﻿The diversity and taxonomy of Tomentella (Thelephoraceae, Thelephorales) with descriptions of four new species from Southwestern China

﻿Abstract Taxonomy plays a central role in understanding the diversity of life, translating the products of biological exploration and discovery specimens and observations into systems of names that settle a “classification home” to taxa. Up to this point, studies on the taxonomy and phylogeny of the basidiomycetous genus Tomentella stemmed mainly from the temperate to boreal zones of the North Hemisphere, but were scarce in tropical Asia. In this study, four new species, viz. Tomentellaolivaceobasidiosa, T.velutina, T.wumenshanensis and T.yunnanensis from China, are described and illustrated based on the morphological characteristics and molecular phylogenetic analyses, in which the sequences of ITS+nLSU+mtSSU+RPB2 genes were used for the phylogenetic analyses using Maximum Likelihood, Maximum Parsimony and Bayesian Inference methods. All the new species can be well recognised by their macroscopical and anatomical characteristics. The four new species, closely related taxa in the phylogenetic tree and morphologically similar species are discussed.


Introduction
The genera Amaurodon J. Schröt., Odontia Pers., Pseudotomentella Svrcek, Tomentella Pers.ex.Pat. and Tomentellopsis Hjortstam, belong to the family Thelephoraceae Chevall. of the order Thelephorales Corner ex Oberw.and the phylum Basidiomycota R.T. Moore (Reid and Larsen 1976;Kõljalg 1996).As their common morphological characteristics are resupinate and thin basidiomata, they were recognised as the resupinate thelephoroid fungi by Kõljalg (1996).Species of the group have their own typical characteristics, such as the light blue basidiomata of Amaurodon, the granulose or hydnoid hymenial surface of Odontia, the basidiospores with bifurcate warts or spines of Pseudotomentella and the absence of rhizomorphs in the genus Tomentellopsis.However, the genus Tomentella has diverse and complex morphological features, such as basidiomata with various colours and smooth to granulose surfaces, and basidiospores with diverse shapes and ornamentations (Reid and Larsen 1976;Kõljalg 1996).
The genus Tomentella was sister to Thelephora in which both are nested within the family Thelephoraceae, while the morphological limits between Tomentella and Thelephora are not yet clear (Patouillard 1887;Ezhov and Zmitrovich 2017).In previous scientific research, studies about wood-inhabiting fungal molecular systematics revealed that within the species with different macroscopic characteristics located in the same family or even within the same genus, similar microscopic characteristics can be seen (Cui et al. 2019;Guan and Zhao 2021;Guan et al. 2021;Wu et al. 2022;Guan et al. 2023;Wang et al. 2023;Zhao et al. 2023bZhao et al. , 2024;;Luo et al. 2024).The studies showed that ITS or nLSU sequences alone could not resolve the phylogenetic relationships in this complex group (Thelephora/Tomentella clade) (Patouillard 1887; Vizzini et al. 2016;Ezhov and Zmitrovich 2017).The research mentioned that genera Thelephora and Tomentella will be considered one genus, based on the morphological and phylogenetic results.
This work describes four new species of Tomentella, which were found in southwest China, based on the morphology and phylogeny.It provides full descriptions, colour photographs, a detailed comparison of four new species with closely related taxa and phylogenetic trees showing the placement of four new species within the genus Tomentella.

Morphology
Fresh fruiting bodies of the fungi were collected from Kunming, Lincang and Zhaotong of Yunnan Province, P.R. China.Specimens were dried in an electric food dehydrator at 40 °C (Hu et al. 2022), then sealed and stored in an envelope bag and deposited in the herbarium of the Southwest Forestry University (SWFC), Kunming, Yunnan Province, P.R. China.Macromorphological descriptions are based on field notes and photos captured in the field and lab.Colour terminology follows Petersen (Petersen 1996).Micromorphological data were obtained from the dried specimens when observed under a light microscope following the previous study (Guan et al. 2023;Zhao et al. 2023a).The following abbreviations are used: KOH = 5% potassium hydroxide water solution, 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 specimens studied and n = a/b (number of spores (a) measured from given number (b) of specimens).

Molecular phylogeny
The EZNA HP Fungal DNA Kit (Omega Biotechnologies Co., Ltd., Kunming, China) was used to extract DNA with some modifications from the dried specimens.The nuclear ribosomal ITS region was amplified with primers ITS5 and 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, 58 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min.The nuclear nLSU region was amplified with primer pair LR0R and LR7 (Vilgalys and Hester 1990;Rehner and Samuels 1994).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 nuclear mt-SSU region was amplified with primer pair MS1 and MS2 (White et al. 1990).The PCR procedure for mt-SSU was as follows: initial denaturation at 94 °C for 2 min, followed by 36 cycles at 94 °C for 45 s, 52 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min.The nuclear RPB2 region was amplified with primer pair bRPB2-6F and bRPB2-7.1R(Matheny 2005).The PCR procedure for RPB2 was as follows: initial denaturation at 95 °C for 2.5 min, denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, extension at 72 °C for 1 min (add 1 °C per cycle), repeat for 40 cycles starting at step 2, extension at 72 °C for 1.5 min, repeat for 40 cycles starting at step 6, leave at 72 °C for 5 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 (https://www.ncbi.nlm.nih.gov/genbank/) (Table 1).
The sequences were aligned in MAFFT v. 7 (Katoh et al. 2019) using the G-INS-i strategy.The alignment was adjusted manually using AliView v. 1.27 (Larsson 2014).The sequence alignments were deposited in TreeBase (ID 31627).Sequences of Phellinotus neoaridus Drechsler-Santos & Robledo.Parmasto retrieved from GenBank was used as an outgroup in the ITS+nLSU+mtS-SU+RPB2 analysis (Fig. 1; Salvador-Montoya et al. (2022)).The sequence alignments were deposited in TreeBase (ID 31628).Sequences of Odontia sparsa Yuan Yuan, Y.C. Dai & H.S. Yuan retrieved from GenBank were used as the outgroups in the ITS+nLSU analysis (Fig. 2; Yuan et al. (2018)).Maximum parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were applied to the combined three datasets following a previous study (Zhao et al. 2023a).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.Maxtrees 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. 2006).Branch support (BS) for the ML analysis was determined by 1000 bootstrap pseudo replicates.
MrModelTest 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each dataset for Bayesian inference (BI), which was performed using MrBayes 3.2.7awith a best 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 2 million generations for ITS+nLSU+mtSSU+RPB2 (Fig. 1) and 12 million generations for ITS+nLSU (Fig. 2) with trees 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+mtSSU+RPB2 dataset (Fig. 1) comprised sequences from 38 fungal specimens representing 26 taxa.The dataset had an aligned length of 6,608 characters, of which 5,402 characters were constant, 318 were variable and parsimony-uninformative and 888 were parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 2,559, CI = 0.6444, HI = 0.3566, RI = 0.7784 and RC = 0.5016).The best model of nucleotide evolution for the ITS+nLSU+mtSSU+RPB2 dataset estimated and applied in the Bayesian analysis was GTR+I+G.Bayesian analysis and ML analysis resulted in a similar topology to the MP analysis.The Bayesian analysis had an average standard deviation of split frequencies = 0.009434 (BI) and the effective sample size (ESS) across the two runs is double the average ESS (avg.ESS) = 950.The phylogram, based on the ITS+nLSU+mtSSU+RPB2 rDNA gene regions (Fig. 1), included seven genera within the family Thelephoraceae (Thelephorales), including Amaurodon, Lenzitopsis Malençon & Bertault, Phellodon P. Karst, Polyozellus Murrill, Thelephora Ehrh.ex Willd., Tomentella and Tomentellopsis, in which four new species were nested into the genus Tomentella.
The ITS+nLSU dataset (Fig. 2) comprised sequences from 115 fungal specimens representing 69 taxa.The dataset had an aligned length of 2117 characters, of which 1664 characters were constant, 71 were variable and parsimony-uninformative and 340 were parsimony-informative.Maximum parsimony analysis yielded one equally parsimonious tree (TL = 2,501, CI = 0.2735, HI = 0.7265, RI = 0.5884 and RC = 0.1609).The best model of nucleotide evolution for the ITS dataset estimated and applied in the Bayesian analysis was GTR+I+G.Bayesian analysis and ML analysis resulted in a topology similar to that of the MP analysis.The Bayesian analysis had an average standard deviation of split frequencies = 0.009654 (BI) and the effective sample size (ESS) across the two runs is double the average ESS (avg.ESS) = 360.The phylogenetic tree (Fig. 2) showed that the new species Tomentella olivaceobasidiosa formed a monophyletic lineage in the ITS+nLSU phylogetic tree.Furthermore, the new species Etymology.Olivaceobasidiosa (Lat.):refers to the olivaceous basidiomata.Description.Basidiomata annual, resupinate, separable from the substrate, arachnoid, without odour or taste when fresh, and up to 3 cm long, 2.5 cm wide, 0.3-0.6 mm thick.Hymenial surface smooth, slightly olivaceous when fresh, olivaceous to slightly brown upon drying.Sterile margin narrow, olivaceous, up to 1 mm.

A B
Description.Basidiomata annual, resupinate, adnate, cotton to floccose, without odour and taste when fresh and up to 4.5 cm long, 3 cm wide, 0.3-0.5 mm thick.Hymenial surface cotton to floccose, fawn to reddish brown when fresh, turn reddish brown to vinaceous brown when dry. Sterile margin narrow, fawn to reddish brown, up to 2 mm.Hyphal system: Monomitic; generative hyphae with clamp connections, yellowish-brown, slightly thick-walled, 3-7 mm in diameter, IKI-, CB-, brown-black to black in KOH.

A B Discussion
Recently, many wood-inhabiting fungal taxa have been reported worldwide (Cui et al. 2019;Guan et al. 2023;Luo et al. 2024;Zhou et al. 2024) and, in the present study, four new species of the genus Tomentella are reported, based on a combination of morphological features and molecular evidence.The macroscopical and anatomical characteristics can well recognise all of them; T. olivaceomarginata is characterised by the olivaceous margin of the basidiomata and the presence of the clavate basidia measuring 39-48.5 × 7-8.5 µm.Tomentella velutina can be recognised by having adnate cotton to floccose basidiomata and the presence of the clavate basidia and broadly ellipsoid basidiospores
Molecular phylogenetic analyses of the previous studies revealed that the taxa of both genera, Thelephora and Tomentella were non-monophyletic groups, in which they were intermixed in molecular phylogeny (Stalpers 1993;Kõljalg 1996;Larsson 2014;Ramírez-López et al. 2015;Vizzini et al. 2016;Li et al. 2020).In the present study, the four-genes (ITS+nLSU+mtSSU+RPB2) phylogenetic analysis provided an improved resolution at the family level and showed that the genera Thelephora and Tomentella grouped together, which is consistent with previous results (Stalpers 1993;Kõljalg et al. 2001;Yorou et al. 2008;Vizzini et al. 2016;Zmitrovich et al. 2018;Li et al. 2020), and four new species were nested into the genus Tomentella.The phylogenetic tree divided Tomentella into several distinct clades and most of the clades are consistent with the previous ITS phylogenetic analyses (Yuan et al. 2020;Lu et al. 2022).This study identifies and describes four new Tomentella species from China, based on morphological characteristics and phylogenetic analyses combining ITS+nLSU sequences (Fig. 2).
The phylogenetic tree reveals that individual species of Tomentella can form ectomycorrhiza with different host tree species in different families and closely related species in the same clade can be restricted to the same host tree family, in which the investigated forests were dominated by the coniferous trees Pinus kesiya mixed with families such as Ericaceae, Fagaceae, Lentibulariaceae, Orchidaceae and Rosaceae (Nguyen et al. 2012;Pócs et al. 2019).The present study found four new taxa in broad-leaved forests (Fagaceae and/or Pinaceae) mixed with coniferous trees.China is one of the most biodiverse countries in the world and more Tomentella species remain to be discovered here.Therefore, further studies are needed to enrich the species diversity of Tomentella.

Figure 2 .
Figure 2. Maximum parsimony strict consensus tree illustrating the phylogeny of the three new species and related species in Tomentella, based on ITS sequences.Branches are labelled with maximum likelihood bootstrap values ≥ 70%, parsimony bootstrap values ≥ 50% and Bayesian posterior probabilities ≥ 0.95, respectively.The type species are marked with *.