Phylogeny and diversity of Bjerkandera (Polyporales, Basidiomycota), including four new species from South America and Asia

Abstract Four new species of Bjerkandera, viz. B. ecuadorensis, B. fulgida, B. minispora, and B. resupinataspp. nov., are described from tropical America and Asia. B. ecuadorensis is characterised by dark grey to black pore surface, a monomitic hyphal system, hyaline to yellowish-brown generative hyphae, and ellipsoid basidiospores measuring 3.9–4.5 × 2.7–3 μm. B. fulgida is distinguished from the other species in the genus by clay buff to pale brown and shiny pore surface. B. minispora is characterised by white tomentose pore mouth and small basidiospores measuring 3.1–4.2 × 2–2.8 μm. B. resupinata is characterised by resupinate basidiomata, pinkish buff to pale brownish pore surface, and ellipsoid to broadly ellipsoid basidiospores measuring 4.5–6 × 3.2–4.1 µm. All these new species grow on angiosperm trunks or rotten wood, and cause a white rot. The closely related taxa to four new species are discussed. An identification key to the ten accepted species of Bjerkandera is provided, and a phylogeny comprising all known Bjerkandera species is provided.

During a study on polypores collected from China, Ecuador, and Thailand, four unknown species of Bjerkandera were distinguished by both morphological and molecular data. They are described and illustrated in this study. In this study, nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. Furthermore, an identification key to all the accepted species in the genus is provided.

Morphological studies
The studied specimens are deposited in the herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC), and the private herbarium of Josef Vlasák (JV), which will later be deposited at the National Museum Prague of Czech Republic (PRM). Morphological descriptions are based on field notes and herbarium specimens. Microscopic analyses follow Miettinen et al. (2018). In the description: KOH = 5% potassium hydroxide, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, CB = Cotton Blue, CB+ = cyanophilous in Cotton Blue, CB-= acyanophilous in Cotton Blue, L = arithmetic average of all spore length, W = arithmetic average of all spore width, Q = L/W ratios, and n = number of spores/measured from given number of specimens. Colour terms are cited from Anonymous (1969) and Petersen (1996).

Molecular studies and phylogenetic analysis
A CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain DNA from dried specimens, and to perform the polymerase chain reaction (PCR) according to the manufacturer's instructions with some modifications (Shen et al. 2019;Sun et al. 2020). Two DNA gene fragments -internal transcribed spacer (ITS) and large subunit nuclear ribosomal RNA gene (nLSU)were amplified using the primer pairs ITS5/ITS4 and LR0R/LR7 (White et al. 1990;Hopple and Vilgalys 1999) (http://www.biology.duke.edu/fungi/mycolab/primers. htm). The PCR procedures for ITS and nLSU followed Zhao et al. (2013) in the phylogenetic analyses. DNA sequencing was performed at Beijing Genomics Institute and the newly-generated sequences were deposited in GenBank (Sayers et al. 2021). Sequences generated for this study were aligned with additional sequences downloaded from GenBank using BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997). The final ITS and nLSU datasets were subsequently aligned using MAFFT v.7 under the E-INS-i strategy with no cost for opening gaps and equal cost for transformations (command line: mafft -genafpair -maxiterate 1000) (Katoh and Standley 2013) and visualised in BioEdit (Hall 1999).
In this study, nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. The sequence alignment was deposited at TreeBase (submission ID 27872). Sequences of Tyromyces chioneus (Fr.) P. Karst, obtained from GenBank, was used as outgroup (Westphalen et al. 2015).
The phylogenetic analyses followed the approach of Han et al. (2016) and Zhu et al. (2019). Maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) analyses were conducted for the datasets of ITS and nLSU sequences. The best-fit evolutionary model was selected by hierarchical likelihood ratio tests (hLRT) and Akaike Information Criterion (AIC) in MrModeltest 2.2 (Nylander 2004) after scoring 24 models of evolution in PAUP* version 4.0b10 (Swofford 2002).
The MP topology and bootstrap values (MP-BS) obtained from 1000 replicates were computed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted, and gaps were treated as missing. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Maxtrees were set to 5,000 branches of zero length were collapsed, and all parsimonious trees were saved. Descriptive tree statistics tree length (TL), composite consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree (MPT) generated. Sequences were also analysed using Maximum Likelihood (ML) with RAxML-HPC2 through the CIPRES Science Gateway (www.phylo.org; Miller et al. 2009). Branch support (BT) for ML analysis was determined by 1000 bootstrap replicates.
Bayesian phylogenetic inference and Bayesian posterior probabilities (BPP) were computed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Four Markov chains were run for 3,500,000 generations until the split deviation frequency value was less than 0.01 and trees were sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian posterior probabilities (BPP) of the clades.

Phylogeny
The combined ITS and nLSU dataset contained sequences from 75 specimens, comprising a total of 40 species (Table 1). The dataset had an aligned length of 2158 characters, of which 1410 (65%) characters are constant, 208 (0.1%) are variable and parsimony-uninformative and 540 (25%) are parsimony informative. Maximum parsimony analysis yielded eleven equally-parsimonious tree (TL = 2701, CI = 0.439, RI = 0.751, RC = 0.330, HI = 0.561), and a strict consensus tree of these trees is shown in Fig. 1. The best model-fit applied in the Bayesian analysis was GTR+I+G, lset nst = 6, rates = invgamma, and prset statefreqpr = dirichlet (1, 1, 1, 1). Bayesian analysis resulted in the nearly congruent topology with an average standard deviation of split frequencies = 0.006804 to MP and ML analysis, and thus only the MP tree was provided.
Etymology. Ecuadorensis (Lat.): referring to the species being found in Ecuador. Basidiomata. Annual, pileate, soft corky, without odor or taste when fresh, becoming corky when dry, projecting up to 4 cm, 5 cm wide and 1.3 mm thick at base. Pileal surface pinkish-buff to buff, glabrous, faintly zonate, margin blunt. Pore surface grey to dark-brown, becoming almost black when touched or bruised; sterile margin distinct, up to 2 mm wide; pores round to angular, 7-9 per mm; dissepiments thin, entire. Context buff-yellow, slightly fibrous to corky, up to 1 mm thick. Tubes concolorous with the pore surface and darker than context, corky, up to 0.3 mm long, and with a distinct dark line between tubes and context. Hyphal structure. Hyphal system monomitic; generative hyphae with clamp connections, smooth, hyaline to yellowish-brown, CB+, IKI-; tissues becoming dark in KOH. Context. Generative hyphae thick-walled with a wide lumen, occasionally branched, densely compacted, and more or less regularly arranged to loosely interwoven, up to 3.8-6 μm in diam.
Etymology. Fulgida (Lat.): referring to the species having the shiny pore surface. Basidiomata. Annual, effused-reflexed, soft corky, without odor or taste when fresh, becoming corky upon drying, resupinating up to 5.5 cm long, 3 cm wide and 1.3 mm thick, with a pileal projection up to 0.6 cm, 2.3 cm wide and 1.3 mm thick at base. Pileal surface pinkish buff to clay-buff, glabrous and faintly zonate when dry; margin acute. Pore surface clay-buff to pale brown, bruised part becoming dark brown to black when dry, shiny; sterile margin up to 2 mm wide; pores round or sometimes angular, 6-8 per mm; dissepiments thin, entire. Context pale cream, slightly fibrous to corky, up to 0.5 mm thick. Tubes concolorous with the pore surface, darker than context, corky, up to 0.8 mm long, with a distinct dark line between tubes and context. Hyphal structure. Hyphal system monomitic; generative hyphae with clamp connections, smooth, hyaline to yellowish, CB+, IKI-; tissues becoming dark in KOH.
In our phylogenetic analysis, Ceriporiopsis carnegieae (D.V. Baxter) Gilb. & Ryvarden is phylogenetically close to the genus Bjerkandera. Ceriporiopsis Domański is a polyphyletic genus, which is nested in the families Irpicaceae, Meruliaceae (the type species C. gilvescens (Bres.) Domański belongs to Meruliaceae), and Phanerochaetaceae (Justo et al. 2017). Meanwhile, Ceriporiopsis carnegieae resembles Bjerkandera by having a monomitic hyphal system, generative hyphae with abundant clamps, and oblong to short-cylindric basidiospores (Baxter 1941;Gilbertson and Ryvarden 1985). However, the former has basidiomata with sharp and pungent odor when fresh, unchanged pore surface when touched or bruised, and seem to lack any dark line between tubes and subiculum (Gilbertson and Ryvarden 1985). One specimen -JV1512-13J -from Costa Rica forms a sister group to the three sequences annotated as Ceriporiopsis carnegieae, and we treat this specimen as Ceriporiopsis sp. There is ongoing controversy regarding for the generic affiliation of C. carnegieae (Nobles 1965;Justo et al. 2017;Motato-Vásquez et al. 2020), because the black line is absent from Ceriporiopsis carnegieae. For the time being, we are reluctant to combine them in Bjerkandera although the two taxa are phylogenetically related. To solve this problem more specimens should be examined and analysed phylogenetically.
Tyromyces vivii Homble ex Ryvarden was described from Norway (Ryvarden et al. 2003), and later it was treated as a synonym of B. fumosa (Ryvarden and Melo 2017). The type material of T. vivii was analyzed, and it nested in B. fumosa (Fig. 1). We confirm this conclusion by molecular evidence.
Previously, the well-known Bjerkandera adusta and B. fumosa have been reported from the northern hemisphere and South America. However, the diversity of Bjerkandera was underestimated, B. centroamericana, B. mikrofumosa and B. albocinerea were recently described in the neotropics (Westphalen et al. 2015;Ryvarden 2016), and new species in our study have a distribution in the neotropics and tropical Asia. So, the genus has a wide distribution from boreal to tropical areas.