Four New Species of Tomentella (Thelephorales, Basidiomycota) from Subtropical Forests in Southwestern China

Species of the basidiomycetous genus Tomentella are widely distributed throughout temperate forests. Numerous studies on the taxonomy and phylogeny of Tomentella have been conducted from the temperate zone in the Northern hemisphere, but few have been from subtropical forests. In this study, four new species, T. casiae, T. guiyangensis, T. olivaceomarginata and T. rotundata from the subtropical mixed forests of Southwestern China, are described and illustrated based on morphological characteristics and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the large subunit of the nuclear ribosomal RNA gene (LSU). Molecular analyses using Maximum Likelihood and Bayesian analysis confirmed the phylogenetic positions of these four new species. Anatomical comparisons among the closely related species in phylogenetic and morphological features are discussed. Four new species could be distinguished by the characteristics of basidiocarps, the color of the hymenophoral surface, the size of the basidia, the shape of the basidiospores and some other features.

Ectomycorrhizal fungi (EMF) play a significant role in recycling nutrients, digesting plant and insect remnants and maintaining biodiversity in natural ecosystems.EMF produce obviously different types of basidiocarps, which are conspicuous or inconspicuous basidiocarps.The genus Tomentella is a widely distributed ectomycorrhizal lineage [9,10] and can form ectomycorrhiza with different host tree families, e.g., Asteropeiaceae, Dipterocarpaceae, Fabaceae, Gnetaceae, Pinaceae, Proteaceae, Sapotaceae, Sarcolaenaceae and Phyllanthaceae [2,[11][12][13][14][15][16][17][18][19][20].Since the first finding of an ectomycorrhiza formed by a Tomentella species [11], many studies have confirmed the Tomentella-Thelephora to play an important role in receiving energy and transporting nutrients to their host plants [11][12][13][21][22][23][24][25][26][27].Tomentella play a protective and promoting role for growth and development in extreme environments [28].In addition, the Tomentella-Thelephora lineage reading from the mycorrhizal root tips of Betulaceae, Fagaceae, Pinaceae and Tiliaceae account for 38.2% of the total EMF [29].The Tomentella-Thelephora lineages are also common in pine forests and nurseries and are intensively studied as they are often used for seedling inoculation in reforestation programs [30].Although these studies have revealed the presence of a large number of Tomentella OTUs in different type of forests, most sequences are difficult to identify at the species level.
There are many studies on species diversity and taxonomy of Tomentella reported in temperate forests, but few have been conducted in subtropical and tropical regions [1,2,6,30].Guizhou Province, known as the "Karst Province", is located on the Yunnan-Guizhou Plateau, Southwestern China, and has a subtropical humid monsoon climate [31].The unique terrain, landforms, climate and vegetation create excellent conditions for the growth of macrofungi [32,33].In 2023, dozens of Tomentella specimens were collected from two subtropical forests in Guiyang, Guizhou Province.Changpoling National Forest Park is mainly dominated by coniferous trees such as Pinus spp., and a number of broad-leaved trees are scattered in the forests [34].Qianlingshan Park is the area characterized by karst landform, and dominated by a subtropical evergreen secondary broad-leaved forest, with the domination of Broussonetia popyifera, Cunninghamia lanceolate, Pinus massoniana, Platycladus orientalis, Polygonum barbatum and Quercus acutissima [35].
In this study, four new species were described using morphological and phylogenetic analyses of DNA sequences.The main aim of this study is to update the species diversity of Tomentella in subtropical forests of China.

Specimen Collections
Specimens were collected from Changpoling National Forest Park (106 • 39 ′ 10 ′′ E-106 • 40 ′ 10 ′′ E, 26 • 38 ′ 45 ′′ N-26 • 40 ′ 00 ′′ N, Altitude: 1202-1370 m) and Qianlingshan Park (106 • 41 ′ 32 ′′ E, 26 • 35 ′ 53 ′′ N, Altitude: 1100-1396 m), Guizhou Province, Southwestern China.The regions belong to the subtropical humid monsoon climate with the obvious precipitations in summer.The average annual temperature is 13.6 • C, with average annual precipitation of 1200 mm.The specimen information, host trees, ecological habits, collector and date were recorded, and the photos of the basidiocarps and growth environment were taken.Then, the specimens were dried and bagged in time for preservation.The specimens were deposited at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP).

Morphological Studies
Macromorphological characteristics, including the color, texture and thickness of basidiocarps, hymenophoral surface and sterile margin, were examined under a stereomicroscope (Nikon SMZ 1000: Tokyo, Japan) at 4× magnification.The color terms follow Kornerup and Wanscher [36] for the macromorphological description.The microscopic procedure follows Lu [37] with some minor amendments.Microscopic measurements were made from thin sections of basidiocarps mounted in Cotton Blue (abbreviated as CB): 0.1 mg aniline blue dissolved in 60 g pure lactic acid; cyanophilous or acyanophilous reactions were assessed using CB.Amyloid and dextrinoid reactions were tested in Melzer's reagent (IKI): 1.5 g KI (potassium iodide), 0.5 g I (crystalline iodine), 22 g chloral hydrate, aq.dest.20 mL, inamyloid = neither amyloid nor dextrinoid reaction.Micromorphological descriptions were studied at magnifications up to 1000× with a light microscope (Nikon Eclipse E600: Tokyo, Japan) with phase contrast illumination, and dimensions were estimated subjectively with an accuracy of 0.2 mm.The following abbreviations are used: L = mean spore length (arithmetical average of all spores), W = mean spore width (arithmetical average of all spores), Q = extreme values of the length/width ratios among the studied specimens and n = the number of spores measured from a given number of specimens.Drawings were made with the aid of a drawing tube.The surface morphology for the basidiospores was observed with a QUANTA 250 scanning electron microscope (ESEM, QUANTA 250, FEI, Eindhoven, The Netherlands) at an accelerating voltage of 25 kV.The working distance was 12.2 mm.A thin layer of gold was coated onto the samples to avoid charging.

DNA Extraction, Amplification and Sequencing
Total genomic DNA was extracted from the dried specimens with a Thermo Scientific Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA).PCR reactions were performed in 30 µL of reaction mixtures containing 15 µL of 2× Phire ® Plant PCR buffer, 0.6 µL of Phire ® Hot Start II DNA Polymerase, 1.5 µL of each PCR primer (10 mM), 10.5 µL of doubly deionized H 2 O (ddH 2 O) and 0.9 µL of template DNA.For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced for all specimens.The BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 29 April 2024) was used to compare the resulting sequences with those in GenBank (Table 1).After confirmation of Tomentella species, additional gene region coding for the large subunit of nuclear ribosomal RNA gene (LSU) was sequenced.The ITS region was amplified with the primers ITS5 (5 ′ -GGAAGTAAAAGTCGTAACAAGG-3 ′ ) and ITS4 (5 ′ -TCCTCCGCTTATTGATATGC-3 ′ ) [38,39].The LSU gene was amplified with the primers LROR (5 ′ -ACCCGCTGAACTTAAGC-3 ′ ) and LR7 (5 ′ -TACTACCACCAAGATCT-3 ′ ) [40,41].The PCR thermal cycling program conditions were as follows: initial denaturation at 95 • C for 5 min, followed by 39 cycles at 95 • C for 30 s, × • C (the annealing temperatures for ITS4/ITS5 and LROR/LR7 were 54 • C and 48 • C, respectively) for 30 s [40,42], at 72 • C for 20 s and a final extension at 72 • C for 10 min.All amplified PCR products were estimated visually with 1.4% agarose gels stained with ethidium bromide and sequenced at the Beijing Genomics Institute (BGI) with the same primers.

Phylogenetic Analyses
The new sequences generated in this study were deposited in GenBank.These new sequences, together with the reference sequences of all samples used in the present study, are listed in Table 1.The sequences were edited and condensed with SeqMan v.7.1.0.The sequences generated in this study were supplemented with additional sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank,accessed on 29 April 2024) and UNITE (https://unite.ut.ee/index.php,accessed on 29 April 2024) based on blast searches and recent publications of the genus Tomentella.The sequences were aligned with MAFFT v.7, after which the alignments were manually corrected using MEGA v. 7.0 [43,44].Phylogenetic analyses including Maximum Likelihood (ML) and Bayesian inference (BI) methods were conducted for the single gene sequence datasets of the ITS and LSU, and the combined dataset of two gene regions.ML analyses were conducted using RAxML-HPC BlackBox 8.2.10 on the CIPRES Science Gateway portal (https://www.phylo.org/portal2, accessed on 29 April 2024) [45], employing a GTRGAMMA substitution model with 1000 bootstrap replicates [46].BI analyses were conducted using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v.3.0 [47].Two Markov chains were run from a random starting tree for 1,000,000 generations, resulting in a total of 10,000 trees.The first 25% of trees sampled were discarded as burn-in, and the remaining trees were used to calculate the posterior probabilities.Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7500 trees.Phylogenetic trees were viewed with FigTree v. 1.4 and processed by Adobe Illustrator CS5.

Phylogenetic Analyses
The combined two-gene sequences dataset (ITS and LSU) was analyzed to determine the phylogenetic positions of the new samples obtained in this study.A total of 2217 characters, including gaps (811 for ITS and 1406 for LSU), were included in the dataset used in the phylogenetic analyses.Of these characters, 928 were constant, 470 were parsimony-uninformative variable and 819 were parsimony-informative.The multiple sequence alignment included the following: nine sequences of the four new species, 211 sequences of Tomentella species [51][52][53][54][55][56][57][58][59][60][61][62][63] and one outgroup sequence of Odontia ferruginea from Estonia [38,64].
A similar topology was obtained using ML and Bayesian analyses with one sample of Odontia ferruginea as the outgroup [65], and only the ML tree is shown in Figure 1 with the ML bootstrap values and Bayesian posterior probabilities.In the phylogenetic tree, 18 clades with moderate to strong support were marked, of which six clades (clade 2, 4, 6, 8, 10 and 16) were consistent with the previous ITS + LSU phylogenetic analyses [65].Five of the six clades were more strongly supported (100% ML/1.00 BPP for clade 2, 89% ML/1 BPP for clade 4, 100% ML/1.00 BPP for clade 6, 90% ML/0.98 BPP for clade 8 and 84% ML for clade10, respectively), and clade 16 lacked significant support.The phylogenetic tree shows that the nine specimens formed four single clades with strong support (100% ML/1.00 BPP for T. casiae, T. guiyangensis, T. olivaceomarginata and T. rotundata) and clustered in the clade that was composed of most species of Tomentella used in this study.Basidiocarps: annual, resupinate, separable from the substrate, mucedinoid, without odor or taste when fresh, 0.5-1 mm thick, continuous.Hymenophoral surface granulose, grayish to gray (7D1-7E1) and concolorous with the subiculum; sterile margin often indeterminate, byssoid, lighter than hymenophore, light gray.

Discussion
In this study, four new Tomentella species distributed in Guizhou of Southwestern China were identified by morphological characteristics and phylogenetic analyses combining ITS and LSU sequences.Phylogenetic analyses and morphological features allowed distinguishing the four new species from other known species.
In the phylogenetic tree, 18 clades with moderate to strong support were obtained.Some of these clades were consistent with the previous ITS + LSU phylogenetic analyses [65].The new species in the clade with high support may share some consistent characteristics.For instance, Tomentella casiae fell in clade 7, and the basidiocarps of the species in this clade have an indeterminate sterile margin; T. rotundata clustered in clade 16, and species in this clade have clamped and rarely simple-septate subicular hyphae; T. oli-vaceomarginata clustered in clade 17, and species in this clade have separable basidiocarps with an arachnoid surface.
The forests investigated in this study are dominated by coniferous trees Pinus massoniana and P. armandii, and broad-leaved trees mainly included Bothrocaryum controversum, Celtis sinensis, Ligustrum lucidum and Robinia pseudoacacia.The specimens of these four new species were collected from rotten angiosperm wood debris, and their symbiotic plant hosts could not yet be determined.However, the development of resupinate basidiocarps represents an adaptive advantage for species of Tomentella growing in primary successional habitats.
In recent years, high-throughput sequencing technology has made it possible to explore unculturable taxa from environmental samples, for example soil or plant root tips, in different regions of the world.However, the BLAST of Tomentella species by the ITS sequences in the international gene database (NCBI and UNITE) normally results in a large number of unidentified environmental samples as best matches.It may result from insufficient taxonomic study of this group of fungi.Many studies by high-throughput sequencing have revealed that there are a large number of Tomentella spp. in China [62, [70][71][72][73][74], and the ectomycorrhizal samples of Tomentella have been collected and reported in a previous study from Guizhou Province [75].Therefore, the species diversity of Tomentella in the Karst subtropical forests of Guizhou needs further exploration.

Figure 1 .
Figure 1.Maximum likelihood tree illustrating the phylogeny of Tomentella casiae, T. guiyangensis, T. olivaceomarginata and T. rotundata related taxa based on ITS + LSU nuclear rDNA sequences dataset.Branches are labeled with Maximum likelihood bootstrap equal to or higher than 50% and Bayesian posterior probabilities equal to or higher than 0.9.Vouchers and regions are indicated after the species names.New species in bold (black).

Table 1 .
Species, vouchers, GenBank/UNITE accessions and localities of specimens used in this study.
Note: NA, not applicable.Strains from present study are in bold.