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The Phylogenetic Relationship Revealed Three New Wood-Inhabiting Fungal Species From Genus Trechispora

Wood-inhabiting fungi play a significant role in wood degradation and the cycle of matter in the ecological system. In the present study, three new wood-inhabiting fungal species, Trechispora bambusicola, Trechispora fimbriata, and Trechispora fissurata spp. nov., are nested in Trechispora, which are proposed based on a combination of morphological features and molecular evidence. Sequences of internal transcribed spacer (ITS) and large subunit (nLSU) regions of the studied samples were generated, and the phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic analyses inferred from ITS showed that T. bambusicola was sister to Trechispora stevensonii, T. fimbriata grouped with Trechispora nivea, and T. fissurata grouped with Trechispora echinospora. The phylogenetic tree based on ITS + nLSU sequences demonstrated that T. bambusicola formed a single lineage and then grouped with Trechispora rigida and T. stevensonii. T. fimbriata was sister to T. nivea. T. fissurata grouped with Trechispora thelephora.

During the studies on wood-inhabiting fungi in southern China, three species of Trechispora could not be assigned to any described species. Obtaining sequences from the new taxa, the authors examine taxonomy and phylogeny of three new species within the genus Trechispora, based on the ITS and nLSU sequences.

Morphology
The studied specimens are deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, China. Macromorphological descriptions were based on field notes. Color terms follow Petersen (1996). Micromorphological data were obtained from the dried specimens and observed under a light microscope following Dai (2012). The following abbreviations were used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB− = acyanophilous, IKI = Melzer's reagent, IKI− = both inamyloid and indextrinoid, L = mean spore length (arithmetic average for all spores), W = mean spore width (arithmetic average for all spores), Q = variation in the L/W ratios between the studied specimens, n (a/b) = number of spores (a) measured from given number (b) of specimens, spore measurements do not include ornamentation.

Molecular Phylogeny
Cetyltrimethylammonium bromide (CTAB) rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain genomic deoxyribonucleic acid (DNA) from dried specimens, according to the manufacturer's instructions following Zhao and Wu (2017). ITS region was amplified with primer pair ITS5 and ITS4 (White et al., 1990). Nuclear LSU region was amplified with primer pair LR0R and LR7 3 . The polymerase chain reaction (PCR) procedures for ITS and nLSU following Zhao and Wu (2017). The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company, Kunming, Yunnan Province, China. All newly generated sequences were deposited at GenBank (Table 1).
Sequencher 4.6 (GeneCodes, Ann Arbor, United States) was used to edit the DNA sequence. Sequences were aligned in MAFFT 7 4 using the "G-INS-I" strategy and manually adjusted in BioEdit (Hall, 1999). The sequence alignment was deposited in TreeBase (submission ID 25879). Sequences of Fibrodontia alba Yurchenko and Sheng H. Wu and Fibrodontia gossypina Parmasto retrieved from GenBank were used as an outgroup in the ITS + nLSU analyses by following Ordynets et al. (2015).
Maximum parsimony (MP) analyses were applied to the ITS + nLSU dataset sequences. Approaches to phylogenetic analysis followed Zhao and Wu (2017), and the tree construction procedure was performed 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 reconnection (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 (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. Datamatrix was also analyzed using maximum likelihood (ML) approach with RAxML-HPC2 through the Cipres Science Gateway with GTR + I + G molecular evolution model 5 (Miller et al., 2009). Branch support (BS) for ML analysis was determined by 1000 BT replicates. MrModeltest 2.3 (Nylander, 2004) was used to determine the best-fit evolution model (GTR + I + G) for each data set for Bayesian inference (BI) of the phylogeny. BI was calculated with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Four Markov chains were run for two runs from random starting trees for 1 million generations and trees were sampled every 100 generations; the first one-fourth of generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received ML BT values > 75%, MP BT values > 75%, or Bayesian posterior probabilities (PP) > 0.95.

Molecular Phylogeny
In the ITS dataset, the sequences from 43 fungal specimens representing 25 species were included. The dataset had an aligned length of 1034 characters, of which 521 characters  1,1,1,1). Bayesian analysis and ML analysis resulted in a similar topology to MP analysis, with an average standard deviation of split frequencies = 0.009985 (BI).
In the ITS + nLSU dataset, it included sequences from 49 fungal specimens representing 27 species. The dataset had an aligned length of 2256 characters, of which 1387 characters are constant, 188 are variable and parsimony-uninformative, and 681 are parsimony-informative. MP analysis yielded 100 equally parsimonious trees (TL = 2811, CI = 0.4963, HI = 0.5037, RI = 0.6409, RC = 0.3180). Best model for the ITS dataset estimated and applied in the Bayesian analysis: GTR + I + G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). Bayesian analysis and ML analysis resulted in a similar topology to MP analysis, with an average standard deviation of split frequencies = 0.009991 (BI).

Basidiomata
Annual, adnate, soft, and fragile, without odor or taste when fresh, becoming granulose upon drying, up to 15 cm long and 5 cm wide, 50-300 µm thick. Hymenial surface odontioid, aculei cylindrical to conical, blunt, 0.3-0.5 mm long, white to cream when fresh, turn to cream to buff upon drying. Margin white to cream.

Basidiomata
Annual, adnate, without odor or taste when fresh, becoming fragile upon drying, up to 10 cm long and 3 cm wide, 100-200 µm thick. Hymenial surface hydnoid, with aculei, cylindrical, blunt, 0.4-0.7 mm long, white to pink when fresh, turn to pink to buff upon drying. Margin white to cream, thinning out, fimbriate.

Type of rot
White rot.

Basidiomata
Annual, adnate, without odor or taste when fresh, becoming cracking upon drying, up to 8 cm long and 4.5 cm wide, 400-800 µm thick. Hymenial surface hydnoid, with aculei, cylindrical to conical, sharp, 0.5-0.9 mm long, cream to straw yellow when fresh, turn to cream to yellow upon drying. Margin cream to yellow.

Type of rot
White rot.
In the present study, three new species, T. bambusicola, T. fimbriata, and T. fissurata spp. nov. are found from rotten wood. Morphologically, T. bambusicola is similar to T. cyatheae Ordynets, Langer and K.H. Larss. by sharing the characteristics of soft and fragile basidiomata. However, T. cyatheae differs from T. bambusicola by having farinaceous to grandinioid hymenophore and thin-walled generative hyphae (Ordynets et al., 2015).
Currently, eight species of Trechispora have been reported from China (Dai, 2011;Xu et al., 2019), Trechispora alnicola, Trechispora cohaerens, T. farinacea, T. microspora, T. nivea, Trechispora polygonospora Ryvarden, Trechispora subsphaerospora (Litsch.) Liberta, and T. yunnanensis, and one species of T. yunnanensis was found in Yunnan Province of China and it differs from three new species by having a smooth to farinaceous hymenial surface and larger basidiospores (7-8.5 × 5-5.5 µm; Xu et al., 2019). Three new taxa do not closely group together in phylogenetic trees, and morphologically, T. bambusicola differs from T. fimbriata and T. fissurata by having granulose basidiomata with cream to buff hymenial surface and growth on dead bamboo. T. fimbriata differs in its fimbriate margin of the basidiomata with pink to buff hymenial surface.
In addition, the ectomycorrhizal fungi (EcM) play an important role in ecosystems based on their mutualistic association with many groups of plants (Heijden et al., 2015). Vanegas-León et al. (2019) discovered the Trechisporales basidiomes and root colonization from T. thelephora basidiome. In the present study, T. fissurata was sister to T. thelephora based on ITS + nLSU phylogenetic analysis (Figure 2), which implied that both species have close evolutionary relationship. However, T. fissurata grows on deeply decayed wood, and T. thelephora is a soil-inhabiting fungus. Therefore, future investigations in both inhabiting types are needed to determine whether the natural selection or other factors pushes the different direction on inhabiting soil/wood among Trechispora.
In the habitat and distribution, Hibbett et al. (2014) revealed that most species of Trechispora is considered as soil-inhabiting. Later, some species were found on deeply decayed wood fungi (Bernicchia and Gorjón, 2010;Dai, 2011). However, some species in Trechispora are a typical feature of ectomycorrhizal fungi as frequently forming basidiomes on soil (Dunham et al., 2007;Vanegas-León et al., 2019). In the neotropical and subtropical region, the ectomycorrhizal basidiomes are found; however, the researches on the new taxa related to wood-decaying fungi of Trechispora from China are poorly reported. Further studies may focus on the relationships between the plants and species from Trechispora and try to better understand the evolutionary directions between soil-inhabiting and decayed wood fungi of Trechispora; many fungal studies on phylogeny and application were from these areas, which will be useful to push future researches for the genus Trechispora (Dai, 2011;Cui et al., 2019;Shen et al., 2019;Zhu et al., 2019;Richter et al., 2019;Angelini et al., 2020;Bao et al., 2020).

DISCLOSURE
All the experiments undertaken in this study comply with the current laws of the People's Republic of China.

AUTHOR CONTRIBUTIONS
C-LZ collected the species. WZ performed the molecular phylogenetic analyses. Both authors were responsible for the morphological analysis and description of the collections, planned, organized, and evaluated critically the experimental parts, wrote the manuscript, contributed to the article, and approved the submitted version.