Crassisporus gen. nov. (Polyporaceae, Basidiomycota) evidenced by morphological characters and phylogenetic analyses with descriptions of four new species

Abstract A new poroid wood-inhabiting fungal genus, Crassisporus gen. nov., is proposed on the basis of morphological characters and molecular evidence. The genus is characterized by an annual growth habit, effused-reflexed to pileate basidiocarps with pale yellowish brown to yellowish brown, concentrically zonate or sulcate, and velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues turning to dark in KOH, oblong to broadly ellipsoid, hyaline, smooth, and slightly thick-walled basidiospores. Phylogenetic analysis based on ITS+nLSU sequences indicate that Crassisporus belongs to the core polyporoid clade. The combined ITS+nLSU+mtSSU+EF1-α+RPB2 sequences dataset of representative taxa in the Polyporaceae demonstrate that Crassisporus is grouped with Haploporus but forms a monophyletic lineage. In addition, four new species of Crassisporus, C. imbricatus, C. leucoporus, C. macroporus, and C. microsporus are described.


Introduction
Polyporales is one of the most diverse orders of Basidiomycota including more than 1800 described species in 216 genera and 13 families (Kirk et al. 2008). In the last 10 years, many new genera in Polyporales have been established, such as Datroniella B.K.  (Li and Cui 2013), Pseudofibroporia Yuan Y. Chen & B.K. Cui (Chen et al. 2017), and Pseudomegasporoporia X.H. Ji & F.Wu (Ji and Wu 2017).
During the investigations of species diversity and phylogeny of Polyporales, four new species were found that did not belong to any known genus, for which reason, a new genus is established to accommodate them. Morphologically, these four taxa do not fit any of the known polypore taxa. To confirm the position of the new genus, phylogenetic analyses of the new genus and related taxa within Polyporales were carried out based on the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (EF1-α), and the second largest subunit of RNA polymerase II (RPB2).

Morphological studies
The studied specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions are based on the field notes and measurements of herbarium specimens. Color terms follow Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Shen et al. (2019). Sections were studied at magnifications up to 1000× using a Nikon E80i microscope and phase contrast illumination (Nikon, Tokyo, Japan). Drawings were made with the aid of a drawing tube. Microscopic features, measurements, and drawings were made from slide preparations stained with cotton blue and Melzer's reagent. Spores were measured from sections cut from the tubes. In presenting the variation of spore size, 5% of measurements were excluded from each end of the range, and were given in parentheses. The following abbreviations were used: KOH, 5% potassium hydroxide; IKI, Melzer's reagent; IKI-, neither amyloid nor dextrinoid; CB, cotton blue; CB+, cyanophilous; CB-, acyanophilous; L, mean spore length (arithmetic average of all spores); W, mean spore width (arithmetic average of all spores); Q, variation in the L/W ratios between the specimens studied; n (a/b), number of spores (a) measured from given number (b) of specimens.

DNA extraction and sequencing
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co. Ltd, Beijing) was used to extract total genomic DNA from dried specimens, and performed the polymerase chain reaction (PCR) according to the manufacturer's instructions with some modifications . The ITS region was amplified with primer pairs ITS5 and ITS4 (White et al. 1990). The nLSU region was amplified with primer pairs LR0R and LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm). The mtSSU region was amplified with primer pairs MS1 and MS2 (White et al. 1990). Part of EF1-α was amplified with primer pairs EF1-983F and EF1-1567R (Rehner and Buckley 2005). RPB2 was amplified with primer pairs fRPB2-5F and fRPB2-7cR or bRPB2-6F and bRPB2-7R (Liu et al. 1999;Matheny 2005). The PCR procedure for ITS, mtSSU and EF1-α was as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for ITS and mtSSU, 54-56 °C for EF1-α for 45 s, 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min, 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. The PCR procedure for RPB2 was as follows: initial denaturation at 94 °C for 2 min, followed by 10 cycles at 94 °C for 40 s, 60 °C for 40 s and 72 °C for 2 min, then followed by 37 cycles at 94 °C for 45 s, 55-57 °C for 1.5 min and 72 °C for 2 min, and a final extension of 72 °C for 10 min. The PCR products were purified and sequenced at Beijing Genomics Institute, China, with the same primers. All newly generated sequences were submitted to GenBank (Table 1).
Phylogenetic analyses used in this study followed the approach of Zhu et al. (2019) and Song and Cui (2017). Maximum parsimony (MP) analysis was performed in PAUP* v. 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates a Newly generated sequences for this study (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each maximum parsimonious tree generated.
RAxML v. 7.2.6 (Stamatakis 2006) was used to perform maximum likelihood (ML) analysis involved 200 ML searches under the GTR+GAMMA model and only the best tree from all searches was kept. In addition, 200 rapid bootstrap replicates were run with the GTR+CAT model to assess the reliability of the nodes.
MrModeltest v. 2.3 (Posada and Crandall 1998;Nylander 2004) was used to determine the best fit evolution model for the combined multi-gene dataset for Bayesian inference (BI). Bayesian inference was calculated with MrBayes v. 3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for two runs from random starting trees for 2 million generations (ITS+nLSU), for 5 million generations (ITS+nLSU+mtSSU+EF1-α+RPB2) until the split deviation frequency value <0.01, and trees were sampled every 100 generation. The first quarter generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated.

Molecular phylogeny
The combined 2-gene dataset included sequences from 68 fungal samples representing 59 taxa. The dataset had an aligned length of 2111 characters, of which 1249 characters were constant, 196 were variable and parsimony-uninformative, and 666 were parsimony-informative. MP analysis yielded 37 equally parsimonious trees (TL = 4143, CI = 0.345, RI = 0.617, RC = 0.213, HI = 0.655). Best model for the combined 2-gene dataset estimated and applied in the BI was GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). MP, ML and BI analyses yielded similar tree topologies with an average standard deviation of split frequencies = 0.006293 (BI), and the ML topology is shown in Figure 1. The phylogeny (Fig. 1)   length of 4306 characters, of which 2521 characters were constant, 258 were variable and parsimony-uninformative, and 1527 were parsimony-informative. MP analysis yielded 1 equally parsimonious tree (TL = 8989, CI = 0.339, RI = 0.620, RC = 0.210, HI = 0.661). Bayesian and ML analyses resulted in a similar topology as the MP analysis, with an average standard deviation of split frequencies = 0.006328 (BI); and the ML topology is shown in Figure 2. A further phylogeny (Fig. 2) inferred from the combined 5-gene dataset was obtained for more representative taxa in the Polyporaceae and showed that the new genus grouped with Haploporus clade but distinctly formed a monophyletic lineage. Notes. Differs from other genera by the combination of effused-reflexed to pileate basidiocarps, pale yellowish brown to yellowish brown, concentrically zonate or sulcate, velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues darkening in KOH, and oblong to broadly ellipsoid, hyaline, smooth and slightly thick-walled basidiospores.
Context yellowish brown, leathery, up to 2.5 mm thick. Tubes concolorous with context, corky, up to 1.5 mm long.
Etymology. Leucoporus (Lat.): referring to the white pore surface when fresh. Description. Fruitbody: Basidiocarps annual, effused-reflexed to pileate, corky, without odor or taste when fresh, soft leathery to corky upon drying. Pilei semicircular or elongated, projecting up to 1.5 cm, 3 cm wide, and 6 mm thick at base. Pileal surface yellowish brown to umber-brown, finely velutinate, concentrically sulcate. Pore surface white when fresh, becoming cream, clay buff to pale yellowish brown when dry; sterile margin distinct, cream to pale yellowish brown, up to 1.5 mm wide; pores round to angular, 3-4 per mm; dissepiments slightly thick, entire. Context pale yellowish brown to fulvous, leathery, up to 3 mm thick. Tubes pale yellowish brown, corky, up to 2.5 mm long.
Type of rot. White rot. Notes. Crassisporus macroporus is characterized by cream-buff to cinnamon-buff colored pore surface with distinct sterile margin when fresh, large pores (2-3 per mm) with thin dissepiments, a trimitic hyphal system with cyanophilous skeletal hyphae, the presence of fusoid cystidioles, and oblong ellipsoid basidiospores (9.5-13.2 × 4-6.2 μm).  Etymology. Macroporus (Lat.): referring to the large pores. Description. Fruitbody: Basidiocarps annual, effused-reflexed to pileate, corky to leathery, without odor or taste when fresh, soft leathery upon drying. Pilei flabelliform, semicircular or elongated, projecting up to 1.5 cm, 4 cm wide and 5 mm thick at base; resupinate part up to 7 cm long, 4 cm wide, and 5 mm thick at center. Pileal surface buff to yellowish brown when fresh, becoming yellowish brown upon drying, finely velutinate, concentrically sulcate. Pore surface cream, buff to cinnamon-buff when fresh, becoming buff, pale yellowish brown to yellowish brown when dry; sterile margin distinct, buff to pale yellowish brown, up to 2 mm wide; pores round to angular, 2-3 per mm; dissepiments thin, entire to lacerate. Context yellowish brown to pale yellowish brown, leathery, up to 1.5 mm thick. Tubes pale yellowish brown, corky, up to 2 mm long.
Etymology. Microsporus (Lat.): referring to the small basidiospores. Description. Fruitbody: Basidiocarps annual, pileate, sessile, corky, without odor or taste when fresh, soft leathery to corky upon drying. Pilei semicircular, projecting up to 2 cm, 4 cm wide, and 4.5 mm thick at base. Pileal surface pale yellowish brown to yellowish brown, finely velutinate, concentrically sulcate. Pore surface cream, buff to cinnamon-buff when fresh, buff, pale yellowish brown to yellowish brown when dry; sterile margin distinct, buff, up to 1 mm wide; pores round to angular, 5-7 per mm; dissepiments slightly thick, entire. Context pale yellowish brown to yellowish brown, leathery to corky when dry, up to 1.5 mm thick. Tubes concolorous with context, soft corky to corky, up to 3 mm long.
Hyphal structure: Hyphal system trimitic; generative hyphae bearing clamp connections; skeletal and binding hyphae IKI-, CB-; tissues turning to deep brown in KOH.
Type of rot. White rot.

Discussion
In the present study, Crassisporus is proposed based on morphological characters and phylogenetic analyses. In the ITS+nLSU analysis, Crassisporus was nested in the core polyporoid clade with strong support (100% MP, 100% ML, 1.00 BPP; Fig. 1). A further study based on combined ITS+nLSU+mtSSU+EF1-α+RPB2 sequences data indicated that Crassisporus grouped with Haploporus with low support, but formed a monophyletic lineage with a strong support (100% MP, 100% ML, 1.00 BPP; Fig. 2). Morphologically, Crassisporus is characterized by the combination of an annual growth habit, effused-reflexed to pileate basidiocarps, pale yellowish brown to yellowish brown, concentrically zonate or sulcate pilei, velutinate pileal surface, a trimitic hyphal system with clamped generative hyphae, tissues turning to dark in KOH, oblong to broadly ellipsoid, hyaline, smooth and slightly thick-walled basidiospores.
Morphologically, the four Crassisporus species can be easily distinguished from each other. Crassisporus microsporus differs from other species by its small pores (5-7 per mm), and small broadly ellipsoid basidiospores (4-5 × 3-3.7 μm). Except for C. imbricatus, C. leucoporus, C. macroporus, and C. microsporus, all have fusoid cystidioles in the hymenium; moreover, C. imbricatus produces imbricate basidiocarps. Previously, the type specimen of C. imbricatus was identified as Coriolopsis byrsina (Mont.) Ryvarden based on morphological characters (Li and Cui 2010). After careful examination of the basidiospores along with DNA sequences analyses, the specimen was found to represent an unknown taxon. Here, we describe it as a new species of Crassisporus based on morphological characters and phylogenetic analysis. Crassisporus macroporus may be confused with C. leucoporus due to their similarity in effused-reflexed to pileate basidiocarps and oblong ellipsoid basidiospores, but C. leucoporus is distinguished from C. macroporus by its smaller pores (3-4 per mm), white pore surface when fresh, and acyanophilous skeletal and binding hyphae.
Crassisporus is similar to Hexagonia Fr. and Neofomitella Y.C. Dai, Hai J. Li & Vlasák, because these genera share pileate brown basidiocarps, a trimitic hyphal system with clamped generative hyphae, and tissues becoming dark in KOH. However, Hexagonia is distinguished from Crassisporus by its larger hexagonal pores and thin-walled basidiospores (Núñez and Ryvarden 2001). Neofomitella differs from Crassisporus in having crusted basidiocarps with the cuticle developing from base to margin, and smaller, thin-walled basidiospores (Li et al. 2014b).
Truncospora Pilát is similar to Crassisporus in having pileate basidiocarps and variable presence of cystidioles. However, Truncospora is distinguished from Crassisporus by variable dextrinoid and cyanophilous skeletal hyphae and truncate, strongly dextrinoid basidiospores Cui et al. 2019).
Grammothelopsis Jülich is similar to Crassisporus in having thick-walled basidiospores; however, it differs from Crassisporus in its resupinate to effused basidiocarps with shallow irregular pores, and variable dextrinoid skeletal hyphae (Robledo and Ryvarden 2007;Zhao and Cui 2012).