Two new species of Exidia sensu lato (Auriculariales, Basidiomycota) based on morphology and DNA sequences

In the present study, fourteen Exidia-like specimens were collected from China, France, and Australia. Based on morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS) and the large subunit of nuclear ribosomal RNA gene (nLSU), four species in Exidia sensu lato, including Exidia saccharina and Tremellochaete atlantica, and two new species, Exidia subsaccharina and Tremellochaete australiensis, were identified. The four species are described and illustrated in detail. E. saccharina and T. atlantica, two known species from China are reported for the first time. E. subsaccharina and T. australiensis, two new species from France and Australia, respectively are also described. E. subsaccharina is characterized by its reddish brown to vinaceous brown basidiomata, slightly papillate hymenial surface, and narrowly allantoid basidiospores without oil drop measuring 12.5–17.5 × 4.2–5.5 μm. It differs from the similar species, E. saccharina, by distinctly larger basidiospores (12.5–17.5 × 4.2–5.5 vs. 10–14.2 × 3.2–4.5 μm). Tremellochaete australiensis is characterized by its white to grayish blue basidiomata, obviously and densely papillate hymenial surface, and allantoid basidiospores with oil drop measuring 13.8–16.2 × 4.8–6.5 μm. It also can be distinguished from the similar species, T. atlantica and T. japonica, by its distinctly larger basidiospores (13.5–17.8 × 4–5.2 vs. 10–11.8 × 4–4.8 μm in T. atlantica; 9.4–11.8 × 3.5–4.2 μm in T. japonica).

In the present study, fourteen Exidia-like specimens were collected from China, France, and Australia. After morphological examinations and phylogenetic analyses using the internal transcribed spacer regions (ITS) and the large subunit of the nuclear ribosomal RNA gene (nLSU), four species were identified in Exidia and Tremellochaete, among which two are new to science, and a detailed description of these species is given in the present study.

Morphology
The studied specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), with color terms following those outlined by Petersen (1996). Sections mounted in 5% KOH and 2% phloxine B (C 20 H 2 Br 4 C l4 Na 2 O 5 ) were studied at a magnification of up to 1,000× using a Nikon Eclipse 80i microscope and phase contrast illumination. A Nikon Digital Sight DS-L3 camera was used to photograph microscopic structures. We also used other reagents, including Cotton Blue and Melzer's reagent to observe micromorphology following Wu et al. (2022b). To show the variation in spore sizes, 5% of measurements were excluded from each end of the range and shown in parentheses. At least thirty basidiospores from each specimen were measured. Stalks were excluded from basidia measurements, and the hilar appendage was excluded from basidiospore measurements. The following abbreviations were used: KOH, potassium hydroxide (5%); L, mean length (arithmetic average of all basidiospores length); W, mean width (arithmetic average of all basidiospores width); Q, L/W ratio for each specimen studied; n (a/b), number of basidiospores (a) measured from a given number of specimens (b).
DNA extraction, PCR reaction, and sequencing DNA was extracted from dried specimens using a rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) and modified following Wu et al. (2021). The internal transcribed spacer regions (ITS) and the large subunit of the nuclear ribosomal RNA gene (nLSU) were amplified with primer pairs ITS 4 and ITS 5 (White et al., 1990) and LR0R and LR7 (Vilgalys and Hester, 1990), respectively. The PCR (polymerase chain reaction) procedure for ITS was 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 at 72 • C for 10 min. The PCR procedure for nLSU was initial denaturation at 94 • C for 1 min, followed by 35 cycles at 94 • C for 1 min, 48 • C for 1 min, and 72 • C for 1.5 min, and a final extension at 72 • C for 10 min (Wu et al., 2018). The PCR products were purified and sequenced at the BGI (Beijing Genomics Institute, China), with the same primers that are used in the PCR reactions. The nLSU    sequences were obtained by splicing bidirectional sequences because LR0R-LR7 is >1,000 bp.

Phylogenetic analyses
The new sequences generated in this study and reference sequences retrieved from GenBank (Table 1) were aligned with MAFFT (version 7; Katoh and Standley, 2013) and then manually adjusted in BioEdit and Mesquite version 3.04 software (Hall, 1999;Maddison and Maddison, 2017). A dataset composed of concatenated ITS+nLSU sequences was used in the phylogenetic analyses using the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) methods. Bourdotia galzinii (Bres.) Trotter was selected as the outgroup in the phylogenetic analyses because the species was closer to species of Auriculariaceae than others but not closely related to species in Exidia sensu lato (Spirin et al., 2019a). Except for the outgroup, sequences from the other eleven Auriculariales genera were added to the phylogenetic analyses because Exidia was previously shown to be polyphyletic (Yuan et al., 2018;Spirin et al., 2019a).

Phylogenetic analyses
The combined ITS+nLSU dataset included 81 fungal specimens representing 44 species in the Auriculariales. The dataset had an aligned length of 1,898 characters, including 1,406 constants, 156 parsimony-uninformative characters, and 336 parsimony-informative characters. MP analysis yielded four equally parsimonious trees (tree length = 1,607, consistency index = 0.432, retention index = 0.725, rescaled consistency index = 0.313, and homoplasy index = 0.568). The average standard deviation of split frequencies in BI analysis was 0.005425. The topology of the ML tree with bootstrap values for BP, BS, and BPP was chosen to represent the phylogenetic relationship of species in the Auriculariales since ML, MP, and BI resulted in similar topologies (Figure 1). The phylogeny demonstrated our fourteen Exidia-like specimens were clustered into four different lineages with high support, including two new lineages that represented two new species, E. subsaccharina (100% BS, 1.00 BPP, and 100% BP; Figure 1) and Tremellochaete australiensis (100% BS, 1.00 BPP, and 100% BP; Figure 1). The four Chinese specimens and one German sample (Roki 88) identified as E. saccharina by Weiß and Oberwinkler (2001) were nested in the same lineage with high support in the phylogeny (Figure 1), so these specimens were treated as E. saccharina, and this represents the first record of the species in China.  Basidiomata: When fresh, the basidiomata are gelatinous, fawn to orange-brown, suborbicular to cerebriform, sessile, usually remaining separate, occasionally coalescing; are up to 10 cm wide and 1.5 cm thick; have free margins; have a hymenial surface that is clearly ridged, with sparse papillae, becoming vinaceous brown when dry; and are absent of mineral inclusions.
Basidiomata: When fresh, the basidiomata are gelatinous, white to ash-gray or brownish, suborbicular to slightly cerebriform, sessile, usually remaining coalescing, occasionally separate; are fused, with up to 10 cm width and 1 cm thickness; have free margins; have occasionally ridged hymenial surface, are obviously and densely studded with irregular papillae, becoming dark gray or grayish brown when dry; and are absent of mineral inclusions.
Basidiomata: When fresh, the basidiomata are gelatinous, white to grayish blue, suborbicular to slightly cerebriform, sessile, usually remaining coalescing, occasionally separate; are fused together with up to 20 cm in width and 0.5-1 cm in thickness; have free margins; have a hymenial surface occasionally ridged, is clearly and densely studded with irregular papillae, becoming dark gray to black when dry; and are absent of mineral inclusions.
Additional specimens examined (

Discussion
Exidia sensu lato is a genus of wood-inhabiting fungi that grows on dead branches and logs and is best known in the temperate regions of Europe, America, and Asia (Malysheva, 2012;Spirin et al., 2018;Wu et al., 2020a;Ye et al., 2020;Wang and Thorn, 2021). Although nearly eighty taxa were recorded in Exidia sensu lato, most species were described in the 20th century (Fries, 1822;Lowy, 1964Lowy, , 1971. In recent years, four species in Exidia and two species in Tremellochaete were described based on morphology and phylogenetic analyses (Alvarenga et al., 2019;Wu et al., 2020a;Ye et al., 2020;Wang and Thorn, 2021), which improved knowledge of Exidia sensu lato across the world. However, since the demarcation of Exidia and Tremellochaete is still ambiguous, multilocus analyses based on taxonomically and geographically broad sampling are needed.

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.

Author contributions
FW and Y-CD coordinated the project, designed the experimental plan, and acquired funding. AT and FW analyzed the data and prepared the original draft. BR and Y-CD collected the samples from the field. S-XH, GG, BR, and Y-CD reviewed .
/fmicb. . and edited the manuscript. All authors contributed to the study and approved the submitted version.

Funding
This study was supported by the National Natural Science Foundation of China (Project Nos. 32070006 and 32270011), the Tibet Autonomous Region Science and Technology Project (XZ202201ZY0006N), and the Fundamental Research Funds for the Central Universities (No. 2021ZY91).