﻿Morphological and molecular identification for four new wood-inhabiting species of Trechispora (Basidiomycota) from China

﻿Abstract Four new wood-inhabiting fungi, Trechisporaalbofarinosa, T.bisterigmata, T.pileata and T.wenshanensisspp. nov., are proposed based on a combination of morphological features and molecular evidence. Trechisporaalbofarinosa is characterized by the farinose basidiomata with flocculence hymenial surface, a monomitic hyphal system with clamped generative hyphae, and ellipsoid, warted basidiospores. Trechisporabisterigmata is characterized by the membranous basidiomata with odontioid hymenial surface, rhizomorphic sterile margin, barrelled basidia and subglobose to broad ellipsoid, smooth basidiospores. Trechisporapileata is characterized by the laterally contracted base, solitary or imbricate basidiomata, fan shaped pileus, radially striate-covered surface with appressed scales, odontioid hymenophore surface, and subglobose to broad ellipsoid, thin-walled, smooth basidiospores. Trechisporawenshanensis is characterized by a cottony basidiomata with a smooth hymenial surface, and ellipsoid, thin-walled, warted basidiospores. Sequences of ITS and LSU marker of the studied samples were generated, and phylogenetic analyses were performed with the maximum likelihood, maximum parsimony, and Bayesian inference methods. The phylogenetic tree inferred from the ITS+nLSU sequences highlighted that four new species were grouped into the genus Trechispora.

There have been many studies on the phylogeny of this genus in recent years.A high phylogenetic diversity on the corticioid Agaricomycetes based on two genes, 5.8S and 28S showed that nine taxa of Trechispora nested into trechisporoid clade (Larsson et al. 2004).The molecular systematics suggested that Trechispora belonged to Hydnodontaceae and was related to genera Brevicellicium K.H. Larss.& Hjortstam, Porpomyces Jülich, Sistotremastrum J. Erikss., and Subulicystidium Parmasto (Telleria et al. 2013).Based on the ITS and nLSU datasets, the phylogenetic study of Trechispora reported two new Trechispora species as T. cyatheae Ordynets, Langer & K.H. Larss. and T. echinocristallina Ordynets, Langer & K.H. Larss., on La Réunion Island (Ordynets et al. 2015).The phylogeny of Trechisporales was inferred from a combined ITS-nLSU sequences, which revealed that two related genera Porpomyces, Scytinopogon Singer, grouped closely together with Trechispora and all of them nested within Hydnodontaceae (Liu et al. 2019).Based on ITS dataset, the three new species of Trechispora were described and used to evaluate the phylogenetic relationship with other species of this genus, in which T. murina was retrieved as a sister to T. bambusicola with moderate supports, and 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 (Luo and Zhao 2022).Recently, based on the morphological features and molecular evidence, three new species of Trechispora have been reported from Northern and Northeastern Thailand (Sommai et al. 2023).
During investigations into the wood-inhabiting fungi in the Yunnan-Guizhou Plateau of China, samples representing four additional species belonging to genus Trechispora were collected.To clarify the placement and relationships of the four species, we carried out a phylogenetic and taxonomic study on Trechispora, based on the ITS+nLSU.

Morphology
The specimens studied were deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, China.The macromorphological descriptions were based on field notes and photos captured in the field and laboratory.Color, texture, taste and odor of basidiomata were mostly based on authors' field trips.Color terminology followed Kornerup and Wanscher (1978).All materials were examined under a Nikon 80i microscope.Drawings were made with the aid of a drawing tube.The measurements and drawings of the microscopic structures were made (Wu et al. 2022).The following abbreviations were used: KOH = 5% potassium hydroxide water solution, CB = cotton blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= both inamyloid and indextrinoid, L = spore length (arithmetic average for all spores), W = spore width (arithmetic average for all spores), Q = L/W ratios of the specimens studied, and n = a/b (a = total number of spores measured, from b = number of specimens).

Molecular phylogeny
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from the dried specimens following the manufacturer's instructions (Zhao and Wu 2017).The nuclear ribosomal ITS region was amplified with the primers ITS5 and ITS4 (White et al. 1990).The nuclear ribosomal LSU gene was amplified with the primers LR0R and LR7 (Vilgalys and Hester 1990;Rehner and Samuels 1994).The 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.The PCR products were purified and directly sequenced at Kunming Tsingke Biological Technology Limited Company, Yunnan Province, China.All newly-generated sequences were deposited in NCBI GenBank (Table 1).
The sequences were aligned in MAFFT version 7 (Katoh et al. 2019) using the G-INS-i strategy.The alignment was adjusted manually using AliView version 1.27 (Larsson 2014).Each dataset was aligned separately at first and then the ITS+nLSU regions were combined with Mesquite version 3.51.The combined dataset was deposited in TreeBASE (submission ID 31349).Sequences of Fibrodontia alba Yurchenko & Sheng H. Wu and F. brevidens (Pat.)Hjortstam & Ryvarden retrieved from GenBank were used as an outgroup in the ITS analysis (Luo and Zhao 2022).
Maximum parsimony analysis in PAUP* version 4.0a169 (http://phylosolutions.com/paup-test/) was applied to ITS+nLSU following a previous study (Zhao and Wu 2017).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 1,000 random sequence additions.Max-trees were set to 5,000, branches of zero length were collapsed and all parsimonious trees were saved.Clade robustness was assessed using bootstrap (BT) analysis with 1,000 pseudo replicates (Felsenstein 1985).Descriptive tree statistics -tree length (TL), composite consistency index (CI), composite retention index (RI), composite rescaled consistency index (RC) and composite homoplasy index (HI) -were calculated for each maximum parsimonious tree generated.The combined dataset was also analysed using Maximum Likelihood (ML) in RAxML-HPC2 through the CIPRES Science Gateway (Miller et al. 2012).Branch support (BS) for the ML analysis was determined by 1000 bootstrap pseudoreplicates.MrModeltest 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each dataset for the purposes of Bayesian inference (BI), Bayesian inference was performed using MrBayes 3.2.7awith a GTR+I+G model of DNA substitution and a gamma distribution rate variation across sites (Ronquist et al. 2012).A total of four Markov chains were run for two runs from random starting trees for 1.7 million generations for ITS+nLSU with tree and parameters sampled every 1,000 generations.The first quarter of all of the generations were discarded as burn-ins.A majority rule consensus tree was computed from the remaining trees.Branches were considered as significantly supported if they received a maximum likelihood bootstrap support value (BS) of > 70%, a maximum parsimony bootstrap support value (BT) of > 70% or a Bayesian posterior probability (BPP) of > 0.95.

Molecular phylogeny
The ITS+nLSU dataset comprised sequences from 88 fungal specimens representing 64 taxa.The dataset had an aligned length of 2271 characters, of which 1376 characters were constant, 190 were variable and parsimony-uninformative and 705 were parsimony-informative.Maximum parsimony analysis yielded 300 equally parsimonious tree (TL = 5543, CI = 0.2979, HI = 0.7021, RI = 0.5278 and RC = 0.1572).The best model of nucleotide evolution for the ITS+nLSU dataset estimated and applied in the Bayesian analysis was found to be GTR+I+G.Bayesian analysis and ML analysis resulted in a similar topology as in the MP analysis.The Bayesian analysis had an average standard deviation of split frequencies = 0.012925 (BI) and the effective sample size (ESS) across the two runs is double the average ESS (avg.ESS) = 389.The phylogenetic tree inferred from the ITS+nLSU sequences highlighted that four new species were grouped into the genus Trechispora (Fig. 1).Etymology.Albofarinosa (Lat.):referring to the farinose basidiomata with white hymenial surface.Description.Basidiomata annual, resupinate, farinose, without odor or taste when fresh, up to 3.5 cm long, 1.5 cm wide, and 300-500 µm thick.Hymenial surface flocculence, white when fresh, white to cream on drying.Sterile margin indistinct, white, and up to 0.5 mm wide.

Discussion
Many recently described wood-inhabiting fungal taxa have been reported in the subtropics and tropics, including in the genus Trechispora (Ordynets et al. 2015;Phookamsak et al. 2019;Xu et al. 2019;Chikowski et al. 2020;Haelewaters et al. 2020;Crous et al. 2021;de Meiras-Ottoni et al. 2021;Zhao and Zhao 2021;Liu et al. 2022a, b;Luo and Zhao 2022;Deng et al. 2023;Sommai et al. 2023).The present study reports four new species in Trechispora, based on a combination of morphological features and molecular evidence.

Figure 1 .
Figure 1.Maximum parsimony strict consensus tree illustrating the phylogeny of the four new species and related species in Trechispora, based on ITS+nLSU sequences.Branches are labelled with maximum likelihood bootstrap values > 70%, parsimony bootstrap values > 50% and Bayesian posterior probabilities > 0.95, respectively.

Table 1 .
List of species, specimens and GenBank accession numbers of sequences used in this study.