Two new species of Fuscoporia (Hymenochaetales, Basidiomycota) from southern China based on morphological characters and molecular evidence

Abstract Fuscoporia (Hymenochaetaceae) is characterized by annual to perennial, resupinate to pileate basidiocarps, a dimitic hyphal system, presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Phylogenetic analyses based on the nLSU and a combined ITS, nLSU and RPB2 datasets of 18 species of Fuscoporia revealed two new lineages that are equated to two new species; Fuscoporia ramulicolasp. nov. grouped together with F. ferrea, F. punctatiformis, F. subferrea and F. yunnanensis with a strong support; Fuscoporia acutimarginatasp. nov. formed a strongly supported lineage distinct from other species. The individual morphological characters of the new species and their related species are discussed. A key to Chinese species of Fuscoporia is provided.

In our study, phylogenetic analyses were carried out based on the nLSU and combined ITS, nLSU and RPB2 datasets including 99 (60 newly generated) sequences representing 18 species of Fuscoporia. From the analyses, two new species of Fuscoporia were found and described. In addition, a key to Chinese species in the genus was provided.

Morphological studies
The studied specimens are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions are based on field observations and notes and dry herbarium specimens. The microscopic analyses followed that described by Cui et al. (2019). Sections were studied at ultimate magnification ×1000 applying Nikon Eclipse 80i microscopy and phase contrast illumination. Drawings were made with the aid of a drawing tube. The measurements and drawings were made from slide preparations stained with Cotton Blue. In recording spore size variation, 5% of measurements were excluded from each end of the range and given in parentheses. The following abbreviations are used in the article: 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). Special color terms are cited from Petersen (1996).

DNA extraction and sequencing
Extract total genomic DNA was extracted from dried specimens by CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's instructions with some modifications Han et al. 2016).
To generate PCR amplicons, the following primer pairs were used: ITS4 and ITS5 (White et al. 1990) for the internal transcribed spacer (ITS), LR0R and LR7 (Vilgalys and Hester 1990) for nuclear large submit (nLSU) and bRPB2-6F and bRPB2-7.1R (Matheny 2005) for partial RNA polymerase II, second largest submit (RPB2). The PCR procedures followed Song and Cui (2017) and Zhu et al. (2019). DNA sequencing was performed at Beijing Genomics Institute and the sequences are deposited in GenBank and listed in Table 1.

Phylogenetic analyses
Sixty new sequences (nineteen ITS, seventeen nLSU and twenty-four RPB2) of Fuscoporia species were newly generated ( Table 1). All sequences of ITS+nLSU+RPB2 analysis (Fig. 2) were shown in Table 1. Additional sequences of representatives genera of Hymenochaetaceae included in nLSU analysis ( Fig. 1) were downloaded from GenBank to explore the phylogenetic relationships of Fuscoporia, which were used in the previous phylogenetic study (Zhou et al. 2016;Chen et al. 2019), thus not shown in Table 1. Oxyporus corticola (Fr.) Ryvarden, Oxyporus populinus (Schumach.) Donk, and Hyphodontia pallidula (Bres.) J. Erikss. were included as outgroups in nLSU analysis based on previous studies (Zhou et al. 2016;Chen et al. 2019). The outgroups selected for ITS+nLSU+RPB2 analysis were Sequences were aligned with BioEdit (Hall 1999) and ClustalX (Thompson et al. 1997). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps were manually adjusted to optimize the alignment. Sequence alignment was deposited at TreeBase (http://purl.org/phylo/treebase; submission ID 22620). Phylogenetic analysis was carried out according to previous studies (Zhou 2015;Shen et al. 2019;Zhu et al. 2019). Maximum parsimony (MP), bayesian inference (BI) and maximum likelihood (ML) methods were employed to perform phylogenetic analysis of the two aligned datasets. MP analysis were performed using PAUP* 4.0b10 (Swofford 2002); BI was calculated with MrBayes3.1.2 (Ronquist and Huelsenbeck 2003); RAxML v.7.2.6 (Stamatakis 2006) was used for ML analysis. The three phylogenetic methods resulted in similar topologies for each dataset, and, thus, only the topology from the MP tree is presented along with statistical values from the ML/BI/MP algorithms (simultaneous MP/BI not less than 75 % and BPP not less than 0.9) at the nodes.

Results
The nLSU datasets included 23 representatives genera of Hymenochaetaceae and the combined ITS+nLSU+RPB2 datasets included 41 fungal specimens representing 20 species. In addition to sequences of new species, 14 new sequences of three species without published DNA sequences were uploaded -F. punctatiformis (Murrill)  China (BI) resulted in a similar consensus tree to that of the Maximum Parsimony (MP) and Maximum Likelihood (ML) analysis, with 1 million generations and an average standard deviation of split frequencies = 0.009570.
The three-gene dataset had an aligned length of 2950 characters, of which 1990 were constant, 90 were variable but parsimony-uninformative, and 870 were parsimony-informative. Maximum Parsimony (MP) analysis yielded 4 most parsimonious trees with near-identical topologies (TL = 2631, CI = 0.569, RI = 0.807, RC = 0.459, HI = 0.431). Bayesian (BI) resulted in a similar consensus tree to that of the Maximum Parsimony (MP) and Maximum Likelihood (ML) analysis, with 1 million generations and an average standard deviation of split frequencies = 0.005640.
Description. Basidiocarps annual, effused-reflexed to pileate, broadly attached, without taste or odor and soft corky when fresh. Pilei conchate, laterally fused, convex towards margin, projecting up to 1.5 cm, 7 cm wide and 6 mm thick at base. Pileal surface yellowish brown to dark brown, velutinate, concentrically sulcate with zoned; margin acute, yellowish brown. Pore surface yellowish brown when dry, glancing; margin distinct, yellowish, up to 2 mm wide; pores circular to angular, 5-7 per mm; dissepiments thin, entire. Context yellowish brown to dull brown, corky, up to 3 mm thick. Tubes yellowish brown, paler than context, corky, up to 3 mm long.
Additional specimens examined (paratypes   Etymology. "Ramulicola" (Latin) referring to the species growing on branches. Description. Basidiocarps annual, resupinate, effused, inseparable, without taste or odor and corky when fresh, light-weight and hard corky when dry, up to 10 cm long, 2.2 cm wide and 1 mm thick at center. Pore surface grayish brown, fawn, cracked with age; sterile margin yellowish brown to olivaceous buff, distinctly paler than tubes, up to 1 mm wide; pores more or less angular, 6-7 per mm; dissepiments thin, sometimes irregular to slightly lacerate; abundant setae seen in tube cavities (under lens). Subiculum reddish brown, corky, very thin, about 0.1 mm thick. Tubes olivaceous buff, paler contrasting with pores and subiculum, hard corky, up to 0.9 mm long.
Tubes. Generative hyphae rare, mostly present at dissepiment edges and subhymenium, hyaline, thin-walled, occasionally branched and frequently simple septate, 1.8-2.8 μm in diam, some of them at dissepiment edges and in the hymenium encrusted with small crystals; skeletal hyphae dominant, yellowish brown, thick-walled with a wide lumen, unbranched, aseptate, flexuous to more or less straight, subparallel along the tubes, 2.5-3.8 μm in diam. Irregular crystals usually present among trama and hymenia.