapalopilus and Rhizochaete. The genera Phlebiopsis and Australohydnum were published simultaneously (Jülich, 1978) but the former is favored to avoid unnecessary name changes. So, we propose that Australohydnum is a synonym of Phlebiopsis. Twenty-four lineages were resolved in the ITS tree of Phlebiopsis, among which 18 are accepted species, including the P. crassa species complex and six new species described herein. Further study is required to identify the taxa named P. cf. dregeana, Irpex vellerus, Phlebiopsis sp. FP-102937 and Phlebiopsis sp. ECS-1971. Among the 24 names of Phlebiopsis in Index Fungorum (accessed on 21 January 2021), we accept 17 taxa in Phlebiopsis s.s., including 11 that are supported by molecular data. Five taxa, P. himalayensis Dhingra, P. mussooriensis, P. peniophoroides Gilb. & Adask., P. ravenelii (Cooke) Hjortstam, and P. roumeguerei (Bres.) Jülich & Stalpers were transferred to Phaeophlebiopsis based on morphology and sequence data. Phlebiopsis lacerata and P. lamprocystidiata are synonyms of P. griseofuscescens and P. darjeelingensis, respectively, as discussed above. Thus, 27 species of Phlebiopsis worldwide are accepted, including the six new species and four new combinations reported herein. An emended description of Phlebiopsis and an identification key to all species in the genus worldwide are presented below.
Phlebiopsis ( Description: Basidiomata annual, resupinate, effused, effusedreflexed or pileate, ceraceous, membranaceous to coriaceous. Pilei, when present, tomentose, gray to brown. Hymenophore smooth, tuberculate, odontoid, hydnoid to poroid, white, gray, grayish brown, purplish brown or brown, turning purple in KOH in two species. Hyphal system monomitic or dimitic; generative hyphae simple-septate, colorless or rarely pale brown, in dimitic species with skeletal or, in one species, micro-binding hyphae. Subiculum absent to well-developed, colorless, brown, agglutinated or not, compact to loosely interwoven. Skeletocystidia absent or present, colorless or brown, distinctly thick-walled, smooth or encrusted. Hymenial cystidia or lamprocystidia typically present, colorless or light brown, thick-walled, usually encrusted. Dendrohyphidia present in one species, colorless, thin-walled, smooth, branched. Basidia clavate or subcylindrical, with four sterigmata and a basal simple septum. Basidiospores cylindrical, ellipsoid, broadly ellipsoid or subglobose, colorless, thin-walled, smooth, negative in Melzer's reagent, acyanophilous. Type species: Phlebiopsis gigantea (Fr.) Jülich Notes -The terminology relating to the cystidia observed in Phlebiopsis species is varied in the literature and thus confusing. There are up to three kinds of cystidia, but intermediate forms can develop to blur their distinctiveness. Lamprocystidia are found in most species of Phlebiopsis in the hymenium, often projecting, and may become embedded as the basidiomata thickens. They are typically conical or subfusiform with thick walls that are lightly to heavily encrusted in the upper half or apex. Skeletocystidia are found in dimitic or pseudodimitic species in which thick-walled hyphae in the subiculum curve toward the hymenium but remain embedded in the subiculum or subhymenium. The terminal ends may or may not be differentiated and usually lack encrustations. Hymenial cystidia are those structures that are similar to skeletocystidia but terminate in the hymenium and may be encrusted. In other cases, they are formed in the subhymenium and are smaller than lamprocystidia and not conical or heavily encrusted. Lamprocystidia

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

AUTHOR CONTRIBUTIONS
S-HH designed the research, collected most of the specimens, and wrote the text. Y-NZ performed the phylogenetic analyses and did most of the measurement, descriptions and illustrations. KN loaned and examined type specimens of some related species, and revised language of the text. C-CC provided with some specimens and sequences. S-LL helped in field trips and species illustrations. KLWK and H-XM helped in field trips and collected some specimens. M-RH collected some specimens and helped in specimen preservation. All authors contributed to the article and approved the submitted version.

FUNDING
Financial support was provided by the National Natural Science Foundation of China (Grant Nos. 31870011 and 31750001).