Taxonomy and Phylogeny Reveal Two New Potential Edible Ectomycorrhizal Mushrooms of Thelephora from East Asia

: The ectomycorrhizal basidiomycetes genus Thelephora has been understudied in subtropical ecosystems. Many species of Thelephora are important edible and medicinal fungi, with substantial economic value. Two new Thelephora species, T. grandinioides and T. wuliangshanensis spp. nov. are proposed here based on a combination of morphological features and molecular evidence. Thelephora grandinioides is characterized by laterally stipitate basidiocarps with a grandinoid hymenial surface, a monomitic hyphal system with clamped generative hyphae, and the presence of tubular and septated cystidia and subglobose to globose basidiospores measuring as 5.3 – 7.4 × 4 – 6.5 µm. Thelephora wuliangshanensis is characterized by infundibuliform basidiocarps, radially black striate on the pileus, a smooth, umber to coffee hymenial surface, a monomitic hyphal system with thick-walled generative hyphae, and basidiospores that turn greenish grey to buff in 5% KOH. Phylogenetic analyses of rDNA internal transcribed spacer region (ITS) and nuclear large subunit region (nrLSU) showed that the two new Thelephora are phylogenetically distinct: T. grandinioides is sister to T. aurantiotincta and T. sikkimensis , while T. wuliangshanensis is sister to a clade comprising T. austrosinensis and T. aurantiotincta with high support as well.

Thelephora share similar characteristics with Tomentella Pers.ex Pat.especially in the form, size, and type of spore ornamentations [3,29,30].Based on phylogenetic analyses using rDNA internal transcribed spacer region (ITS) sequences showed that the species of Thelephora mixed with Tomentella, revealing that both genera are closely related, but it is well-known that the phylogenetic analyses of ITS loci are insufficient to resolve phylogenetic relationships among closely related taxa [4,5,31,32].Based on ITS and nrLSU analyses, Vizzini et al. [25] showed that Thelephora and Tomentella species do not separate to two monophyletic groups but they are intermixed and form a well-supported monophyletic clade (Thelephora/Tomentella clade).Back to traditional method, the most important characteristic for distinguishing Thelephora and Tomentella is the form of the basidiocarps (resupinate in Tomentella; erect, with varied forms, to partially resupinate in Thelephora) [24,29,33].Das et al. [26] proposed that other features such as the hymenophore surface needed to be observed to determine whether it could act as a more informative characteristic than the highly variable stipitate/resupinate configuration of basidiocarps.Phylogenetic analyses of combined ITS and nrLSU dataset in Basidiomycota revealed that Thelephora is sister to Tomentella nested in Thelephoraceae while the limits between both genera are not yet clear [5,26,34].While ITS and nrLSU sequences alone cannot resolve phylogenetic relationships in this complex group of species [5,34].Vizzini et al. [25] mentioned that in the future Thelephora and Tomentella will be considered as one genus merging Tomentella into Thelephora.
With this work we intend to identify two Thelephora species found in southern China based on morphology and phylogeny, and provide full descriptions, color photographs, a detailed comparison of two new species with closely related taxa and a phylogenetic tree to show the placement of two new species.

Specimens Collection and Herbarium Specimen Preparation
Four samples of Thelephora were collected in Yunnan (Figure 1A-C) viz.CLZhao 3406 (Holotype) from the Wuliangshan National Nature Reserve, Huangcaoling, Jingdong County, Puer, at latitude 24°18′ N and longitude 101°05′ E, at 2113 m above sea level, 1 October 2017; CLZhao 3408 from the Xieqipo Forest Park, Zhenyuan County, Puer, at latitude 24°18′ N and longitude 101°05′ E, at 1350 m above sea level, 1 October 2017; CLZhao 4107 (Holotype) from the Wuliangshan National Nature Reserve, Huangcaoling, Jingdong County, Puer, at latitude 24°23′ N and longitude 100°45′ E, at 2313 m above sea level, 5 October 2017; CLZhao 21020 from the Wuliangshan National Nature Reserve, Huangcaoling, Jingdong County, Puer, at latitude 23°57′ N and longitude 100°57′ E, 8 October 2020.The fruiting bodies were observed growing on the ground of pinebroadleaved mixed forest.Photographs of the fruiting bodies were taken in the field, macromorphological characteristics were recorded and then the fruiting bodies were collected.The collected fruiting bodies were dried in an electric food dehydrator at 40 °C, then sealed and stored in an envelope bag.They were then transported to mycology laboratory of Southwest Forestry University, Kunming where microscopic morphology and phylogeny were studied.

Morphology
The specimens studied are deposited at the herbarium of Southwest Forestry University (SWFC), Kunming, Yunnan Province, China.Macromorphological descriptions were based on field notes and photos captured in the field and lab.Color terminology followed Petersen [35].Micromorphological data were obtained from the dried specimens, and were observed under a light microscope following Dai [36].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 = 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, n = a/b (number of spores (a) measured from given number (b) of specimens).

Molecular Phylogeny
A conventional cetyl trimethylammonium bromide (CTAB) plant genome rapid extraction kit (DN14, Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain genomic DNA from dried specimens, according to the manufacturer's instructions [37].Amplification reactions were performed in a 30 μL reaction volume composed of 15 μL 2 × FastTaq Premix (a mixture of FastTaq TM DNA Polymerase, buffer, dNTP Mixture, and stabilizer) (Beijing Qingke Biological Technology Co., Ltd., Beijing, China), 1 μL of each of the reverse and forward primers (Beijing Kinco Biotechnology Co., Ltd., Kunming Branch, China), 8.5 μL doble distilled water (ddH2O), and 1-1.2 μL DNA.ITS region was amplified with primer pair ITS5 and ITS4 [38].The nrLSU was amplified with primer pairs LR0R and LR7 [39].PCR procedure for ITS followed: 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.For the nrLSU regions, PCR amplification conditions were used as follows: initial denaturation of 1 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 s, 1 min of annealing at 48 °C, 90 s extension at 72 °C, and a final extension of 10 min at 72 °C.PCR products were purified and sequenced at Kunming Tsingke Biological Technology Limited Company, Kunming, Yunnan Province, China.All newly generated sequences were deposited in NCBI GenBank/UNITE (Table 1).
Sequences were aligned in MAFFT 7 (https://mafft.cbrc.jp/alignment/server/,accessed on 3 December 2021) using G-INS-i strategy for ITS combined dataset, and manually adjusted in BioEdit [40].Aligned dataset was deposited in TreeBase (submission ID 28432).Odontia fibrosa (Berk.and M.A. Curtis) Kõljalg and O. ferruginea Pers.were selected as outgroup for phylogenetic analyses of combined dataset [25,26].Maximum parsimony (MP) analysis was applied to the ITS and nrLSU dataset followed Zhao and Wu [37].Tree construction procedure was performed in PAUP* version 4.0b10 [48].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 analysis with 1000 replicates [49].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.Datamatrix was also analyzed using Maximum Likelihood (ML) approach with RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org,accessed on 3 December 2021) [50].Branch support for ML analysis was determined by 1000 bootstrap replicates.
MrModeltest 2.3 [51] was used to determine the best-fit evolution model for the data set for Bayesian inference (BI).BI was calculated with MrBayes 3.1.2[52].Four Markov chains were run for 2 runs from random starting trees for 160 thousand generations for ITS.The first one-fourth of all generations was discarded as burn-in.The majority rule consensus tree of all remaining trees was calculated.Branches were considered as significantly supported if they received maximum likelihood bootstrap value > 60%, maximum parsimony bootstrap value > 50%, or Bayesian posterior probabilities > 0.90.

Pairwise Homoplasy Test
The Genealogical concordance phylogenetic species recognition analysis (GCPSR) is a tool used to check significant recombinant events.The data were analyzed using SplitsTree 4 with the pairwise homoplasy Фw, PHI test to determine the recombination level within closely related species [53][54][55].One-locus dataset (ITS and nrLSU) with closely related species were used for the analyses.PHI results lower than 0.05 (Φw < 0.05) indicates a significant recombination is present in the dataset.The relationships between closely related taxa were visualized by constructing split graphs from the concatenated datasets, using the LogDet transformation and splits decomposition options.
The phylogram inferred from ITS+nrLSU, ITS, and nrLSU sequences (Figures 2-4) demonstrated that our specimens formed two isolated branches within Thelephora while T. grandinioides is sister to T. aurantiotincta Corner, and T. sikkimensis K. Das  Bootstrap support values for maximum likelihood (ML) equal to or higher than 60%, Bayesian Probability (PP) equal to or higher than 0.90, and maximum parsimony (MP) bootstrap proportions equal to or higher than 50% are mentioned above the branches (MP/ML/PP).Strains of the newly described species are depicted in blue.

Figure 3.
Maximum parsimony consensus tree based on the ITS sequences.Bootstrap support values for maximum likelihood (ML) equal to or higher than 60%, Bayesian Probability (PP) equal to or higher than 0.90, and maximum parsimony (MP) bootstrap proportions equal to or higher than 50% are mentioned above the branches (MP/ML/PP).Strains of the newly described species are depicted in blue.Bootstrap support values for maximum likelihood (ML) equal to or higher than 60%, Bayesian Probability (PP) equal to or higher than 0.90, and maximum parsimony (MP) bootstrap proportions equal to or higher than 50% are mentioned above the branches (MP/ML/PP).Strains of the newly described species are depicted in blue.
Basidiocarps-Annual, laterally stipitate, gregarious.Pilei medium-sized, coriaceous, infundibuliform, up to 9 cm long, 7 cm wide, 1.5 mm thick; fawn to isabelline when fresh, greyish brown on drying; proliferous from a central common base, rosulate, usually with several to many laterally confluent spathulate to flabelliform or valves, uplifted; the surface radially striate; margin thin, wavy.Hymenial surface grandinoid, olivaceous buff to clay-buff when fresh, clay-buff to slightly greyish brown on drying.Stipe cylindrical, up to 4 cm long, up to 1.5 cm in diameter.Context fleshy tough to leathery in fresh condition, corky to leathery in dried condition, up to 1 mm thick at the thickest portion of pileus, thinner at margin and thicker toward the base, pinkish buff to buff.Aculei, 6-8 per mm, 0.1-0.2mm long, greyish brown.Odor mild when fresh, somewhat smelly when dried, or with the beef jerky flavor.
Basidiocarps-Annual, laterally stipitate, gregarious.Pilei small to medium-sized, coriaceous, infundibuliform, up to 5.5 cm long, 4.5 cm wide, 1 mm thick; buff to salmon when fresh, pinkish buff to cinnamon-buff on drying; proliferous from a central common base, usually with several to many laterally confluent spathulate to flabelliform, uplifted; the surface radially black striate; margin thin, serrulate.Hymenial surface smooth, umber to coffee when fresh, coffee on drying.Stipe cylindrical, up to 2 cm long, up to 5 mm in diameter.Context fleshy tough in fresh condition, leathery in dried condition, up to 0.7 mm thick at the thickest portion of pileus, thinner at margin and thicker towards the base, pinkish buff.Odor mild when fresh, somewhat with the beef jerky flavor.

Discussion
In the present study, two new species, Thelephora grandinioides and T. wuliangshanensis are described based on phylogenetic analyses and morphological characteristics.In addition, the PHI test (Figures 5 and 6) was carried out to confirm there is no recombination present in the two new species compared with closely related taxa.
Thelephora is closely related to Tomentella both in morphology and phylogeny [5,10,14,25,29,74].Molecular phylogenetic analyses of previous studies showed that the taxa of Thelephora and Tomentella are non-monophyletic groups, and they are intermixed in molecular phylogeny [4,5,25,29,31,33].Traditionally, the form of basidiocarps is the most important characteristic in distinguishing Thelephora and Tomentella, which are resupinate in Tomentella, but erect, with varied forms, to partially resupinate in Thelephora [3,14,24,25,29,34,74].The variations in basidiocarp form may also complicate the characteristics of taxa [75,76], and the results of the morphological investigations and molecular phylogenetic analyses suggested that basidiocarp reduction happened several times independently across the evolution of thelephoroid fungi [14,25].Taxa with reduced basidiocarps should be taken into account in the diagnoses of genera for which the initial descriptions did not cover a real spectrum of polymorphism and trends of morphological rationalization in connection with colonization of specific habitats [14,25,76].According to molecular data, only one genus may be recognized, and Tomentella will be merged into Thelephora [25].
, Hembrom and Kuhar, and T. wuliangshanensis are sister to a clade comprising T. austrosinensis T.H. Li and T. Li and T. aurantiotincta.

Figure 2 .
Figure 2. Maximum parsimony strict consensus tree based on the ITS+nrLSU sequences.Bootstrap support values for maximum likelihood (ML) equal to or higher than 60%, Bayesian Probability (PP) equal to or higher than 0.90, and maximum parsimony (MP) bootstrap proportions equal to or higher than 50% are mentioned above the branches (MP/ML/PP).Strains of the newly described species are depicted in blue.

Figure 4 .
Figure 4. Maximum parsimony strict consensus tree based on the nrLSU sequences.Bootstrap support values for maximum likelihood (ML) equal to or higher than 60%, Bayesian Probability (PP) equal to or higher than 0.90, and maximum parsimony (MP) bootstrap proportions equal to or higher than 50% are mentioned above the branches (MP/ML/PP).Strains of the newly described species are depicted in blue.

Figure 5 .
Figure 5. Split graphs showing the results of PHI test for the ITS data of Thelephora grandinioides and T. wuliangshanensis and closely related taxa using LogDet transformation and splits decomposition.
PHI test results Φw ≤ 0.05 indicate that there is significant recombination within the dataset.New taxa are in red while, closly related species to new species are in other colors.

Figure 6 .
Figure 6.Split graphs showing the results of PHI test for the nrLSU data of Thelephora grandinioides and T. wuliangshanensis and closely related taxa using LogDet transformation and splits decomposition.PHI test results Φw ≤ 0.05 indicate that there is significant recombination within the dataset.New taxa are in red while, closly related species to new species are in other colors.

Table 1 .
Names, vouchers, location, and corresponding GenBank/UNITE accession numbers of taxa used in this study.The newly generated sequences are shown in black bold and T indicates the type.

Table 2 .
The nucleotide differences of phylogenetically similar species to Thelephora grandinioides and T. sikkimensis.

Table 3 .
The top ten species results of Blast search of Thelephora grandinioides and T. sikkimensis sequences.

Table 4 .
Comparison of Basidiospore, Basidia, Cystidia, Host, Substrate, Location and corresponding references of Thelelphora species.The characteristics of newly generated taxa are shown in black bold.

Table 5 .
Edible and medicinal species of Thelelphora."Y" means have edible or medicinal value; "P" means have edible or medicinal potential.