Morphology and phylogeny identify two new species and one new subspecies of Podoscypha from Yunnan Province, Southwest China

In this study, Podoscypha was taxonomically and phylogenetically evaluated. In total, five specimens collected from the tropical areas of Yunnan Province in Southwest China were studied. In combination with morphological observations and phylogenetic analyses based on ITS and LSU loci, two new species and one new subspecies, Podoscypha subinvoluta, P. tropica, and P. petalodes subsp. cystidiata, respectively, were discovered. The illustrated descriptions of the new species and subspecies are provided. Moreover, the main morphological differences between related species are discussed.

By the end of 2022, only four Podoscypha species: Podoscypha brasiliensis D.A. Reid, P. elegans (G. Mey.) Pat., P. simulans (D.A. Reid) Sheng H. Wu, and P. yunnanensis C.L. Zhao had been discovered in China (Wu, 2003;Dai, 2011;Wu et al., 2019). During field investigations on macrofungi diversity in the tropical areas in Yunnan Province, China, several collections of Podoscypha were obtained. Morphological and phylogenetic analyses showed that these represented two new species and one new subspecies. In this study, the species are described with illustrated morphological description, phylogeny, and comparisons with morphologically similar or phylogenetically related species.

Morphology
All the studied specimens were processed and deposited in the National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control (NIOHP, China CDC). Macromorphological descriptions were based on the field notes and color photographs of basidiocarps. Color codes were matched based on Petersen (1996). The microscopic procedure was performed according to Reid (1965), Douanla-Meli and Langer (2004), and Wu et al. (2019). Sections were studied at a magnification of ×1,000 with a Nikon E 80i microscope and phase-contrast illumination. Line drawings were made with the aid of a drawing tube. When describing basidiospores, the abbreviation n/m/p means that "n" basidiospores from "m" basidiomata of "p" collections were measured. The dimensions for basidiospores are given using the following notation form (a−) b−c (−d). The range "b−c" contains a minimum of 90% of the measured values, and extreme values (a, d) are given in parentheses. L means the spore length (arithmetic average of all spores). W means the spore width (arithmetic average of all spores). Q is the "length/width ratio" of a spore in side view; Q m means the average Q of all basidiospores measured ± sample standard deviation. In the text, the following abbreviations were used: IKI = Melzer's reagent, IKI− = both in amyloid and non-dextrinoid, KOH = 5% potassium hydroxide, CB = cotton blue, CB+ = cyanophilous, and CB− = acyanophilous.

DNA extraction, PCR amplification, and phylogenetic analysis
The Phire ® Plant Direct PCR Kit (Finnzymes Oy, Finland) was used to obtain PCR products from dried specimens, according to the manufacturer's instructions with some modifications (Li et al., 2015). ITS5/ITS4 were used to amplify the internal transcribed spacer (ITS) regions (White et al., 1990). LR0R/LR5 (Vilgalys and Hester, 1990) were used to amplify nuclear large subunit (LSU) rDNA. The PCR procedure for amplifying ITS and LSU sequences was as follows: initial denaturation at 98°C for 5 min; 35 cycles at 98°C for 5 s, 58°C and 52°C for 5 s, and 72°C for 5 s; and a final extension at 72°C for 10 min, respectively. The PCR products were qualified by electrophoresis and sequenced at Sangon Biotech, China, with the same primers. All newly generated sequences were first aligned and then deposited in GenBank. The results indicated that they were all Podoscypha species.
In addition to the sequences obtained from this study, the sequences of other taxa in Podoscypha were downloaded from GenBank, and Abortiporus biennis was selected as the outgroup for phylogenetic analysis, mainly based on the reports of Binder et al. (2013) and Wu et al. (2019). The detailed information is shown in Table 1. The sequences were aligned using ClustalX 1.83 (Chenna et al., 2003) and optimized manually in BioEdit 7.0.5.3 (Hall, 1999) prior to the phylogenetic analysis.
For analyzing BI, the best-fit models of nucleotide substitution were selected by the hierarchical likelihood ratio tests (hLRT; Huelsenbeck and Crandall, 1997;Posada and Crandall, 2001) using MrModelTest 2.2 (Posada and Crandall, 1998;Nylander, 2004). The best-fit models were GTR + G for ITS and LSU. Four Markov chains were set to run for 2 and 4 million generations for the ITS and LSU datasets, respectively, and then, automatically terminated using the stoprul and stopval commands when the standard deviation of the split frequencies fell below 0.01, with sampling for every 100th generation. The first 25% of trees were discarded (Ronquist and Huelsenbeck, 2003). Phylogenetic trees were visualized using TreeView (Page, 1996). Branches that received bootstrap supports for MP (≥75%), BPP (≥0.95), and ML (≥75%) were considered significantly supported.

Phylogenetic analyses
The ITS dataset consisted of 44 sequences representing 22 taxa, of which 21 were Podoscypha. The aligned length of the ITS sequence was 835 bp, which contained 447 parsimony informative sites. MP analysis revealed two equally parsimonious trees (tree length = 1,541, CI = 0.635, RI = 0.816, RC = 0.518, and HI = 0.316). BI and ML analyses showed almost identical tree topologies as MP analysis, with an average standard deviation of split frequencies of 0.007834 for BI analysis. Additionally, the MP tree is presented with the support values from MP, BI, and ML analyses ( Figure 1).
The LSU dataset consisted of 44 sequences, representing approximately 18 taxa, of which 17 were Podoscypha. The aligned length of the LSU sequence was 926 bp, which contained 72 parsimony informative sites. MP analysis revealed 44 equally parsimonious trees (tree length = 161, CI = 0.745, RI = 0.917, RC = 0.684, HI = 0.255). BI and ML analyses resulted in almost identical tree topologies to MP analysis, with an average standard deviation of split frequencies of 0.006556 for BI analysis. Additionally, the MP tree is presented with the support values from MP, BI, and ML analyses ( Figure 2).
Habitat and distribution-Gregarious on angiosperm trees or fallen angiosperm branches, at present only discovered from its type locality, summer.
Known distribution-China (Yunnan). Description-Basidiocarps annual, gregarious, stipitate, without odor or taste, corky when fresh and hard corky upon drying. Pilei Frontiers in Microbiology 07 frontiersin.org spathulate to flabelliform, up to 4 cm high and 5 cm wide. Pileal surface glabrous, concentrically and radially zonate; cinnamon, yellowish brown, pinkish buff to clay-buff at the center and near the stipe when fresh, cinnamon, yellowish brown to orange-brown when dry, and gradually lightening toward the margin. The margin is cream to buff when fresh, pinkish buff to cinnamon-buff when dry and wavy. Hymenophore surface smooth, concentrically and radially zonate, cream, buff, to cinnamon-buff when fresh, flesh-pink to clay-buff when dry. Stipe surface glabrous, cinnamon, yellowish brown, pinkish buff to clay-buff near the cap, and dark brown to black toward the base when fresh; the color is similar to the pileal surface when dry, up to 3 cm long and 1.5-4 mm in diameter.
Hyphal structure-dimitic, generative hyphae with clamps, hyaline, thin-to thick-walled, branched; skeletal hyphae colorless, thick-walled with a wide to narrow lumen, unbranched to rarely branched; IKI−, CB−, tissues unchanged in KOH. Generative hyphae in the pileal context dominant, hyaline, thin-to thick-walled, branched, almost parallel along the pileal surface, 2-5 μm in diameter; skeletal hyphae in the pileal context hyaline, thick-walled, with a wide to narrow lumen, unbranched to rarely branched, 2-4 μm in diameter; generative hyphae in the stipe trama similar to that in the pileal context, 3-4 μm in diameter; skeletal hyphae in the stipe trama similar to that in the pileal context, 1.5-3.5 μm in diameter.
Habitat and distribution-Gregarious on fallen angiosperm trunks, at present only discovered from its type locality, summer.
Known distribution-China (Yunnan). Description-Basidiocarps annual, gregarious, stipitate, without odor or taste, corky when fresh and hard corky upon drying. Pilei flabelliform or reniform, up to 4.5 cm high and 6.5 cm wide. Pileal surface tomentose near the stipe and glabrous near the margin, concentrically and radially zonate; most parts white to cream, buff, cinnamon, yellowish brown to clay-buff near the stipe when fresh, flesh-pink to clay-pink to cinnamon when dry. Hymenophore surface smooth, cream, buff, to cinnamon when fresh, pinkish buff to claybuff when dry. Stipe surface glabrous, cinnamon to yellowish brown when fresh, color similar to the pileal surface when dry, up to 1.2 cm long and 1.5-3 mm in diameter.
Hyphal structure-dimitic, generative hyphae with clamps, hyaline, thin-to thick-walled, branched; skeletal hyphae colorless, thick-walled with a wide to narrow lumen, unbranched to rarely branched; IKI−, CB−, tissues unchanged in KOH. Generative hyphae in the pileal context common, hyaline, thin-to thick-walled, branched, 2-5 μm in diameter; skeletal hyphae in the pileal context dominant, hyaline, thick-walled, with a wide to narrow lumen, unbranched to rarely branched, more or less parallel along the pileal surface, 3-6 μm in diameter; generative hyphae in the stipe trama similar to that in the pileal context, 3-4 μm in diameter; skeletal hyphae in the stipe trama similar to that in the pileal context, 4-7 μm in diameter, branched skeletal hyphae rare, thick-walled to subsoil, 2-3.5 μm in diameter, may be easily misinterpreted as binding hyphae.
Etymology-tropica referring to the species being distributed in the tropics.
Habitat and distribution-Gregarious on fallen angiosperm trunks or fallen angiosperm branches, at present only discovered from its type locality, summer.
Known distribution-China (Yunnan). Description-Basidiocarps annual, gregarious, stipitate, without odor or taste, corky when fresh, and hard corky upon drying. Pilei spathulate, reniform, flabellate to pseudoinfundibuliform, up to 5 cm high and 6 cm wide. Pileal surface glabrous, yellowish brown, pinkish buff to brownish orange at the center and near the stipe when fresh, clay-buff to orange-brown when dry. The margin cream, buff to pinkish buff when fresh, clay-buff to orange-brown when dry, and strongly dentate. Hymenophore surface smooth, cream to buff when fresh, cinnamon to clay-buff when dry. Stipe surface glabrous, buff to straw-yellow on the upper part, and dark brown to fuscous toward the base when fresh, and the color slightly paler when dry, up to 5 cm long and 2-4 mm in diameter.
Hyphal structure-dimitic, generative hyphae with clamps, hyaline, thin-walled, branched; skeletal hyphae colorless, thickwalled with a wide to narrow lumen, unbranched to rarely branched; IKI−, CB−, tissues unchanged in KOH. Generative hyphae in the pileal context common, hyaline, thin-walled, branched, interwoven, 2-4 μm in diameter; skeletal hyphae in the pileal context dominant, hyaline, thick-walled, with a wide to the narrow lumen, unbranched to rarely branched, more or less parallel along the pileal surface, 2-5 μm in diameter; generative hyphae in the stipe trama similar to that in the pileal context, 2-4.5 μm in diameter; skeletal hyphae in the stipe trama similar to that in the pileal context, 2-4 μm in diameter.
Additional specimen examined (
Podoscypha subinvolutais similar to P. involuta by its partly tomentose concentrically and radially zonate pileal surface, mostly lanceolate cystidia, pilocystidia clavate to cylindrical, thick-walled, and small basidiospores; however, P. subinvoluta lacks gloeocystidia but has caulocystidia and larger basidiospores (2.5-3.7 × 2-2.7 μm vs. 2-3 × 1.75-2 μm, Reid, 1965). The partial ITS1 sequence of P. subinvoluta was obtained. Among the 279 bp aligned sequences, it clustered with one sequence of P. involuta (JN649357) from Thailand (only 2 bp sites were different), whereas it differed from the other two P. involuta sequences (JN649358 and MH861804) by 23 bp sites. Thus, we speculate that the specimen of P. involuta (JN649357) may be P. subinvoluta. The LSU sequence from the type specimen of P. subinvoluta grouped with two P. involuta sequences, and only 5 bp sites were different in the 915 bp aligned sequences.
Frontiers in Microbiology 10 frontiersin.org its smaller basidiospores (4-5 × 2.75-3.2 μm, Reid, 1965). Similar to P. tropica, P. disseminata also has gloeocystidia, pilocystidia, and caulocystidia; however, P. disseminata has pilocystidia and caulocystidia with secondary septata, chlamydospores in the context, and slightly narrower basidiospores (4.5-6 × 3-4 μm, Douanla-Meli and Langer, 2004). Podoscypha yunnanensis was a new species described in Yunnan Province, China (Wu et al., 2019). It has only caulocystidia without gloeocystidia and pilocystidia, which can be easily distinguished from the two new species and one new subspecies discovered in this study. The diversity of Podoscypha in China is poorly understood, especially in the subtropical and tropical regions. Further studies are required to reveal the species diversity in China.

Author contributions
H-JL designed the research. H-JL, JS, Y-ZZ, and J-QL prepared the samples, conducted molecular experiments, and analyzed the data. H-JL and JS discussed the results and drafted and edited the manuscript. JS carried out the project administration and funding acquisition. All authors contributed to the manuscript and approved the submitted version.

Funding
The research was financed by the National Natural Science Foundation of China (32270016 and 32070016).