Four New Species and One New Record of Thelephora from China

Species of the genus Thelephora (Thelephorales, Thelephoraceae) are ectomycorrhizal symbionts of coniferous and broad-leaved plants, and some of them are well-known edible mushrooms, making it an exceptionally important group ecologically and economically. However, the diversity of the species from China has not been fully elucidated. In this study, we conducted a phylogenetic analysis based on the internal transcribed spacer (ITS) regions, using Maximum Likelihood and Bayesian analyses, along with morphological observations of this genus. Four new species from China are proposed, viz., T. dactyliophora, T. lacunosa, T. petaloides, and T. pinnatifida. In addition, T. sikkimensis originally described from India is reported for the first time from China. Thelephora dactyliophora, T. pinnatifida, and T. sikkimensis are distributed in subtropical forests and mainly associated with plants of the families Fagaceae and Pinaceae. Thelephora lacunosa and T. petaloides are distributed in tropical to subtropical forests. Thelephora lacunosa is mainly associated with plants of the families Fagaceae and Pinaceae, while T. petaloides is mainly associated with plants of the family Fagaceae. Line drawings of microstructures, color pictures of fresh basidiomes, and detailed descriptions of these five species are provided.


Introduction
Thelephora Ehrh.ex Willd.was originally described by Ehrhart and validly published by Willdenow with T. terrestris Ehrh.as its type species [1,2].The genus is characterized by the diverse shapes of basidiomes which are stereoid, imbricate, rosette, infundibuliform, coralloid, or resupinate; the sulcate, zonate, glabrous to strigose, somewhat radially rugulose or wrinkled abhymenial surfaces; the smooth, slightly rugose to warty hymenial surfaces; the monomitic, clamped hyphal systems; the verruculose or echinulate basidiospore ornamentations; and the presence or absence of cystidia [1][2][3][4][5][6].Species of the genus Thelephora are associated with a variety of plants of the families Pinaceae, Casuarinaceae, Ericaceae, Fagaceae, and Betulaceae.The mycelia of these species around the plant roots can help plants obtain essential minerals and water from the soil and resist diseases and drought [7], making significant contributions to plant health and ecosystem stability.In addition, some species in the genus play an essential role in the edible and medicinal fungal industry; e.g., Thelephora aurantiotincta Corner and T. ganbajun M. Zang have anticancer and anti-allergic effects [8][9][10].Meanwhile, T. ganbajun is also one of the most popular edible mushrooms in China and some East Asian countries [11][12][13][14].
According to our preliminary statistics, 64 species of Thelephora have been accepted (http://www.indexfungorum.org/Names/Names.asp,accessed date: 25 February 2024), and they are mainly distributed in the northern temperate and tropical regions from Europe [15], North America [16][17][18][19][20], and Asia [21,22].In China, 24 species have been reported [12][13][14][23][24][25].During our recent research on Thelephora across China, we encountered five impressive species that have not been reported from this country.These species can be easily recognized by their conspicuous colors in the field.Molecular phylogenetic analyses of this genus based on the nuclear ribosomal internal transcribed spacer (ITS) indicated that they represent five distinct species.Combined with morphological characteristics, five species including four new to science: T. dactyliophora Yan C. Li

Specimen Collections
Nineteen specimens of Thelephora were collected from China, including seventeen from southwestern China (Yunnan Province) and two from eastern China (Zhejiang Province).Photographs of these fresh specimens were taken in the field.Macroscopic characteristics and field notes including date, location, and habitat were made for each specimen, and then these specimens were dried using a fungal dryer and deposited in the fungal herbarium of the Herbarium KUN (Kunming Institute of Botany, Chinese Academy of Sciences) for taxonomic studies.Small pieces of each specimen were also dried in silica gel for molecular studies.

Morphological Studies
Macroscopic characteristics were obtained from specimen records and photos captured in the field.Color codes followed Kornerup and Wanscher [26,27].Microscopic characteristics that include the structure of the pileipellis and the morphology of context hyphae, subhymenium hyphae, basidia, cystidia, and basidiospores were observed under a ZEISS Axiostar Plus microscope (Carl Zeiss AG, Oberkochen, Germany).Tissues were sectioned and mounted in 10% KOH, Cotton Blue (test for cyanophily), and Melzer's reagent (test for amyloidity and dextrinoidity) [13,25,28,29].Microscopic structures of pileipellis, hymenium, and subhymenium were drawn freehand; basidiospores and hyphae were measured at 1000× g magnification.The observations of basidiospore ornamentations were obtained with a ZEISS Sigma 300 scanning electron microscope (SEM) (Carl Zeiss AG, Oberkochen, Germany).For the measurement of basidiospores, at least 20 basidiospores were measured for each specimen; the notation "basidiospores (n/m/p)" is used to mean n basidiospores measured from m basidiomes of p specimens.L denotes spore length (arithmetic average of all spores); W denotes spore width (arithmetic average of all spores); Q denotes variation in the length/width ratios of basidiospores in side view; Q m denotes the average Q of all basidiospores ± sample standard deviation [30][31][32].Since the shape of basidiospore is mainly inferred by its length/width ratio, we have defined the terms of basidiospore shapes as follows: globose (length/width ratio = 1.01-1.05),subglobose to broadly ellipsoid (length/width ratio = 1.05-1.3),ellipsoid (length/width ratio = 1.3-1.6), and elongated (length/width ratio = 1.6-2).The abbreviation masl means meters above sea level [33].

DNA Extraction, PCR and DNA Sequencings
The extraction of genomic DNA from dried specimens was conducted using the CTAB method [34] and Ezup Column Fungi Genomic DNA Purification Kit (Shanghai Jinban Biotechnology Co., Ltd., Shanghai, China).PCR reactions contained 1 µL DNA solution (adjusted to approximately 20 ng), 1 µL of each primer, and 15 µL 2× Taq PCR Master Mix including Taq DNA Polymerase, MgCl 2 , and dNTP mix (Beijing Biomed Gene Technology Co., Ltd., Beijing, China).The final volume was adjusted to 30 µL with distilled sterile H 2 O.The amplification conditions were set as follows: denaturation at 95  C [35,36].In this study, the internal transcribed spacer (ITS) regions were amplified with the primers ITS1F/ITS4 [30,34,37,38].Some full lengths of ITS sequences that failed to be amplified were divided into two parts for amplification with primer pairs ITS1F/5.8S and 5.8SR/ITS4, respectively [39], and then sequences of these two parts were spliced together to obtain the full length of the ITS sequence.Sequences newly generated in this study were deposited in GenBank (Table 1).

Phylogenetic Analyses
DNA sequences were compiled with SeqMan (DNASTAR Lasergene 9).Sequences related to Thelephora downloaded from GenBank (https://www.ncbi.nlm.nih.gov/,accessed on 6 April 2024) and UNITE (https://unite.ut.ee/, accessed on 25 February 2024), and newly generated in this study were aligned with Mafft V7.490 [42] and manually adjusted with PhyDE when necessary.Then we performed phylogenetic analyses by Maximum Likelihood (ML) and Bayesian Inference (BI) approaches.The ML analysis was inferred with RaxmlGUI 2.0.10 [43] with model set as GTRGAMMAI for 1000 bootstrap replicates combined with an ML search.The BI analysis was conducted using MrBayes 3.2 [44] running 2,000,000 bootstrap replicates; the best-fit likelihood model employed for ITS dataset was HKY + I + G + F based on the Akaike Information Criterion (AIC) with ModelFinder [45,46]; the posterior probabilities (PP) were determined twice by running one cold and three heated chains in parallel mode, saving trees every 1000th generation.Other parameters were kept at their default settings.Runs were terminated once the average standard deviation of split frequencies went below 0.01.Genetic distances among ITS sequences were calculated using the Maximum Composite Likelihood model [47] in MEGA 11 [48].

Phylogenetic Analyses
Phylogenetic analyses were conducted based on 53 ITS sequences of which 16 were newly generated in this study and 37 were downloaded from UNITE and GenBank (Table 1).Odontia fibrosa (Berk.& M.A. Curtis) Kõljalg and O. ferruginea Pers.were used as the outgroup taxa [14,18,22,47].The alignment was submitted to TreeBASE (S30371).ML and BI approaches showed minimal differences in evaluation results; thus, only an ML tree was used for display (Figure 1).Our phylogenetic analyses indicated that T. sikkimensis, T. petaloides, and T. lacunosa cluster together with high support values (98/1).T. dactyliophora is sister to T. aquila S.R. Yang, Y.L. Wei & H.S. with moderate support values (93/1).Thelephora pinnatifida forms a separate branch with high support values (100/1).

Analyses of Genetic Distances
In this study, the Maximum Composite Likelihood model was used to calculate the genetic distances within and between species of Thelephora used in this study.As shown in Table S1, the genetic distances of the ITS sequences within species ranged from 0 to 0.9%, with an average distance of 0.2%; the genetic distances between species ranged from 1.2% to 22.3%, with an average of 12.8%.MycoBank: 852625.Etymology: dactyliophora refers to the finger-like branches of the basidiomes.Diagnosis: Basidiomes coralloid; branch multiple ranks, finger-like to narrow flabelliform when young, spathulate to narrow petaloid when mature; abhymenial surface slightly rugulose, non-zonate, brownish gray to gray; hymenial surface concolorous with abhymenial surface, rugulose and non-zonate.Basidiospores nodulose to verrucose.This species is similar to T. palmata (Scop.)Fr. in its coralloid basidiomes, but differs in its slightly rugulose brownish gray to gray hymenial surface and relatively small basidiospores.
Habitat: In subtropical evergreen broad-leaved forests dominated by plants of Fagaceae and Betulaceae, or in mixed forests dominated by plants of Fagaceae and Pinaceae.
Habitat: In subtropical evergreen broad-leaved forests dominated by plants of Fagaceae, or in subtropical mixed forests dominated by plants of Fagaceae and Pinaceae.
Monomitic hyphal system.Pileipellis: generative hyphae 4-5 µm wide, hyaline, subparallel to loosely interwoven, thin-walled, moderately branched, with clamp connections and a few simple septa, yellowish in Melzer's reagent, non-amyloid, non-dextrinoid, acyanophilic.Context: generative hyphae 4-5 µm wide, hyaline, subparallel to loosely interwoven, thin-walled, moderately branched, with clamp connections and a few simple septa, yellowish in Melzer's reagent, non-amyloid, non-dextrinoid, acyanophilic.Subhymenium: generative hyphae 2-4 µm wide, pale yellow to pale yellowish brown, subparallel to slightly divergent, thin-walled, frequently branched near clamp connections, with a few simple septa, yellowish in Melzer's reagent, non-amyloid, non-dextrinoid, acyanophilic.Notes: In our phylogenetic analyses, T. petaloides, T. sikkimensis, T. lacunosa, and T. wuliangshanensis clustered together with high support values (85/1) (Figure 1).However, T. wuliangshanensis is characterized by its infundibuliform basidiomes, the buff to salmon abhymenial surface with radially black striations, and the umber to coffee and nearly glabrous hymenial surface [14].Morphologically, T. petaloides and T. lacunosa share similar characteristics in the flat, flabelliform branches of basidiomes and warty hymenial surface.However, T. lacunosa can be distinguished by its imperceptibly wavy margin of basidiomes; lacunary, sulcate, irregularly ridged, non-zonate, and warty abhymenial surface which is brown, grayish brown to grayish orange but white to pale orange-gray at the margin; and brownish orange hymenial surface with orange-white tint at the margin.In addition, the sulcate abhymenial surface, the flat and flabelliform branches of basidiomes, the white context, and the warty hymenial surface make T. petaloides similar to T. sikkimensis.However, T. sikkimensis is obviously distinguished from T. petaloides in its relatively small basidiomes (45-67 × 50-70 mm in T. sikkimensis vs. 40-100 × 55-220 mm in T. petaloides); grayish black to purplish gray or grayish green abhymenial surface which is chalky white to orange-white at margin; smooth to irregularly plicate hymenium surface which is pale orange to purplish gray or grayish green, but orange-white to almost white at the margin; and rugose and warty stipe which is pale orange or sometimes purplish gray.
Thelephora pinnatifida Yan C. Li & Zhu L. Yang, sp.nov., Figures 2h,i, 3j-l and 7a,b.MycoBank: 852628.Etymology: pinnatifida refers to the pinnatifid margin of branches of the basidiomes.Diagnosis: Basidiomes clavarioid; branch clavate to pinnatifid or ramiform with tips needle-like; surface sulcate, non-zonate, visibly ribbed, brownish orange to brown.Basidiospores nodulose to verrucose.This species is similar to T. iqbalii Khalid & Hanif in its clavarioid basidiomes and needle-like tips of branches but differs in its brownish orange to brown basidiomes with chalky white to orange-white margin and relatively long basidia.
Thelephora sikkimensis K. green (13E2-25E5) elsewhere, but chalky white to orange-white (1A1-5A2) at margin.Hymenial surface rugose, warty, gradually from smooth to irregularly plicate near the margin, non-zonate, pale orange (5A4) when young, purplish gray (13E2) to grayish green (24C1) when mature, but orange-white to almost white (5A2-1A1) at margin.Context 1-2 mm thick, relatively thin at margin and thick towards center, white, odor mild when fresh, yeast powder flavor when drying.Stipe 12-14 × 2-9 mm, irregularly cylindrical, surface rugose and warty, pale orange (5A3), sometimes purplish gray (13E2).Description: Basidiomes 45-67 mm high, 50-70 mm wide, gregarious to caespitose, humid and leathery when fresh, corky to hard and brittle when drying, branches arising nearly glabrous abhymenial surface which is brownish gray at the center becoming orangeyellow to grayish orange or pale orange elsewhere but chalky white at the margin; rugose and warty hymenium which is orange-gray at the center, bright orange elsewhere, and chalky white at the margin; rugose and glabrous brownish gray stipe; and a relatively thick context which is up to 4 mm thick.Thelephora lacunosa has medium-sized basidiomes (67-80 × 80-150 mm); lacunary, irregularly ridged, non-zonate, and warty abhymenial surface which is brown, grayish brown to grayish orange at the center, much paler towards the margin, and white to pale orange-gray at the margin; rugose and warty hymenium surface, brownish orange to gray or purple-gray at the center, pale orange towards the margin, and orange-white at the margin; and subconical stipe which is broadened or flatted at the base with a brownish red to brownish yellow surface.

Discussion
In this study, five species, T. sikkimensis new to China and T. petaloides, T. lacunosa, T. dactyliophora, and T. pinnatifida new to science, are reported based on a combination of phylogenetic analyses, genetic differences, and morphological features.In our phylogenetic analyses, T. lacunosa, T. petaloides, and T. sikkimensis clustered together with high support values, but the relationships among the three taxa are not well solved by the ML and Bayesian analyses.In addition, the diagnostic morphological characters among them are often subtle.It seems that these species might have started diverging from each other relatively recently.Genetic distance measurements have been used frequently as a tool to investigate species boundaries and to identify cryptic diversity in fungi [51][52][53].Based on the Maximum Composite Likelihood model, the genetic variations between the three closely related species T. lacunosa/T.sikkimensis, T. petaloides/T.sikkimensis, and T. lacunosa/T.petaloides are 1.9-3%, 2.7-3.4%, and 2.1-3%, respectively, which are much higher than the within-species genetic variation (ranging from 0 to 0.9%).Morphologically, all of these five species have nodulose to verrucose ornamentations on the surface of basidiospores.But T. sikkimensis, T. petaloides, and T. lacunosa have imbricate basidiomes, while T. dactyliophora and T. pinnatifida have clavarioid basidiomes.It is evident that the shapes of basidiomes and the morphology of the hymenial surface are important characteristics distinguishing species of Thelephora.However, for the precise identification of Thelephora species, the size of basidiomes, basidia, and basidiospores; the shapes and size of the stipe; the color and zonation of abhymenial and hymenial surfaces; and the thickness of the context are also important characteristics.Detailed morphology comparisons among these five species are shown in Table 2.The combination of phylogenetic analyses, genetic differences, and morphological features can enhance the reliability and objectivity of species delimitation [54].
To date, 285 species of this genus have been reported and described (Index Fungorum: http://www.indexfungorum.org/Names/Names.asp,accessed on 6 April 2024), of which 29 (including the 5 species reported in this study) have been reported from China.China is one of the most biodiverse countries in the world.Like other groups of fungi, there are likely to be many Thelephora species that have not been scientifically described or remain to be discovered [55].Further studies are needed to enrich the species diversity of this genus.

Figure 1 .
Figure 1.Phylogenetic relationships between species of Thelelphora inferred from ITS dataset using the Maximum Likelihood and Bayesian inference approaches (ML topology is shown).ML bootstrap support (≥50) and Bayesian posterior probability (≥0.95) are shown at the branches (BS/PP).Sequences newly generated in this study are highlighted in red.Vouchers are indicated after the species names.Odontia fibrosa and O. ferruginea were used as the outgroup taxa.

Figure 1 .
Figure 1.Phylogenetic relationships between species of Thelelphora inferred from ITS dataset using the Maximum Likelihood and Bayesian inference approaches (ML topology is shown).ML bootstrap support (≥50) and Bayesian posterior probability (≥0.95) are shown at the branches (BS/PP).Sequences newly generated in this study are highlighted in red.Vouchers are indicated after the species names.Odontia fibrosa and O. ferruginea were used as the outgroup taxa.
• C for 4 min, 35 cycles of 30 s at 94 • C, 40 s at 53 • C, 1 min at 72 • C, a final extension of 8 min at 72 • C, and then coolant at 14

Table 1 .
Species, vouchers, GenBank/UNITE accessions, and localities of specimens used in this study.Sequences newly generated in this study are shown in bold.