Molecular phylogeny and morphology reveal four new species of Thelephora (Thelephorales, Basidiomycota) from subtropical China, closely related to T. ganbajun

The genus of Thelephora is a group of cosmopolitan ectomycorrhizal fungi with basidiocarps of morphological diversity that has an extremely scarce species reported from the forest ecosystem in China. In this study, phylogenetic analyses of Thelephora species from subtropical China were carried out based on multiple loci including the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), and the small subunit of mitochondrial rRNA gene (mtSSU). Maximum likelihood and Bayesian analyses were used to construct the phylogenetic tree. The phylogenetic positions of four new species, Th. aquila, Th. glaucoflora, Th. nebula, and Th. pseudoganbajun, were revealed based on morphological and molecular evidence. Molecular analyses demonstrated that the four new species were closely related to Th. ganbajun and formed a clade with robust support in the phylogenetic tree. Regarding morphology, they share some common morphological characteristics, including flabelliform to imbricate pilei, generative hyphae more or less covered by crystals, and subglobose to irregularly lobed basidiospores (5–8 × 4–7 μm) with tuberculate ornamentation. These new species are described and illustrated and are compared to similar morphological or phylogenetically related species. A key to the new and allied species from China is provided.

The variable morphological and anatomical features of Thelephora have been discussed in detail in subsequent studies (Ramírez-López et al., 2013, 2015Khalid and Hanif, 2017;Das et al., 2018;Li et al., 2020). For instance, the basidiocarps range from stereoid, coral-like, merismatoid, spathulate-rosulate; the hyphal system is generally monomitic; the generative hyphae are usually clamped, simple-septate, and smooth to encrusted; cystidia are often absent; the ornamentation of the basidiospores is usually tuberculate or echinulate (Cunningham, 1957;Corner, 1968;Stalpers, 1993;Ramírez-López et al., 2013, 2015Khalid and Hanif, 2017;Das et al., 2018;Li et al., 2020). Basidiocarps of some species exhibit phenotypic plasticity, e.g., Th. versatilis and Th. Pseudoversatilis, can display a sub-resupinate or completely resupinate form over living plants (Ramírez-López et al., 2013, 2015. Generally, species of Thelephora contain a variety of shapes of basidiocarps and basidiospores with tuberculate or echinulate ornamentation, which help differentiate it from other genera in Thelephoraceae. Nevertheless, molecular validations have confirmed that Thelephora and Tomentella have a very close genetic relationship on the phylogenetic tree; the two genera are usually intermixed in the same evolutionary branch and do not form separate monophyletic groups (Vizzini et al., 2016;Lu et al., 2022). Furthermore, some species of the two genera have similar microscopic characteristics, such as tuberculate or echinulate ornamentation, and the same size and form as basidiospores (Stalpers, 1993;Lee et al., 2010;Yorou et al., 2011;Ramírez-López et al., 2015). The subtle classification boundary of the two genera has been controversial and still remains unresolved (Ramírez-López et al., 2015;Li et al., 2020;Lu et al., 2022).
Members of the genus Thelephora play an essential role in ecology, e.g., Th. terrestris is a well-known and rather common ectomycorrhizal symbiont in conifer tree nurseries promoting the growth of conifer seedlings (Weir, 1921;Corner, 1968;Marx and Bryan, 1970). Some species possess edible and medicinal values, for instance, Th. ganbajun M. Zang is a delicious edible fungus with a high economic value in China. Recent studies have documented that the chemically active ingredients, such as p-biphenyl phenolic compounds, polysaccharides, steroids, and fatty acids, extracted from Th. ganbajun have multiple effects such as antioxidant, antitumor, liver protection, and immune system enhancement for humans (Xu et al., 2016;Wang et al., 2017a;Zheng et al., 2020;Lu et al., 2022).
Approximately 52 accepted species of Thelephora have been described worldwide (http://www.indexfungorum.org/Names/ Names.asp). Several studies have provided an ITS or ITS + LSU phylogenetic overview of the genus (Ramírez-López et al., 2015;Vizzini et al., 2016;Das et al., 2018;Li et al., 2020), based on species from the northern temperate and tropical regions of Asia, Europe, and North America (Ramírez-López et al., 2013, 2015Khalid and Hanif, 2017;Das et al., 2018;Li et al., 2020). To date, 21 species of Thelephora have been recorded from China (Teng, 1934;Li et al., 2020;Liu et al., 2021), and most of them were identified based on themorphological comparison in the last century, and reference to taxonomy and phylogeny of this genus is extremely scarce. Meanwhile, most of the identifications are from molecular sequences without morphological study in the new century. For the star species Th. ganbajun, the situation may be even worse, as the name "ganbajun" has been applied to most related sequences in GenBank by some researchers. There are over 600 ITS sequences named "Thelephora ganbajun" in the NCBI database, yet the sequence discrepancies range from 1 to 9.5%. Therefore, it is essential to clarify the relationship between Th. ganbajun and the species with which it can be easily confused.
Investigations of stipitate aphyllophoroid fungi in China have been carried out in recent decades, and numerous Thelephora specimens have been collected. During the study of these specimens, four undescribed species collected from subtropical China were identified by means of morphology and phylogenetic analyses of a three-gene (ITS + nLSU + mtSSU) dataset. In this study, we describe and illustrate these taxa based on morphological and phylogenetic evidence and provide a key to the species of Thelephora from China.

Morphological studies
Specimens were deposited at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP). Microscopic procedures follow Cao et al. (2021). Macromorphological characteristics of basidiocarps were observed under a stereomicroscope (Nikon SMZ 1000: Tokyo, Japan) at 4× magnification. The observations of microscopic characters were performed on freehand sections of dried basidiocarps, mounted in 3% KOH, and stained in Cotton Blue (test for cyanophilous or acyanophilous reactions) and Melzer's reagent (test for amyloid and dextrinoid reactions). All measurements were studied at magnifications up to 1,000× using a Nikon Eclipse E600 microscope (Tokyo, Japan) with phase contrast illumination. The following abbreviations are used: IKI = Melzer's reagent; IKI -= neither amyloid nor dextrinoid; KOH = 3% potassium hydroxide; CB = Cotton Blue; CB + = cyanophilous; L = mean spore length (arithmetic average of all spores); W = mean spore width (arithmetic average of all spores); Q = variation in the L/W ratios between the specimens studied; and n (a/b) = number of spores. The surface morphology for the basidiospores was observed with a Phenom Prox scanning electron microscope (ESEM, Phenom Prox, FEI, Netherlands) at an accelerating voltage of 20 kV. A thin layer of gold was coated on the samples to avoid charging. Special color terms are from Rayner (1970) and Munsell (2015).

Molecular study
Genomic DNA was extracted from the dried specimens with a Thermo Scientific Phire Plant Direct PCR kit (Thermo Fisher Scientific, Waltham, MA, United States). The internal transcribed spacer region (ITS) was amplified with primer pairs ITS4 and ITS1-F (White et al., 1990); the large subunit of nuclear ribosomal RNA gene (nLSU) with LR0R and LR5 (Moncalvo et al., 1993); and the mitochondrial small subunit rDNA gene (mtSSU) with MS1 and MS2 (Matheny, 2005 Mu et al., 2021). The PCR products were purified and sequenced at the Beijing Genomics Institute (BGI), China.

Phylogenetic analyses
The newly generated sequences in this study and related sequences downloaded from GenBank (Table 1) were converted into FASTA format files by ClustalX (Thompson et al., 1997). Then, alignments were performed using MAFFT 7.110 (Katoh et al., 2019) and manually adjusted to allow maximum alignment and minimize gaps; finally, the results of the alignments were saved as the FASTA format files.
The combined ITS + nLSU + mtSSU dataset phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) analysis. All characters were equally weighted, and all gaps were treated as missing data. ModelFinder (Kalyaanamoorthy et al., 2017) on Phylosuite  was used to select the best-fit partition model (Edge-linked) using the AICc criterion for combined ITS + nLSU + mtSSU dataset. Best-fit models according to AICc were as follows: K3Pu + F + I + G4 (ITS), TIM3 + F + R2 (nLSU), TIM + F + I (mtSSU) for ML; SYM + I + G4 (ITS), GTR + F + I + G4 (nLSU), and GTR + F + I (mtSSU) for BI. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al., 2015) under the edge-linked partition model for 1,000 standard bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test (Guindon et al., 2010). Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al., 2012) implementing the Markov Chain Monte Carlo technique, with two parallel runs and eight million replicates. Four simultaneous chains were run beginning from random trees, and sampling one tree for every 100 generations until the average standard deviation of split frequencies was below 0.01. The burn-in was set to discard 25% of the trees. Identity/similarity between two sequences was calculated using the "pairwise alignment, calculation of the similarity/identity" option of BioEdit v. 7.0.5 (Hall, 2005).
In the phylogenetic tree ( Figure 1), 19 sampled specimens representing four new species formed four isolated clades with strong support (100% ML/1 BI for Th. aquila, 99% ML/0.99 BI for Th. glaucoflora, 93% ML/0.98 BI for Th. nebula, and 98% ML/0.99 BI for Th. pseudoganbajun) and clustered in the clade with other four species including Th. austrosinensis, Th. ganbajun, Th. grandinioides, and Th. vialis with strong support (100% ML/1 BI). Eight samples of Th. ganbajun and the type samples formed a fully supported lineage (100% ML/1 BI) that differs from the other samples. The phylogenetic tree also reveals that three taxa of Tomentella and 37 taxa of Thelephora are intermixed in the phylogenetic tree. Basidiocarp: Upright, solitary to concrescent, small to mediumsized, up to 5 cm high and 4 cm wide, coriaceous when fresh, hard and light in weight when dried; taste mild, almost no odor when dry. Pileus flabelliform to applanate-lobate, usually with multiple pilei arising from a central common stipe or base, margin thin (0.1 mm), irregularly lobed to wavy. Abhymenial surface somewhat radially rugulose or wrinkled, zonate, black (GLEY 1 2.5/N) near the center then gradually turning pale toward the outside and becoming white (30A1) at the margin when fresh; hymenial surface concolorous or deeper and white (30A1) at the margin. Stipe central, up to 2 cm long, brown (6E8), glabrous, clavillose to flatted or broadened.
Additional specimen ( Pseudoganbajun, and Th. vialis clustered in a clade with full support based on the molecular evidence ( Figure 1). Morphologically, a special characteristic of Th. aquila is the black abhymenial surface when mature, which makes it distinct from other species in the genus. Furthermore, Th. aquila resembles Th. austrosinensis in having single to concrescent basidiocarps, flabelliform to lobate pilei, absence of cystidia, and tuberculate basidiospores. However, Th. austrosinensis differs from Th. aquila by a grayish black to grayish yellow abhymenial surface and a violet pale grayyellow hymenial surface . Th. aquila and Th. grandinioides share some common features, including the upright basidiocarp, flabelliform to applanate-lobate pilei, and tuberculate basidiospores. Nevertheless, Th. grandinioides can be differentiated by a fawn to isabelline abhymenial surface, a grandinoid, olivaceous buff to clay-buff hymenial surface, as well as the presence of cystidia (Liu et al., 2021). Basidiocarp: Upright, usually solitary, small to medium-sized, up to 4 cm high and 3 cm wide, coriaceous when fresh, tastes mild, almost no odor when dried. Pileus flabelliform, imbricate, usually with multiple pilei arising from a central stipe or base, basidiocarp like a rose flower, margin thin (0.1 mm thick), imperceptibly wavy. Abhymenial surface somewhat radially rugulose or wrinkled, zonate, sulcate near the base, gray to greenish gray (29E1-29E2) near the center then gradually becoming gray to white (29A1-29B1) at the margin when fresh; hymenial surface radial rugulose, zonate, violet-gray (16B2-16E2) at the base then gray (16D1-16E1), .

FIGURE
ML tree illustrating the phylogeny of the new species and related taxa based on ITS + nLSU + mtSSU nuclear rDNA sequences dataset. Branches are labeled with maximum likelihood bootstrap higher than % and Bayesian posterior probabilities higher than . .
Subhymenium: generative hyphae hyaline, slightly thick-walled (<1 µm), frequently branched often near the clamp connections, occasionally isotypical clamp connections symmetrically growing on both sides of the hyphae, hyphal cells partly short to slightly inflated, occasionally covered by dense crystals, subparallel to loosely interwoven, up to 4-8 µm diam.
Additional specimens ( 2022, Yuan 16733 (IFP 19549). Notes: Six samples (Yuan 16780, 16733, 16835, 16771, and 16794) together with two sequences downloaded from GenBank, which were labeled "Thelephora cf. ganbajun" from Xiangyun County and Yunlin County, Yunnan Province, formed a clade in the phylogenetic tree ( Figure 1). In morphology, the flabelliform or spathulate and imbricate pilei of similar size as well as brown to yellowish brown stipe make Th. pseudoganbajun easily confused with Th. ganbajun. However, a brown to yellowish brown abhymenial surface, a light brown to brown hymenial surface, clamps symmetrically growing on subhymenial hyphae, and the absence of cystidia make the former different from Th. ganbajun (Zang, 1987). The phylogenetic tree also shows a close relationship between Th. pseudoganbajun and Th. vialis. They share similar characteristics in having tuberculate ornamentation of basidiospores and flabelliform or spathulate pilei. However, Th. vialis differs from Th. pseudoganbajun by a pallid yellowish to pale dull brown abhymenial surface and a pallid yellowish to grayish brown hymenial surface, as well as no clamps symmetrically growing on the subhymenial hyphae (Corner, 1968). Morphologically, flabelliform or spathulate and imbricate pilei and solitary to concrescent basidiocarps, make Th. pseudoganbajun similar to Th. anthocephala. However, Th. anthocephala differentiates from Th. pseudoganbajun by a ferruginous or purplish abhymenial surface and a dark brown to the grayish violet hymenial surface, a subtomentose stipe, as well as purplish umber and bigger echinulate basidiospores (7-11 × 5-8.5 µm in Th. anthocephala vs. 5.1-7.2 × 4-6.1 µm in Th. pseudoganbajun Corner, 1968).
Basidiocarp: Upright, usually solitary, small to medium-sized, up to 14 cm high and 15 cm wide, coriaceous when fresh, tastes mild, coriaceous, yeast powder flavor when dried. Pileus more or less deeply lacerate becoming flabelliform, often imbricate, sometimes proliferating from the center and becoming imbricate and forming a rosette shape, and margin flush and thin (0.5-2 mm). Abhymenial surface somewhat smooth, distinctly zonate, .

Discussion
The three-gene (ITS + nLSU + mtSSU) phylogenetic analysis provided an improved resolution at the interspecific level. The tree showed that the phylogenetic clades obtained higher support at the species level, but relatively low support in the deeper nodes, which is consistent with previous results (Ramírez-López et al., 2013, 2015Khalid and Hanif, 2017;Das et al., 2018;Li et al., 2020).
The phylogenetic tree revealed the relationships among Thelephora ganbajun, Th. austrosinensis, Th. grandinioides, Th. Vialis, and the four new species. These eight species clustered in a clade and obtained full support, indicating that they have a close phylogenetic relationship. They share some common morphological characteristics, including flabelliform to imbricate pilei proliferating from a common base, zonate abhymenial surface, generative hyphae more or less covered by crystals, and relatively small, tuberculate basidiospores (5-8 × 4-7 µm), but are significantly distinguished in terms of molecular sequences and morphological characteristics. We provide a key to the new and allied species from China. Th. vialis is not included in the key, because our preliminary study shows that the name "Th. vialis" may represent another species in China.
The specimens involved in this study were mainly collected from subtropical forests, where the elevation is relatively high (800-2,200 m) and the aphyllophoroid fungi are very rich (He et al., 2011;Wu et al., 2016Wu et al., , 2020Wu et al., , 2022Cui et al., 2018Cui et al., , 2019Deng C. Y. et al., 2020;Deng W. Y. et al., 2020;Dai et al., 2021;Ma et al., 2022). The forests are primarily dominated by broad-leaved trees such as Fagaceae, Castanopis spp., and a small portion of Pinaceae trees. As ectomycorrhizal fungi, these species may be associated with tree species of Fagaceae and/or Pinaceae.
Up to now, more than 600 ITS sequences named "Thelephora ganbajun" have been submitted to NCBI and UNITE databases. The previous study has shown that some selected sequences formed five distinct clades in the ITS phylogenetic tree , and the reference sequence from the type specimen (HKAS 14735) nested in the clade 1, which represents the true Th. ganbajun. In this study, some sequences named "Th. ganbajun" from GenBank, for instance, KY245247, KY245255, EU696860, and EU696931, have sequence similarities with the type Yuan 16794 ranging from 99.8 to 100% and were identified as Th. pseudoganbajun. Some sequences, for example, EU696831, EU696818, EU696871, and EU696881, have sequence similarities with the type Dai 13623A ranging from 99.69 to 99.08% and were identified as Th. glaucoflora. However, the other two new species, Th. aquila and Th. nebula, were not identified with those known sequences from GenBank. More continuous investigations are needed to understand the species diversity of this group of fungi.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.
organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.