Two new species of Hymenochaetaceae on Dracaena cambodiana from tropical China

Abstract Two new wood-rotting fungi in the family Hymenochaetaceae, Fulvifomes dracaenicolasp. nov. and Hymenochaete dracaenicolasp. nov., are described and illustrated from tropical China based on morphological characteristics and molecular data. It is worth to mention that both of them grow on Dracaena cambodiana which is a kind of angiosperm tree distributed in tropical regions. F. dracaenicola is characterised by perennial, pileate, triquetrous basidioma with yellowish brown fresh pores which becoming honey yellow with silk sheening upon drying, a dimitic hyphal system in trama and monomitic in context, and subglobose basidiospores measuring 4.8–5 × 4–4.1 μm. H. dracaenicola is characterised by annual, resupinate basidioma with a clay buff hymenophore, a dimitic hyphal system, absence of tomentum and cortex, presence of subulate setae, absence of cystidia, presence of cystidioles and simple hyphidia, and oblong ellipsoid basidiospores measuring 5.2–5.8 × 2.5–2.8 µm. The phylogenetic analyses based on ITS + nLSU rDNA sequences confirm the placement of two new species respectively in Fulvifomes and Hymenochaete. Phylogenetically closely related species to the two new species are discussed.


Introduction
Fulvifomes Murrill (Hymenochaetaceae, Hymenochaetales) was erected in 1914 and typified by F. robiniae (Murrill) Murrill (Murrill 1914). Wagner and Fischer (2002) thought that Fulvifomes comprises species with a dimitic hyphal system, absence of setae, and yellowish, thick-walled basidiospores. Hattori et al. (2014) provided a key to worldwide species of Fulvifomes and other species possibly belonging to Fulvifomes. Zhou (2014) treated Aurificaria D.A. Reid as a taxonomic synonym of Fulvifomes and transferred Aurificaria indica (Massee) D.A. Reid to Fulvifomes. However, Fulvifomes indicus (Massee) L.W. Zhou has a monomitic hyphal system, but he thought that the hyphal system might be not a stable character at the generic level within Hymenochaetaceae. Salvador-Montoya et al. (2018) redefined Fulvifomes and thought Fulvifomes should encompass species with a monomitic hyphal system in the context, a dimitic hyphal system in the trama. We agree with Zhou and Salvador-Montoya et al., and consider the genus Fulvifomes has a monomitic or dimitic hyphal system.
During investigations on the diversity of wood-rotting fungi from China, five unknown specimens were collected from Hainan Province, and their morphology corresponds to the concepts of Fulvifomes and Hymenochaete. To confirm their affinity, phylogenetic analyses based on the ITS and nLSU rDNA sequences were carried out. Both morphological characteristics and molecular evidence demonstrated these five specimens represent two new species of Hymenochaetaceae, which we describe in the present paper.

Morphological studies
Macro-morphological descriptions were based on field notes and dry herbarium specimens. Microscopic measurements and drawings were made from slide preparations of dried tissues stained with Cotton Blue and Melzer's reagent following Dai (2010). Pores were measured by subjectively choosing as straight a line of pores as possible and measuring how many fit per mm. The following abbreviations are used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, 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 specimens studied, and n (a/b) = number of spores (a) measured from given number of specimens (b). In presenting spore size variation, 5% of measurements were excluded from each end of the range and this value is given in parentheses. Special color terms follow Anonymous (1969) and Petersen (1996). Herbarium abbreviations follow Thiers (2018). The studied specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC).

Molecular studies and phylogenetic analysis
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract total genomic DNA from dried specimens following the manufacturer's instructions with some modifications Shen et al. 2019). ITS regions were amplified with primers ITS4 and ITS5 (White et al. 1990), and the nLSU with primers LR0R and LR7. The polymerase chain reaction (PCR) procedure for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 58 °C for 45 s, and 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 35 cycles at 94 °C for 30 s, 48 °C for 1 min, and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min (Chen et al. 2015). The PCR products were purified and sequenced in the Beijing Genomics Institute, China, with the same primers used in the PCR reactions.
Maximum parsimony analysis was applied to the ITS + nLSU dataset sequences. Approaches to phylogenetic analysis followed Song et al. (2016), and the tree construction procedure was computed in PAUP* version 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 tree bisection and reconnection (TBR) branch swapping and 1000 random sequence additions maxtrees 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 (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 (MPT) generated. Sequences were also analyzed using maximum likelihood (ML) with RAxML-HPC through the CIPRES Science Gateway (Miller et al. 2009; http://www. phylo.org). Branch support for ML analysis was determined by 1000 bootstrap replicates.   (Posada and Crandall 1998;Nylander 2004) was used to determine the best-fit evolution model for the combined dataset for Bayesian Inference (BI). BI was performed using MrBayes v. 3.2.7a (Ronquist and Huelsenbeck 2003) with four simultaneous independent chains for two datasets, performing 3 million generations (Fulvifomes) and 5 million generations (Hymenochaete) until the split deviation frequency value < 0.01, and sampled every 1000 th generation. The first 25% sampled trees were discarded as burn-in, while the remaining ones were used to calculate Bayesian posterior probabilities (BPP) of the clades.

Fulvifomes
The combined ITS + nLSU dataset included sequences from 50 specimens representing 31 species ( model-fi for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G. Bayesian analysis and ML analysis resulted in a similar topology to the MP analysis, with an average standard deviation of split frequencies of 0.004578 (BI). The phylogeny (Fig. 1) inferred from the ITS and nLSU sequences demonstrated that the new species Fulvifomes dracaenicola nested in the Fulvifomes clade. Moreover, two specimens of F. dracaenicola form a lineage with strong support (100% BP, 100% BS, 1.00 BPP, Fig. 1).

Hymenochaete
The combined ITS + nLSU dataset included sequences from 79 specimens representing 69 species (Table 2). The dataset had an aligned length of 2249 characters, of which 1486 (66%) were constant, 248 (11%) were variable but parsimony-uninformative, and 515 (23%) were parsimony-informative. MP analysis yielded 48 equally parsimonious trees (TL = 3261, CI = 0.365, RI = 0.619, RC = 0.226, HI = 0.635). The best model for the ITS + nLSU dataset estimated and applied in the Bayesian analysis was GTR+I+G. Bayesian analysis and MP analysis resulted in a similar topology to the ML analysis, with an average standard deviation of split frequencies of 0.009996 (BI).
The phylogeny (Fig. 2) inferred from the ITS and nLSU sequences demonstrated that the new species Hymenochaete dracaenicola clustered in the Hymenochaete clade and two specimens of H. dracaenicola form a lineage with strong support (100% BS, 100% BP, 1.00 BPP, Fig. 2).  Fruiting body. Basidioma perennial, pileate, without odor or taste and woody hard when fresh, light in weight when dry. Pilei triquetrous, projecting up to 2.5 cm, 2.3 cm wide and 2.6 cm thick at base. Pileal surface yellowish brown to grayish brown when fresh, vinaceous brown when dry, encrusted, glabrous, zonate, uncracked, mar- gin olivaceous brown. Pore surface yellowish brown when fresh, honey yellow with silk sheening when dry; sterile margin indistinct; pores circular, 5-7 per mm; dissepiments thin, entire. Context cinnamon buff to fawn, corky, often darker near the pileus surface, up to 1.4 cm thick, with a distinct crust (black line) near pileus surface at the basal area, partly with additional crust (black line) within context or above tubes. Tubes cinnamon buff to cinnamon, woody hard, up to 1.2 cm thick, tube layers distinctly stratified, individual tube layer up to 0.5 cm long.
Holotype Fruiting body. Basidioma annual, resupinate, adnate, not separable from substrate, hard corky, up to 7.5 cm long, 2 cm wide, and less than 0.1 mm thick at center. Hymenophore surface smooth or locally verruculose, clay buff, with some scattered crevices; margin cinnamon buff, up to 0.4 mm.  Hyphal structure. Hyphal system dimitic; generative hyphae infrequent, simple septate; skeletal hyphae dominant; tissues darkening but otherwise unchanged in KOH.