Three new species of Junghuhnia (Polyporales, Basidiomycota) from China

Abstract In this study, taxonomic and phylogenetic analyses of Junghuhnia were performed. Three new species were characterised according to morphological characteristics and molecular phylogenetic analysis using ITS and nLSU sequences. They are J. austrosinensissp. nov., J. nandinaesp. nov. and J. subcollabenssp. nov.Junghuhnia austrosinensis is characterised by resupinate, thin basidiomata with white to buff-yellow hymenophore, small pores (9–11 per mm), clamped generative hyphae possessing hymenial cystidia, ellipsoid basidiospores (2.5–3 × 1.7–2 µm) and growth on fallen bamboo or angiosperm branch. Junghuhnia nandinae is characterised by resupinate basidiomata with pink to salmon pores and a distinct white margin, clamp generative hyphae, interwoven tramal hyphae, ellipsoid basidiospores measuring 2.6–3.2 × 1.8–2 µm and growth on Nandina domestica. Junghuhnia subcollabens is characterised by resupinate basidiomata with pale salmon to brownish vinaceous hymenophore, small pores (10–12 per mm), generative hyphae with simple septa and clamp connections, interwoven tramal hyphae, lunate basidiospores measuring 2.9–3.4 × 1.6–1.8 µm and thriving on rotten wood of angiosperms.

During recent studies on wood-inhabiting fungi in China, samples morphologically belonging to Junghuhnia were collected. After microscopic examination and phylogenetic analysis of ITS and nLSU sequences, we identified three new lineages in Junghuhnia and they are different from the existing fungal taxa. Therefore, three novel Junghuhnia species are characterised.

Morphology
The samples were evaluated and submitted at the Institute of Microbiology herbaria of BJFC (Beijing Forestry University) and IFP (Institute of Applied Ecology, Chinese Academy of Sciences). The field notes formed the basis of macro-morphological details. Microscopic examination (magnifications ≤ 1000×; Nikon Eclipse 80i microscope) of the sections in phase contrast illumination was undertaken as per the protocols by Dai (2010) and Cui et al. (2019). A drawing tube was used to prepare the drawings. The sections were stained using Melzer's reagent and Cotton Blue to carry out measurements, assess microscopic features and prepare drawings. Sections from the tubes were used to assess the spores. To show the variation in spore sizes, from both ends of the range, 5% of measurements were excluded and are mentioned in parentheses. Abbreviations include KOH, potassium hydroxide (5%); IKI-, Melzer's reagent negative; IKI, Melzer's reagent; CB+, cyanophilous in Cotton blue; Q, the L/W ratio; W, mean spore width and L, mean spore length (both L and W: arithmetic average of all spores); n = number of spores in a specified number of specimens. The terms used for special colour are as per Rayner (1970) and Petersen (1996).

Molecular phylogenetic study
Genomic DNA was isolated from the dried specimens using the CTAB rapid plant genome extraction kit from Aidlab Biotechnologies (Beijing, China), as per provided guidelines with few alterations. The ITS5 and ITS4 primers were used (White et al. 1990) for the amplification of ITS sequences through PCR and the LR0R and LR7 primers were used for nLSU (Vilgalys and Hester 1990). The PCR process for ITS was: 95 °C for 3 min for initial denaturation; 35 cycles for 40 sec at 94 °C, 45 sec at 54 °C, 1 min at 72 °C, 72 °C for 10 min (final extension). The PCR process for nLSU was: 94 °C for 1 min for initial denaturation, 35 cycles for 1 min at 94 °C, 1 min at 50 °C, 1.5 min at 72 °C and 72 °C for 10 min (final extension). After purification of the products from PCR, they were sequenced at Beijing Genomics Institute (China) using the same set of primers.
Phylogenetic analyses were applied to the combined ITS+nLSU dataset. Sequences generated in this study were aligned with additional sequences downloaded from GenBank (Table 1) referred to Miettinen et al. (2012) and Yuan et al. (2019). The alignment of the dataset with Exidiopsis calcea (Pers.) K. Wells, as the outgroup following Yuan et al. (2016), was done applying MAFFT 7 with the option of G-INS-i (Katoh and Standley 2013) and the outcome was deposited at TreeBase (submission ID 25589). Construction of the ML (Maximum Likelihood) tree was done applying raxmlGUI 1.2 (Stamatakis 2006;Silvestro and Michalak 2012) with the model GTR + I + G and the option of auto FC (Pattengale 2010) in BS (bootstrap) replicates. The determination of the best-fit evolution model was done using MrModeltest2.3 (Posada and Crandall 1998;Nylander 2004) for the combined dataset for estimating BI (Bayesian Inference), which was estimated using MrBayes3.2.5 (Ronquist et al. 2012). From random starting trees, two runs of four Markov chains were run for the combined datasets for 1 million generations and, every 100 generations, trees were sampled. The initial generations (one-fourth) were rejected as burn-in. Then, for all remaining trees, the majority rule consensus tree was calculated. Branches were considered as significantly supported if they received bootstrap support (BS) for Bayesian posterior probabilities (BPP) and Maximum Likelihood ≥ 0.95 (BPP) and 75% (BS), respectively.

Phylogenetic analysis
The dataset included 54 fungal collections representing 48 species. The best model for the dataset estimated and applied in the BI was GTR+I+G. BI resulted in a similar topology with an average standard deviation of split frequencies = 0.006554 to ML analysis, and thus only the BI tree was provided. Both BPPs (≥ 0.95) and BS values (≥ 50 %) are mentioned at the nodes (Fig. 1). The three new species formed three independent lineages with robust support (BS, 100%; BPP, 1.00). Etymology. Refers to the species being collected in the south of China. Basidiomata. Annual, resupinate, soft corky, without odour or taste when fresh, corky when dried, 7 cm length, 4 cm width and 0.4 mm thick at centre. Pore surface white when fresh, cream to buff-yellow when dried; margin distinct, white and nearly 1 mm width; pores round to angular, 9-11 per mm; dissepiments thin, entire. Subiculum cream, paler than tubes, corky when dried, nearly 0.1 mm thick. Tubes concolorous with pore surface, corky, nearly 0.3 mm length.

Discussion
Junghuhnia, Antrodiella and Steccherinum are phylogenetically related and they belong to the family of Steccherinaceae Parmasto in Polyporales (Yuan 2014;Miettinen and Ryvarden 2016;Justo et al. 2017). Our phylogeny also shows similar relationships amongst the species in the three genera (Fig. 1). Morphologically, Junghuhnia is distinguished from the other two genera by its poroid hymenophore and skeletocystidia. Based on phylogenetic analyses, several genera of wood-inhabiting fungi include species with lamellate, poroid and hydnaceous hymenophore at the same time (He and Dai 2012;Cui et al. 2019), but we still keep the traditional concepts for the three genera because their limited taxa were analysed according to morphology and phylogeny.
Three new species of Junghuhnia are described from Southern China in the present paper. Although extensive surveys on wood-decaying fungi in Southern China were carried out, and more than 3000 specimens were collected with 132 new polypore (Dai 2010;Zhao et al. 2015;Chen et al. 2020;Wu et al. 2020), it is expected that more new taxa will be found after additional investigations based on careful morphological examinations and phylogenetic analyses because of the rich woody plant species in subtropical and tropical China.