A Molecular Systematics and Taxonomy Research on Trechispora (Hydnodontaceae, Trechisporales): Concentrating on Three New Trechispora Species from East Asia

Trechispora are an important genus of wood-inhabiting fungi that have the ability to decompose rotten wood in the forest ecosystem. In this study, we reported three new species of Trechispora: T. murina, T. odontioidea, T. olivacea from a subtropical region of Yunnan Province, China. Species descriptions were based on a combination of morphological features and phylogenetic analyses of the ITS and LSU region of nuclear ribosomal DNA. Trechispora murina is characterized by the resupinate basidiomata, grandinioid hymenial surface with a greyish tint, monomitic hyphal system and ellipsoid, thick-walled, ornamented basidiospores; T. odontioidea has an odontioid hymenial surface with cylindrical to conical, blunt aculei and subglobose to globose, colorless, slightly thick-walled, ornamented basidiospores; T. olivacea has a farinaceous hymenial surface with olivaceous tint, basidia clavate and thick-walled, ornamented, broadly ellipsoid to globose basidiospores. Sequences of the ITS and nLSU rDNA markers of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony, and Bayesian inference methods. After a series of phylogenetic analyses, the 5.8S+nLSU dataset was constructed to test the phylogenetic relationship of Trechispora with other genera of Hydnodontaceae. The ITS dataset was used to evaluate the phylogenetic relationship of the three new species with other species of Trechispora. Using ITS phylogeny, the new species T. murina was retrieved as a sister to T. bambusicola with moderate supports; T. odontioidea formed a single lineage and then grouped with T. fimbriata and T. nivea; while T. olivacea formed a monophyletic lineage with T. farinacea, T. hondurensis, and T. mollis.

During the investigations of the corticioid fungi, Yunnan Province, China, we collected three fungal taxa, which could not be assigned to any described species within Hydnodontaceae. We present morphological and molecular phylogenetic evidence that support them as the three new species in Trechispora.

Sample Collection and Herbarium Specimen Preparation
Fresh fruiting bodies of the fungi growing on fallen angiosperm branches were collected in 2019 from the Honghe and Wenshan of Yunnan Province, China. The samples were photographed in situ and macroscopic details were recorded. Field photographs were taken by a Jianeng 80D camera (Tokyo, Japan). All photographs were focus-stacked and merged using Helicon Focus Pro 7.7.5 software. Once the macroscopic details were recorded, the specimens were transported to a field station where the specimens were dried on an electronic food dryer at 45 • C. Once dried, the specimens were labeled and sealed in envelopes and plastic bags. The dried specimens were deposited in the herbarium of the Southwest Forestry University (SWFC), Kunming, Yunnan Province, China.

Morphology
The macromorphological descriptions were based on field notes and photos captured in the field and laboratory. Color, texture, taste and odor of fruit bodies were mostly based on the authors' field trip investigations. Color terminology follows Kornerup and Wanscher [39]. All materials were examined under a Nikon 80i microscope (Nikon Corporation, Tokyo, Japan). Drawings were made with the aid of a drawing tube. The measurements and drawings were made from slide preparations stained with cotton blue (0.1 mg aniline blue dissolved in 60 g pure lactic acid), Melzer's reagent (1.5 g potassium iodide, 0.5 g crystalline iodine, 22 g chloral hydrate, aq. dest. 20 mL), and 5% potassium hydroxide. Spores were measured from the sections of the basidiomata and when presenting spore size data, 5% of the measurements excluded from each end of the range are shown in parentheses [40]. The following abbreviations were used: KOH = 5% potassium hydroxide water solution, CB = cotton clue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= both inamyloid and indextrinoid, L = means spore length (arithmetic average for all spores), W = means spore width (arithmetic average for all spores), Q = variation in the L/W ratios between the specimens studied, and n = a/b ((a) number of spores were measured in total, coming from (b) number of specimen).

Molecular Phylogeny
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain genomic DNA from the dried specimens following the manufacturer's instructions [41]. The nuclear ribosomal ITS region was amplified with the primers ITS5 and ITS4 [42]. The nuclear nLSU region was amplified with the primer pairs LR0R and LR7 (http://lutzonilab.org/nuclear-ribosomal-dna/, accessed on 7 June 2019). The PCR procedure used 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 used 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. The PCR products were purified and sequenced at Kunming Tsingke Biological Technology Limited Company (Kunming, Yunnan Province, China). All the newly generated sequences were deposited in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 November 2021) ( Table 1). T. vermiferum KHL 8714 -AY463477 [35] The sequences and alignment were adjusted manually using AliView version 1.27 [52]. The datasets were aligned with Mesquite version 3.51. The 5.8S+nLSU sequences dataset was used to know the phylogenetic relationship of the three new species in Trechispora and related genera, and the ITS dataset was used to evaluate the phylogenetic relationships of the new species with known species of the genus. Sequences of Porpomyces mucidus (Pers.) that Jülich and P. submucidus   The three combined datasets were analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI), according to Zhao and Wu [41]. Maximum parsimony analyses were constructed using PAUP* version 4.0b10 [53]. 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 bootstrap (BT) analysis with 1000 replicates [54]. 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. Multiple sequence alignment was also analyzed using ML in RAxML-HPC2 through the Cipres Science Gateway [55]. Branch support (BS) for ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 [56] was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI), which was performed using MrBayes 3.2.7a with a GTR+I+G model of DNA substitution and a gamma distribution rate variation across sites [57]. A total of 4 Markov chains were run, each consisting of 1.6 million generations, with random starting trees for 5.8S+nLSU ( Figure 1) and 1.2 million generations for ITS ( Figure 2) with trees and parameters sampled every 1000 generations. The first one-fourth of all generations were discarded as burn-in. The majority rule consensus tree of all remaining trees was calculated. Branches were considered as significantly supported if they received a maximum likelihood bootstrap value (BS) ≥ 70%, maximum parsimony bootstrap value (BT) ≥ 70%, or Bayesian posterior probabilities (BPP) ≥ 0.95.   Etymology-murina (Lat.): Referring to the furry mouse-like hymenial surface. Basidiomata-Annual, resupinate, thin, growing adnate but easily separable, up to 15 cm long, 3 cm wide, 100-500 µm thick. Hymenial surface grandinioid, pale greyish to grey when fresh, turn to greyish upon drying. Sterile margin concolorous with a hymenial surface, up to 2 mm wide.

Conflicts of Interest:
The authors declare no conflict of interest.