Taxonomy and phylogeny of brown-rot corticioid fungi in China: Coniophora beijingensis and Veluticeps subfasciculata spp. nov.

Brown-rot fungi account for a small portion of the wood-decaying fungi. There are a few corticioid genera causing brown rot of wood, and their species diversity is still under investigated and studied, especially in subtropical and tropical areas. Two new brown-rot corticioid fungi, Coniophora beijingensis and Veluticeps subfasciculata were found during the investigation of corticioid fungi in China. Phylogenetic analyses of the two genera were carried out separately based on ITS-28S sequence data. Coniophora beijingensis was collected from Beijing, north China, from different kinds of angiosperm and gymnosperm trees, and is characterized by possessing a monomitic hyphal system with colorless hyphae and relatively small pale yellow basidiospores 7–8.6 μm× 4.5–6 μm. Veluticeps subfasciculata was collected from Guizhou and Sichuan Provinces, southwestern China, on Cupressus and is characterized by the resupinate to effused-reflexed basidiomes with a colliculose hymenophore, nodose-septate generative hyphae, fasciculate skeletocystidia and subcylindrical to subfusiform basidiospores 8–11 μm × 2.5–3.5 μm. Descriptions and illustrations are provided for the two new species, and identification keys to Coniophora and Veluticeps species in China are given. Coniophora fusispora is reported in China for the first time.


Introduction
Modern phylogenetic analyses indicated that corticioid brown-rot fungi distributed in several main lineages of Agaricomycetes, such as, Boletales, Polyporales, Gloeophyllales, and Amylocorticiales (Larsson, 2007;Hibbett et al., 2014). The total number of this group of fungi is small with some relatively small but important genera, among which Coniophora DC. and Veluticeps Cooke are two typical ones.
Veluticeps Cooke, typified by V. berkeleyana Cooke, is characterized by the resupinate, effused-reflexed or pileate basidiomes with a smooth, warted or odontioid hymenophore, a monomitic hyphal system with simple-or nodose-septate hyphae, single or fasciculate cystidia and ellipsoid or cylindrical basidiospores negative in Melzer's reagent (Nakasone, 1990;Bernicchia and Gorjón, 2010). Phylogenetic studies indicated that Veluticeps is a member of the small order Gloeophyllales, which include the famous brown-rot poroid genus, Gloeophyllum P. Karst. (Larsson, 2007;Garcia-Sandoval et al., 2011;He and Li, 2013;. Nakasone (1990Nakasone ( , 2004) did monographic studies on the genus and other similar genera. The genus now comprises about 11 species with several new species were described from China based on morphological and molecular evidence recently (He and Li, 2013;. Although the poroid brown-rot fungi in China have been intensively studied in recent years (Han et al., 2016;Shen et al., 2019;Liu et al., 2021), the species diversity of the corticioid ones are still largely unknown. Preliminary morphological and molecular studies on the corticioid specimens collected from China recently revealed two undescribed species. In this paper, we carried out two independent phylogenetic analyses based on ITS-28S sequence data, and describe and illustrate the two new species as Coniophora beijingensis and Veluticeps subfasciculata.

Specimen collection
In situ photos of specimens were taken with a Canon camera EOS 70D (Canon Corporation, Japan). Specimens were dried with a portable dryer, labelled, then stored in a freezer at minus 40°C for 2 weeks to kill the insects and their eggs before proceeding with morphological and molecular studies. Voucher specimens are deposited at the herbarium of Beijing Forestry University, Beijing, China (BJFC). Herbarium code designations follow Index Herbarium. 2

Morphological studies
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 was 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, Japan) at magnifications up to 1,000 ×. Drawings were made with the aid of a drawing tube. The following abbreviations are used: L = mean basidiospore length, W = mean basidiospore width, Q = L/W ratio, n (a/b) = number of basidiospores (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 (ITS) 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 (28S) was amplified with the primer pair LR0R/LR7 3 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 4 (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 1,000 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 1,000 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 2 million generations for the Coniophora dataset and 1 million generations for the Veluticeps dataset 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.

Discussion
Although the brown-rot corticioid fungi only account for a small portion of the wood-decaying fungi in nature, they also play important roles in the cycling of substances and renewal of ecosystem. Our present and previous studies (He and Li, 2013; indicated that the species diversity of this group of fungi in China is rich and needs more investigations and studies. Future works should be emphasized on the special habitats and hosts, for example, the gymnosperm trees in areas of high altitudes. With more specimens collected and sequenced, the number of this group fungi in China will be largely increased in future.
Coniophora and Veluticeps are two common brown-rot corticoid genera in China. Morphologically, both genera have brown basidiomes, but Coniophora in Boletales can be easily distinguished from Veluticeps in Gloeophyllales by having yellowish-brown, thickwalled basidiospores. Hymenochaete Lév. in Hymenochaetales is similar to Coniophora and Veluticeps by sharing resupinate brown basidiomes, but differs in having characteristic brown setae.
Coniophora fusispora, a characteristic and widely distributed species, is reported from China for the first time based on two specimens collected on the bases of living Pinus tree in Beijing. The species numbers of Coniophora and Veluticeps in China have now become to seven and six with two new species and a new Chinese record reported above. Herein, we present two identification keys to all species of the two genera as follows.
A key to Coniophora species in China

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 at: https://www.ncbi.nlm.nih.gov/genbank/, MW192496-MW192498; MW191807-MW191809; MW192503; MW192502; MW191814; and MW191813.