Global Taxonomy and Phylogeny of Irpicaceae (Polyporales, Basidiomycota) With Descriptions of Seven New Species and Proposals of Two New Combinations

The phylogenetic analyses of the family Irpicaceae were carried out based on a complete global sampling. The dataset that included concatenated ITS1-5.8S-ITS2 and nrLSU sequences of 67 taxa of Irpicaceae from around the world was subjected to the maximum likelihood analyses and Bayesian inference. In the phylogenetic tree, species from 14 genera were distributed in nine clades, among which five genera—Irpex, Phanerochaetella, Byssomerulius, Cytidiella, and Meruliopsis, received high support values. The genus Efibula was shown to be paraphyletic and four subclades could be recognized, while Phanerochaete allantospora, Leptoporus mollis, and several species from Ceriporia and Candelabrochaete formed a large clade with relatively strong support. Based on the molecular and morphological evidence, seven new corticioid species—Candelabrochaete guangdongensis, Efibula grandinosa, E. hainanensis, E. shenghuae, E. taiwanensis, Irpex alboflavescens, and Phanerochaetella sinensis, were revealed from the materials mostly from East Asia. The monotypic genus Flavodontia, newly described from southwestern China, is regarded as a later synonym of Irpex, and the new combination I. rosea is proposed. In addition, Phanerochaetella queletii is proposed for a taxon first described from Italy and newly recorded from China; Phanerochaete jose-ferreirae from Portugal is determined to be a later synonym. Descriptions and illustrations of the new species and the newly combined taxa are presented, and morphological comparisons for the known species of Efibula and Phanerochaetella are provided.


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 photographs 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 at minus 40 • C for two 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, Wisconsin, USA (CFMR), and National Museum of Natural Science, Taichung, Taiwan, China (TNM). Herbarium code designations follow Index Herbarium 1 Thin, freehand sections were made from dried basidiomata and mounted in 2% (w/v) aqueous potassium hydroxide (KOH) and 1% (w/v) aqueous phloxine. Amyloidity and dextrinoidity of basidiospores were checked in Melzer's reagent (IKI). Cyanophily of hyphal and basidiospore walls was observed in 1% (weight/volume) cotton blue in 60% (w/v) lactic acid (CB). Microscopic examinations were carried out with a Nikon Eclipse 80i microscope (Nikon Corporation, Japan) at magnifications up to 1,000 ×. 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 and 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.

Phylogenetic Analyses
The molecular phylogeny was inferred from a concatenated dataset of ITS-nrLSU sequences of species in the Irpicaceae. Gloeoporus dichrous (Fr.) Bres. and G. pannocinctus (Romell) J. Erikss. were selected as the outgroup (Floudas and Hibbett, 2015;Chen et al., 2021). The ITS and nrLSU sequences were aligned separately using MAFFT v.7 4 (Katoh et al., 2017) 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 (Maddison and Maddison, 2018). The datasets were deposited in TreeBase 5 (submission ID: 29610). Maximum likelihood (ML) analyses and Bayesian inference (BI) were carried out by using RAxML v.8.2.10 (Stamatakis, 2014) and MrBayes 3.2.6 (Ronquist et al., 2012), respectively. 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 8,000,000 generations 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
The concatenated ITS-nrLSU dataset contained 120 ITS and 101 nrLSU sequences from 126 samples representing 69 taxa of Irpicaceae ( Table 1). The concatenated dataset had an aligned length of 2220 characters. jModelTest suggested that GTR+I+G was the best-fit model of nucleotide evolution for the concatenated ITS-nrLSU. The average standard deviation of split frequencies of BI was 0.007321 at the end of the run. ML analyses resulted in almost identical tree topology compared to the BI analysis. Only the BI tree is provided in Figure 1 with the likelihood bootstrap values (≥50%, before the slash) and Bayesian posterior probabilities (≥0.95, behind the slash) labeled along the branches.
The topology of the tree is similar to those in previous studies (Justo et al., 2017;Chen et al., 2021). For the ingroups, species from 14 genera were distributed in nine clades: Etymology-Refers to the type locality in Guangdong Province, southern China.
Notes-Efibula hainanensis Figure 4 is characterized by having a smooth hymenophore, thin-walled cystidia, and relatively small ellipsoid basidispores. Until now, E. hainanensis is the only species in the genus with cystidia. In the phylogenetic tree (Figure 1), E. hainanensis and E. intertexta (Sheng H. Wu) C.C. Chen and Sheng H. Wu formed a relatively strongly supported lineage. Morphologically, E. intertexta differs from E. hainanensis by having distinctly thickening hymenial layer and cylindrical basidiospores (5.6-6.4 × 2.2-2.6 µm) and lacking cystidia . Efibula tuberculata (P. Karst.) Zmitr. and Spirin is similar to E. hainanensis by sharing relatively small basidiospores but differs in having a smooth to tuberculate, cracked hymenophore, occasional clamps, and slightly larger basidiospores . Etymology-Named to honor Dr. Sheng-Hua Wu (TNM, Taiwan) who established and contributed much to the genus of Efibula.
Notes-Efibula shenghuae Figure 5 is characterized by its grandinioid hymenophore, indistinct subiculum, and masses of crystals in subhymenium. In the phylogenetic tree (Figure 1), E. shenghuae and E. grandinosa formed a well-supported lineage sister to E. clarkii. However, E. grandinosa can be easily distinguished from E. shenghuae by having hyphal pegs, while E. clarkii differs from E. shenghuae by having extensively cracked basidiomata and less crystals (Floudas and Hibbett, 2015). Efibula tuberculata is similar to E. shenghuae by sharing smooth to tuberculate hymenophore but differs by having cracked basidiomata and less crystals in section . Moreover, the two species formed distinct lineages in the tree.
Notes-Wang and Zhao (2022) built a new monotypic genus Flavodontia Figures 9, 10 C.L. Zhao for the species F. rosea collected from Yunnan Province, southwestern China, mainly based on molecular evidence; however, our phylogenetic analyses using an expanded dataset of Irpicaceae demonstrated that the species was nested within the Irpex clade, which has been shown to include taxa from Emmia, Flavodon, and Hydnopolyporus . Morphologically, I. rosea has effused-reflexed coriaceous basidiomata with smooth, odontioid, or irpicoid hymenophores, simple-septate generative hyphae, broadly ellipsoid basidiospores and lacks cystidia, which fits well with the characters of Irpex. Thus, we propose the new combination and treat Flavodontia as a later synonym of Irpex.
Etymology-Refers to the type locality in China.
Type specimens examined-Italy, Vallombrosa, ad ramos corticates Abietis pectinate, Nov 1899, Martielli (BPI 0282568, branches of woody angiosperms, rarely on gymnosperms. This widely distributed species was first described from Italy but is known throughout Europe and is frequently collected in the upper Midwest in the USA. This is the first record this species in China. There is variability in the hymenophore ranging from smooth to distinctly tuberculate, sparsely to highly rimose, and nearly white and cream to brownish orange. Similarly, the margin may be narrowly adnate, white, and fimbriate to abrupt, slightly detached and incurved. Variability in basidiospore length and width was also observed. The description above is based on the Chinese specimens only that appear to have slightly narrower basidiospores than reported earlier. Despite the basidiospore size difference between the type specimens of C. queletii and C. jose-ferrieriae, their overwhelming similarities in basidiomata texture and color, hymenophore configuration, and other microscopic features indicate that they are conspecific. For additional descriptions and illustrations, see Eriksson et al. (1978), Nakasone (2008), and Bernicchia and Gorjón (2010).

DISCUSSION
The species diversity, taxonomy, and phylogeny of the phlebioid clade in Polyporales were intensively studied recently by many authors, and a large number of new taxa from East Asia were described (Floudas and Hibbett, 2015;Miettinen et al., 2016;Justo et al., 2017;Ma and Zhao, 2019;Chen et al., 2020Chen et al., , 2021Xu et al., 2020;Wang and Zhao, 2021;Zhao et al., 2021;Tian et al., 2022). This study furthers our knowledge of this group with the addition of seven new corticioid species in the Irpicaceae.
There is no doubt that more new taxa will be revealed as more surveys are carried out in areas of Asia with the aid of molecular evidence. Our phylogenetic analysis results supported to place the newly described monotypic genus, Flavodontia, in synonymy under Irpex. We also demonstrated that one species, namely, P. queletti, has a wide distribution throughout the north temperate region from China to Europe and North America. Broadly ellipsoid 6-9 × 3.5-4.5 USA Burdsall, 1985 The results of our phylogenetic analyses of Irpicaceae are consistent with that presented by Chen et al. (2021). In both studies, clades of the five genera-Irpex, Phanerochaetella, Byssomerulius, Cytidiella, and Meruliopsis, received strong support values, whereas Efibula was shown to be paraphyletic with species distributed in four subclades. According to the phylogenetic results, Irpex now includes species with smooth, poroid, labyrinthine, irpicoid, hydnoid to irregular hymenophore configurations. However, there are still many old names in Irpex that need to be studied by using modern taxonomic methods and systems. For Efibula, there are no distinct morphological characters to divide it into small genera at present ( Table 2). The newly erected genus, Phanerochaetella, contains several species with diverse micromorphology: lamprocystidia present or absent and basidiospores from broadly ellipsoid to cylindrical ( Table 3). Three species of Candelabrochaete formed two distinct lineages in the Ceriporia/Candelabrochaete s.l./ Leptoporus/Phanerochaete allantospora clade, but their generic position remains unresolved since the type species, C. africana Boidin, was not nested within the phlebioid clade (Justo et al., 2017;Chen et al., 2021).
The molecular evidence has brought significant changes and increased our understanding in the taxonomy of Irpicaceae. The morphological circumscriptions of some genera became broader, for example, Irpex now contains species with poroid, labyrinthine, irpicoid, hydnoid to irregular hymenophore, and Efibula is shown to contain species with or without horizontally arranged subicular hyphae. Species with simpleseptate hyphae and without cystidia can be found in Efibula, Irpex, and Phanerochaetella. To determine important and useful morphological characters for distinguishing those genera and resolve infra-generic phylogeny, additional taxa from these genera from other regions should be included in the future phylogenetic studies. In addition, comparative morphological analyses of fruitbody features such as subiculum and subhymenium thickness, construction, and texture in addition to basidia, cystidia, and basidiospore shape and size are important areas of consideration in future studies. Information on habitat and distribution may be useful for understanding species delimitation and phylogeny of species within a genus.

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 below: https://www.ncbi.nlm. nih.gov/genbank/, see the Table 1 included in article.