Taxonomy and Phylogeny of Peniophora Sensu Lato (Russulales, Basidiomycota)

Peniophora is an old corticioid genus, from which two small satellite genera, Dendrophora and Duportella, were derived based on morphological differences. Molecular systematic studies showed that they belong to Peniophoraceae, Russulales, but the inter- and intra-generic phylogenetic relationships are still unclear. Moreover, the species diversity of this group in subtropical and tropical Asia has not been sufficiently investigated and studied. In this study, we carried out an intensive taxonomic and phylogenetic study on Peniophora sensu lato based on analyses of concatenated ITS1-5.8S-ITS2 (ITS, Internal Transcribed Spacer) and 28S (D1–D2 domains of nuc 28S rDNA) sequence data of all available species worldwide. In the phylogenetic trees, species of Peniophora s. l. (sensu lato) including types of Peniophora s.s. (sensu stricto), Dendrophora and Duportella were interspersed within a strongly supported clade. It means that the morphological delimitations of the three genera are not reliable, and they should be regarded as a large genus. As a result, eight species of Duportella were transferred to Peniophora, although five of them have not been sequenced. Four new distinct lineages, corresponding to Peniophora cremicolor, P. major, P. shenghuae and P. vietnamensis spp. nov., were recovered in the trees. Peniophora taiwanensis is treated as a later synonym of P. malaiensis based on morphological and molecular evidence. Duportella renispora is excluded from Peniophoraceae, because BLAST results of the ITS and 28S sequences of the holotype showed that it is closely related to Amylostereum. Descriptions and illustrations are provided for the four new species, and an identification key is given for all 25 species of Peniophora in China. Our results indicated that the species diversity of the corticioid fungi in Asia is rich and deserves further study.


Introduction
The genus Peniophora Cooke (Peniophoraceae, Russulales) introduced in 1879, typified by Thelephora quercina Pers. ex Fr., is one of the oldest genera of corticioid fungi. It is a cosmopolitan genus with a wide distribution from boreal to tropical areas, causing a white rot on both angiosperms and gymnosperms. Species of the genus prefer to grow on small branches especially those dead but still attached ones in the exposed and dry environments. At the beginning, mycologists adopted a broad concept, and many unrelated species were described in the genus. It includes 639 names in Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, accessed on 20 November 2022), but most species have been moved to other genera, and the morphological circumscription of Peniophora has been narrowed [1]. Meanwhile, some intrageneric taxa were proposed and used, and two satellite genera Duportella Pat. and Dendrophora (Parmasto) Chamuris were separated from Peniophora s.l. and accepted by many mycologists [2][3][4][5]. In the morphological monograph of Peniophoraceae [5], Andreasen and Hallenberg accepted 70 species of Peniophora s.s., 12 species of Duportella and 2 species of Dendrophora and provided keys and descriptions for all the genera and species.
According to the modern concept, Peniophora s.s. includes species possessing resupinate basidiomes with a smooth hymenophore, a monomitic hyphal system with

Morphological Studies
Voucher specimens and strains are deposited at the herbaria of Beijing Forestry University, Beijing, China (BJFC) and Centre for Forest Mycology Research, U.S. Forest Service, Madison, Wisconsin, USA (CFMR). Freehand sections were made from dried basidiomes and mounted in 2% (w/v) potassium hydroxide (KOH) with 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, Tokyo, Japan) at magnifications up to 1000×. Drawings were made with the aid of a drawing tube. The following abbreviations are used: 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 terms follow Kornerup and Wanscher [17].

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 which were 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 [18] 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 28S D1-D2 region was amplified with the primer pair LR0R/LR7 [19] 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 newly generated sequences were deposited in GenBank (Table 1). BioEdit v.7.0.5.3 [20] was 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 [23], RAxML v.8.2.10 [24] and MrBayes 3.2.6 [25], respectively. In MP analysis, trees were generated using 100 replicates of the random stepwise addition of sequence and 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 used for other parameters. For BI, the best-fit substitution model was estimated with jModeltest v.2.17 [26]. Four Markov chains were run for 0.3 and 20 million generations for the Peniophoraceae and Peniophora s.l. datasets, respectively, until the split deviation frequency values were lower than 0.01. Trees were sampled every 100 generations. 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
Thirty-nine ITS and 39 28S sequences were newly generated in this study (Table 1). The Peniophoraceae dataset contained 33 ITS and 34 28S sequences from 34 samples representing 13 genera and the outgroup, and it had an aligned length of 2313 characters, of which 544 were parsimony-informative. MP analysis yielded 5000 equally parsimonious trees (TL = 2605, CI = 0.528, RI = 0.568, RC = 0.300, HI = 0.472). The Peniophora s.l. dataset contained 113 ITS and 97 28S sequences from 114 samples representing 63 taxa and the outgroup, and it had an aligned length of 1993 characters, of which 350 were parsimonyinformative. MP analysis yielded 5000 equally parsimonious trees (TL = 1973, CI = 0.372, RI = 0.698, RC = 0.260, HI = 0.628). The jModelTest suggested GTR+I+G as the best-fit model of nucleotide evolution for both Peniophoraceae and Peniophora s.l. datasets. The average standard deviation of split frequencies of BI was 0.004389 (for the Peniophoraceae dataset) and 0.004179 (for the Peniophora s.l. dataset) at the end of the run. ML and BI analyses resulted in almost identical tree topologies compared to the MP analysis. The MP trees of Penophoraceae and Peniophora s.l. are shown in Figures 1 and 2, respectively, with the parsimony bootstrap values (≥50%, front), likelihood bootstrap values (≥50%, middle) and Bayesian posterior probabilities (≥0.95, back) labeled along the branches.
In the trees, species of Peniophora s.l. including the type species of Peniophora s.s., Dendrophora and Duportella formed a strongly supported clade (95/95/1 in Figure 1, 100/100/1 in Figure 2). Four distinct lineages corresponding to Peniophora cremicolor, P. major, P. shenghuae and P. vietnamensis spp. nov. were recovered. For other sequences generated in this study, they formed distinct lineages alone or together with sequences from GenBank and represented known species.
Microscopic structures-Hyphal system monomitic; generative hyphae simple-septate. Subiculum indistinct or absent; hyphae colorless, thin-to slightly thick-walled, Notes-Peniophora vietnamensis is characterized by its brownish orange basidiomes on Araceae plant, simple-septate generative hyphae, brown lamprocystidia, subulate gloeocystidia and oblong cylindrical basidiospores. In the phylogenetic trees (Figures 1 and 2), P. vietnamensis and P. trigonosperma Boidin, Lanq. & Gilles formed a strongly supported lineage. Both species grow on palm trees and have similar brown lamprocystidia, however, P. trigonosperma differs in having clamped hyphae, cylindrical to fusiform gloeocystidia and triangular basidiospores [5]. Duportella rhoica Boidin & Lanq. is similar to P. vietnamensis by sharing oblong cylindrical basidiospores (10-14.5 × 4-6 µm) but differs in having a dimitic Notes-The species is characterized by the brown lamprocystidia and lacrimoid to pyriform or subreniform basidiospores. Although the sequences of the species are unavailable, present molecular evidence indicated that Peniophora includes species with differently shaped basidiospores (Figures 1 and 2 Notes-Our phylogenetic analyses indicated that the holotype of P. malaiensis, LY 8292 from Singapore (CBS679.91), formed a strongly supported lineage with three specimens collected from Guangxi Autonomous Region and Taiwan, China, including the paratype of P. taiwanensis, Wu 9206-28 ( Figure 2). The ITS sequence similarity between LY 8292 and Wu 9206-28 is 99.16% (5 base pair differences). Morphologically, the two species are very similar except that P. taiwanensis has narrower basidiospores (1.8-2.2 µm) than P. malaiensis (2-2.7 µm) [3,11]. However, our measurements of the basidiospore width of the specimen He 4870 match that of P. malaiensis (2-3 µm). Therefore, we believe that the difference in the basidiospore width is within the specific range and treat P. taiwanensis as a later synonym of P. malaiensis. Notes-The strain (CBS 733.91) isolated from the holotype of the D. renispora (LY 12699) was sequenced by Vu et al. [9]. The blast results of the ITS (MH862319) and 28S (MH873991) sequences showed that the species does not belong to Peniophora s.l. but is close to Amylostereum Boidin. Morphologically, the thinly encrusted brown cystidia and absence of subiculum of the species do match the characteristics of Amylostereum, which, however, has amyloid basidiospores. In order to confirm the identity of the species, the type specimen should be checked in the future. 23. Lamprocystidia up to 20 µm wide; basidiospores up to 7 µm long P. pithya 23. Lamprocystidia up to 12 µm wide; basidiospores up to 10 µm long 24 24. Gloeocystidia 30-80 × 8-20 µm; basidiospores 2.5-3.5 µm wide P. nuda 24. Gloeocystidia 12.5-58 × 5.5-15.5 µm; basidiospores 3-5.5 µm wide P. yunnanensis

Discussion
The modern molecular phylogenetics have significantly changed the taxonomic systems of wood-inhabiting fungi in recent years. On the one hand, many new lineages and taxa were found and described, and on another hand, some morphologically dissimilar taxa were proved to be closely related in phylogeny [41][42][43][44][45]. Our results indicated that in the Peniophora s.l. group, the morphological characters, such as the color of lamprocystidia and dendrohyphidia used for generic delimitation are not reliable. As already shown in other studies [46][47][48][49][50], the identities of some genera erected based merely on morphology or together with phylogenetic analyses of not well-sampled datasets need to be confirmed.
The Peniophoraceae is a well-supported large family in Russulales, most species of which have resupinate basidiomes and non-poroid hymenophores growing on twigs, branches or trunks of woody plants or bamboos [5,51]. The species diversity, taxonomy and phylogeny of Peniophoraceae in China have been investigated and studied [16,37,[52][53][54][55], and this study is a part of this consecutive research. However, the two large genera in Peniophoraceae, Scytinostroma Donk and Vararia P. Karst., that are closely related and were shown to be polyphyletic [6,7,28], have not been sufficiently studied worldwide. We are going to carry out a complete morphological and phylogenetic analyses of the Peniophoraceae by adding a lot of Scytinostroma-Vararia samples from Asia accumulated by the authors in recent years.