﻿Morphological characteristics and phylogenetic analyses revealed four new wood inhabiting fungi (Agaricomycetes, Basidiomycota) in Xizang Autonomous Region, China

﻿Abstract Four new fungi from Xizang in southwest China, Caloceraramaria, Ceraceomycesrhizomorphus, Leptosporomyceslinzhiensis, and Ramariaxizangensis are described and illustrated based on the morphological and molecular evidence. Caloceraramaria is characterized by the ramal and bright orange basidiomata, a monomitic hyphal system with simple septa generative hyphae, usually 4-septate basidiospores; Ceraceomycesrhizomorphus is characterized by the cream to yellowish basidiomata with rhizomorphs, cylindrical basidiospores; Leptosporomyceslinzhiensis is characterized by white with pink basidiomata, cylindrical to oblong ellipsoid basidiospores; Ramariaxizangensis is characterized by flesh pink basidiomata, branched dichotomously in 4–5 ranks, a monomitic hyphal system with clamped generative hyphae, ellipsoid to cylindrical and densely warted basidiospores.


Introduction
The fruiting bodies of Basidiomycota exhibit complex forms, such as gilled, poroid, toothed, coralloid basidiomata.Numerous taxonomists have endeavored to construct a stable classification system based on these characters (Gäumann 1953).Recently, the analysis of DNA sequences has emerged as a common method for deducing fungal phylogenies and enhancing higher classification frameworks through the integration of genetic traits (Cui et al. 2019;Wijayawardene et al. 2020;Liu et al. 2023).
The abundance of biodiversity in Abies forests can be attributed to the plentiful presence of humus and mycorrhizal fungi, which foster an optimal environment for the proliferation of the macrofungal species.Information regarding the fungal diversity in Abies communities is scattered over a range of publications (Ryvarden and Gilbertson 1993;Dai 2022).Ceraceomyces Jülich, a small genus characterized by yellow rhizomorphic basidiomata, was established by Jülich based on the taxon C. tessulatus (Cooke) Jülich (Jülich 1972).This genus, originally from North America, features annual, resupinate, pellicular basidiomata with a smooth or merulioid hymenial surface, a monomitic hyphal system, narrowly clavate basidia, and subglobose, narrowly ovate, ellipsoid to cylindrical basidiospores (Chikowski et al. 2016).Phylogenetic studies revealed that Ceraceomyces was polyphyletic, comprising three distinct groups.The section of Corticium tessulatum Cooke belonged to Polyporales, and Ceraceomyces serpens (Tode) Ginns and C. eludens K.H. Larss.were part of phlebioid clade (Larsson et al. 2004).A recent study indicated that the type species, Corticium tessulatum is classified under the order Amylocorticiales (Binder et al. 2010;Chikowski et al. 2016), as well as species, C. yunnanensis Qi Yuan & C.L. Zhao and C. borealis (Romell) J. Erikss.& Ryvarden (Yuan et al. 2023).Currently, eleven species are recognized in the genus Ceraceomyces, including C. cystidiatus (J.Erikss.& Hjortstam) Hjortstam, C. eludens, C. microsporus K.H. Larss.and C. sublaevis (Bres.)Jülich were accepted in the genus.A genus, Crystallicutis El-Gharabawy, Leal-Dutra & G.W. Griff.was derived from Ceraceomyces based on the crystals in the hymenium and subiculum of the basidiomata, which includes the brown-rot species C. serpens (El-Gharabawy et al. 2021).Both species C. sulphurinus and C. violascens (Fr.)Jscens were recorded in Ceraceomyces, are considered congeneric with Rhizochaete Gresl., Nakasone & Rajchenb.due to the characteristics like the rhizomorphic margin and the purple reaction in KOH.
The genus Leptosporomyces Jülich is characterized by the resupinate basidiomata, white yellow and smooth hymenial surface, a monomitic hyphal system with clamped connections, and thin-wall, smooth, acyanophilous basidiospores.Recent research has indicated that Leptosporomyces was polyphyletic, with two taxa, L. galzinii (Bourdot) Jülich and L. raunkiaeri (M.P. Christ.)Jülich, grouped in the order Atheliales, while L. septentrionalis (J.Erikss.)Krieglst.was placed in the order Amylocorticiales (Larsson 2007;Hodkinson et al. 2014;Sulistyo et al. 2021).The generic delimitation of Fibulomyces Jülich and Leptosporomyces remains controversial, with both being indistinguishable in phylogenetic and morphological analyses, leading to the former being considered as a synonym of the latter (Bernicchia and Gorjón 2010).
In the present paper, species from four genera are collected from Xizang under forest of Abies, and the phylogenetic relationships of four taxa are still unclear.Thus, to explore the diversity and taxonomic status with different characters for those taxa will be significant for macrofungi in Xizang, and the taxonomy and phylogeny analysis show that they are new to science.

Material and method
The specimens were collected from Xizang which were deposited in the herbarium of the Southwest Forestry University (SWFC), Kunming, Yunnan Province, China.Samples were photographed when fresh in the field, and their habitats were recorded.Microscopic structures were discussed by Zhao et al. (2023).Special color terms were set by Anonymous (1969) and Petersen (1996).A Nikon Digital Sight DS-L3 or Leica ICC50 HD camera (magnification ×1,000) was used to exam hand-cut sections of basidiomata, which were first treated with 5% KOH for a few minutes and then with 1% phloxine B (C 20 H 4 Br 4 Cl 2 K 2 O 5 ).At least 30 basidiospores of each species were examined.The values were expressed as a mean with 5% of the measurements excluded from each end of the range, given in parentheses.Stalks were excluded for basidia measurement, and the hilar appendages were excluded for basidiospore measurement.

DNA extraction, amplification, and sequencing
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing) was used to obtain DNA from dried specimens and PCR was performed according to the manufacturer's instructions with some modifications (Yang et al. 2023).ITS were amplified using the primer pairs ITS5/ITS4 (White et al. 1990).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, 54 °C for 45 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min.The PCR procedure for LSU was as follows: initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 1.5 min; and a final extension at 72 °C for 10 min.All newly generated sequences were submitted to GenBank and are listed in Table 1.
Sequences generated for this study were aligned, with additional sequences downloaded from GenBank.Sequences were aligned using MAFFT v.7 (https:// mafft.cbrc.jp/alignment/server/),adjusting the direction of nucleotide sequences according to the first sequence (accurate enough for most cases), and selecting the G-INS-i iterative refinement method (Katoh et al. 2019).Alignments were manually adjusted to maximize alignment and minimize gaps with BioEdit v.7.0.9 (Hall 1999).A dataset of concatenated ITS and LSU sequences was used to determine the phylogenetic position of the new species.Maximum likelihood (ML) analysis was performed using the CIPRES Science Gateway (Miller et al. 2010) based on the dataset using the RA × ML-HPC BlackBox tool, with setting RA × ML halt bootstrapping automatically and 0.25 for maximum hours and obtaining the best tree using ML search.Other parameters in ML analysis used default settings, and statistical support values were obtained using nonparametric bootstrapping with 1,000 replicates.Bayesian inference (BI) analysis based on the dataset was performed using MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2012).The best substitution model for the dataset was selected by ModelFinder (Kalyaanamoorthy et al. 2017) using a Bayesian information criterion, and the model was used for Bayesian analysis.Four Markov chains were run from random starting trees.
Trees were sampled every 1,000 th generation.The first 25% of sampled trees were discarded as burn-in, whereas other trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).The aligned sequences were deposited in TreeBase (https://www.treebase.org/treebase-web/home.html;submission ID 31437).
Branches of the consensus tree that received bootstrap support for ML were greater than or equal to 75%, Bayesian posterior probabilities more than 0.9, respectively.

Result
The Phylogeny of Calocera BI analysis yielded a similar topology to MP and ML analysis.Only the MP tree is provided here (Fig. 1).Branches that received bootstrap support for ML (ML-BS), and BI (BPP) greater than or equal to 75% (MP-BS and ML-BS) and 0.90 (BPP) were considered as significantly supported, respectively.The ITS and LSU dataset contains sequences from 26 fungal specimens representing twelve Calocera taxa.The average SD of split frequencies in BI analyses is 0.005504 (BI).The phylogenetic tree (Fig. 1) reveals the new species has close relationship with C. tibatica, sister to C. viscosa and C. cornea.

The Phylogeny of Ceraceomyces
The dataset included ITS and LSU from 29 samples representing 22 taxa.The best model for the concatenated ITS+LSU dataset estimated and applied for BI analysis was "GTR+I+G4", datatype = DNA, nucmodel = 4by4, lset nst = 6, rates = invgamma; state frequencies had a Dirichlet prior (1,1,1,1), and the distribution was approximated using four categories.BI analysis yielded a similar topology to ML analysis, with an average standard deviation of split frequencies of 0.006593.The ML tree was provided (Fig. 2).Branches that received bootstrap support for ML and BI ≥ 70%, and 0.75 were considered significantly supported, respectively.
The analysis reveals four clades (Fig. 2

The Phylogeny of Leptosporomyces
BI analysis yielded a similar topology to MP and ML analysis, with an average standard deviation of split frequencies = 0.008841.Only the MP tree is provided here (Fig. 3).Branches that received bootstrap support for ML (ML-BS), and BI (BPP) greater than or equal to 75% (MP-BS and ML-BS) and 0.90 (BPP) were considered as significantly supported, respectively.Four previously accepted species, L. galzinii, L. fuscostratus (Jülich) Krieglst., L. raunkiaeri, and L. mundus (H.S. Jacks.& Dearden) Jülich received strong support in three lineages.The new species L. linzhiensis had a close relationship with L. septentrionalis with full support.The Phylogeny of Ramaria BI analysis yielded a similar topology to MP and ML analysis.Only the MP tree is provided here (Fig. 4).Branches that received bootstrap support for ML (ML-BS), and BI (BPP) greater than or equal to 75% (MP-BS and ML-BS) and 0.90 (BPP) were considered as significantly supported, respectively.Four clades were obtained from our phylogenetic analysis, Ramaria sub.Laeticolora, Ramaria Sub.Ramaria, Ramaria Sub.Echinormaria and Ramaria sub.Holotype.China, Xizang, Linzhi, Sejila Mountain National Forest Park, 29°64'N, 94°71'E, elev.3852 m, gregarious on humus under Abies, 2 August 2023, CLZhao 31166 (SWFC).
Fruiting body.Basidiomata resupinate, athelioid, membranous upon drying, without odor or taste when fresh, up to 10 cm long, 4 cm wide, 200 µm thick.Hymenial surface smooth to cracked, white with pink tint when fresh, turning to yellowish cream upon drying.Margin sterile, white, fimbriate.
Fruiting body.Basidiomata solitary to gregarious, with 8 cm high × 6 cm wide at the widest point, repeat branched dichotomously in 4-5 ranks, flesh pink when fresh, become clay buff with dry; apices obtuse, orange yellow when fresh, becoming fuscous when dry. Stipe ≥ 3 cm high, compound to fasciculate in groups of 5, emerging from a common base, concolorous with the branches.

Discussion
Wood decay fungi encompasses the vast group of aphyllophoroid fungi with corticioid, prioid or jelly form of basidiomata (Herter 1910).This classification has historically been used to define the different families of Basidiomycetes.However, molecular studies have revealed that many of these fungi are distributed across various orders within the Basidiomycetes, including the likes of Amylocorticiales, Atheliales, Dacrymycetales, and Gomphales (Kirk et al. 2018;Wei et al. 2022).As a result, further research is needed to elucidate the relationships and morphological variability of these taxa through phylogenetic analysis.
The Xizang Autonomous Region, situated in the southwest of China, is renowned as one of the most bio-diverse regions in the country.This is attributed to its complex topography and diverse ecosystems, making it a focal point for fungal biodiversity in China.Recently, studies focusing on fungal diversity and the ecology of Basidiomycota in Xizang were carried out (Ke 2016;Pubu et al. 2016;Wang et al. 2023).According to the study (Pubu et al. 2016), 1733 species were collected in Xizang.The fungal research indicated that Sejila Mountain National Forest Park is predominantly composed of spruce and fir trees, which provide an ideal habitat for a rich diversity of macrofungi species to flourish (Zhao and Li 1987).In our study, four species were found from Xizang, Calocera ramaria, Ceraceomyces rhizomorphus, Leptosporomyces linzhiensis, and Ramaria xizangensis.
According to our field inventory, the four Chinese new species were found in alpine zone near the Sejila Mountain, and the coniferous forest dominant by Abies at high altitude with cold and humid environments.Previously, numerous new species have been found in Southwest China (Dai 2022; Zhao et al. 2023), and the present paper confirms the fungal diversity is very rich in the montane forests of Southeast Xizang.

Figure 1 . 2 MH856777MW549778
Figure 1.Phylogeny of species in Calocera generated by maximum likelihood based on ITS+LSU sequence data.Branches are labeled with maximum likelihood bootstrap ≥ 75% and Bayesian posterior probabilities ≥ 0.90, respectively.New species are in bold.0.2

Figure
Figure 2. Phylogeny of species in Ceraceomyces generated by maximum likelihood based on ITS+LSU sequence data.Branches are labeled with maximum likelihood bootstrap ≥ 75% and Bayesian posterior probabilities ≥ 0.90, respectively.New species are in bold.

Table 1 .
Taxa information and the sequences used in this study.*Newly generated sequences for this study.
Figure 3. Phylogeny of species in Leptosporomyces generated by maximum likelihood based on ITS+LSU sequence data.Branches are labeled with maximum likelihood bootstrap ≥ 75% and Bayesian posterior probabilities ≥ 0.90, respectively.New species are in bold.