Morpho-Phylogenetic Evidence Reveals New Species of Fuscosporellaceae and Savoryellaceae from Freshwater Habitats in Guizhou Province, China

During a survey of freshwater fungi in Guizhou Province, China, six hyphomycetous collections were founded on decaying wood from freshwater habitats. These taxa were characterized and identified based on morphology, phylogeny, and culture characteristics. Phylogenetic analysis of combined LSU, SSU, ITS, RPB2 and TEF1α sequence data indicated that our six isolates formed three distinct lineages and were distributed within Fuscosporellaceae and Savoryellaceae. They can be organized as three new species: Fuscosporella guizhouensis, Mucispora aquatica and Neoascotaiwania guizhouensis. Fuscosporella guizhouensis and Neoascotaiwania guizhouensis have sporodochial conidiomata, micronematous conidiophores and dark brown conidia. The former possesses irregularly ellipsoidal conidia with apical appendages, while the latter has fusiform to obovoid conidia. Mucispora aquatica is characterized by macronematous conidiophores, elongating percurrently and dark brown, narrowly obovoid conidia. The detailed, illustrated descriptions and notes for each new taxon are provided, and the species of Fuscosporella is reported for the first time in China.


Introduction
Freshwater fungi were defined as "fungi that the whole or part of their life cycle rely on freshwater" [1]. They are a diverse and heterogeneous group comprising different species and play an essential role in the organic carbon cycle of aquatic ecosystems [2,3]. Freshwater habitats include lentic and lotic water [1,2] and artificial habitats [4,5]. Calabon et al. [6] recently gave a comprehensive review of the freshwater fungal numbers and listed 3870 freshwater fungal species. Two hundred and ninety-eight novel species have been described in China and Thailand from 2015 to 2020 [6]. It is worth noting that the application of molecular techniques coupled with traditional morphology has significantly improved fungal identification and classification, especially the phylogenetic relationships of freshwater taxa.
Six isolates were obtained from submerged decaying wood during the survey of freshwater fungi in Guizhou Province, China. This study aims to describe these new findings and contribute to fungal diversity in China. Morphological comparison coupled with multi-gene phylogeny was carried out to determine the classification of these new collections. As a result, three new species are introduced, and the establishment of these new taxa is justified by morphology and phylogenetic evidence.

Collection and Examination of Specimens
Specimens of submerged decaying wood were collected from a freshwater stream in Guizhou Province, China, in February 2021. Samples were brought to the laboratory in plastic bags and incubated in plastic boxes lined with moistened tissue paper at room temperature for one week. Morphological observations were made using a Motic SMZ (Stereoscopic Zoom Microscope) 168 Series dissecting microscope (Motic, Xiamen, China) for fungal structures on a natural substrate. The fruiting bodies were collected using a syringe needle and transferred to a drop of tap water on a clean slide. The features were examined and photographed using a Nikon ECLIPSE Ni-U compound microscope fitted with a Nikon DS-Ri2 digital camera. Measurements were made with the Tarosoft Image Frame Work v. 0.9.7 software following the procedures outlined by Liu et al. [21], and images used for photo plates were processed with Adobe Photoshop CC 2018 software (Adobe Systems, San Jose, CA, USA). Single-spore isolations were made on potato dextrose agar (PDA) or water agar (WA) and later transferred onto new PDA plates following the methods described in Senanayake et al. [22]. Incubation and cultural growth were observed at 25 • C.
Herbarium specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and Herbarium, University of Electronic Science and Technology (HUEST), Chengdu, China. The pure cultures obtained in this study were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China. The new taxa were registered in MycoBank (2022).

DNA Extraction, PCR Amplification and Sequencing
Isolates grew in PDA medium at 25 • C for one month. Fungal mycelia were scraped off and transferred to 1.5 mL microcentrifuge tubes using a sterilized lancet for genomic DNA extraction. A Tsingke Fungus Genomic DNA Extraction Kit (Tsingke Biotech, Shanghai, China) was used to extract DNA following the manufacturer's instructions. Five gene regions were amplified by Polymerase Chain Reaction (PCR). The nuclear large subunit rDNA (28S, LSU), nuclear small subunit rDNA (18S, SSU), internal transcribed spacer (ITS), RNA polymerase second-largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1α) were selected for the study. The primers used were LR0R/LR5 for LSU [23], NS1/NS4 for SSU [24], ITS5/ITS4 for ITS [24], fRPB2-5F and fRPB2-7cR for RPB2 [25] and TEF1-983F/TEF1-2218R for TEF1α [26]. The amplifications were performed in a 25 µL reaction volume containing 9.5 µL of ddH 2 O, 12.5 µL of 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China), 1 µL of DNA template and 1 µL of each primer. The amplification condition for ITS, LSU, SSU and TEF1α consisted of initial denaturation at 94 • C for 3 min, followed by 40 cycles of 45 s at 94 • C, 50 s at 55 • C and 1 min at 72 • C, and a final extension period of 10 min at 72 • C. The amplification condition for the RPB2 gene consisted of initial denaturation at 95 • C for 5 min; followed by 37 cycles of 15 s at 95 • C, 50 s at 56 • C and 2 min at 72 • C, and a final extension period of 10 min at 72 • C. The PCR product purification and sequencing were performed at Beijing Tsingke Biotechnology (Chengdu) Co., Ltd., Chengdu, China.
ML analyses were performed with RAxML-HPC v.8 on XSEDE (8.2.12) [32,33] through the CIPRES Science Gateway V. 3.3 (https://www.phylo.org/portal2/login!input.action (accessed on 18 May 2022)) [34]. The tree search included 1000 non-parametric bootstrap replicates; the best scoring tree was selected among suboptimal trees from each run by comparing likelihood scores under the GTRGAMMA substitution model. The resulting replicates were plotted on to the best scoring tree obtained previously. ML bootstrap values equal to or greater than 75% were marked near each node.
BI was performed in MrBayes 3.2.6 [35]. The program MrModeltest 2 v. 2.3 [36] was used to determine the best nucleotide substitution model for each data partition. The GTR + I+G substitution model was decided for all LSU, SSU, ITS, RPB2 and TEF1α genes. Posterior probabilities (PP) [37] were determined by Markov chain Monte Carlo sampling (MCMC). Six simultaneous Markov chains were run for 10 million generations, and trees were sampled every 1000th generation. The first 25% of the saved trees, representing the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating posterior probabilities in the majority rule consensus tree [38]. PP values equal to or greater than 0.95 were marked near each node.
MP analyses with the heuristic search were performed in PAUP v. 4.0 b10 [39]. The gaps in the alignment were treated as missing characters, and all characters were unordered. Maxtrees were unlimited, branches of zero length were collapsed, and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa [40]. MP bootstrap values equal to or greater than 75% were marked near each node.
Phylogenetic trees were printed with Fig Tree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ accessed on 18 July 2022)) and the layout was created in Adobe Illustrator CS6 software (Adobe Systems, Inc., San Jose, CA, USA). The sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic Results
Five gene loci, LSU, SSU, ITS, RPB2, and TEF1α, were used to determine the phylogenetic placement of the new collections. The concatenated matrix was comprised of 50 taxa with a total of 4822 characters (LSU: 1-942 bp, SSU: 943-2168 bp, ITS: 2169-2794 bp, RPB2: 2795-3871 bp, TEF1α: 3872-4822 bp) including gaps. Single-gene analyses were carried out to compare the topologies and clade stabilities, respectively. The results showed that ML, MP and Bayesian inference (BI) were similar in topology without significant conflictions, and these results agree with previous studies [7,16,53]. The best scoring RAxML tree (−ln = −38 991.137) is shown in Figure 1.

Phylogenetic Results
Five gene loci, LSU, SSU, ITS, RPB2, and TEF1α, were used to determine the phylogenetic placement of the new collections. The concatenated matrix was comprised of 50 taxa with a total of 4822 characters (LSU: 1-942 bp, SSU: 943-2168 bp, ITS: 2169-2794 bp, RPB2: 2795-3871 bp, TEF1α: 3872-4822 bp) including gaps. Single-gene analyses were carried out to compare the topologies and clade stabilities, respectively. The results showed that ML, MP and Bayesian inference (BI) were similar in topology without significant conflictions, and these results agree with previous studies [7,16,53]. The best scoring RAxML tree (−ln = −38 991.137) is shown in Figure 1.  Culture characteristics: Conidia germinated on WA within 24 h, and germ tubes produced from basal cell. Colonies growing on PDA reached 12−15 mm in diameter after one month at 25 • C, obverse olive to greyish green or dark greyish green in the inner, and light greyish green in the outer ring from above, reverse dark greyish green. Mycelium in culture up to 1-3 µm wide, subhyaline to brown, septate, branched. Conidiophores and conidiogenous cells indistinct. Chlamydospores are apparent in culture, globose to ellipsoidal or irregular shaped, hyaline at the beginning, becoming brown to black with ages, 10−21 × 5−19 µm (x = 16 × 12 µm, n = 30).

Taxonomy
Material examined: China, Guizhou Province, Guiyang City, Wudang District, Xiangsihe scenic spot, undisturbed forests with freshwater habitats, 26 • [7]. The conidia of F. guizhouensis are irregular ellipsoidal, while F. pyriformis has obovoid to pyriform conidia. In addition, F. guizhouensis is distinguished by the hyaline apical appendages, which are absent in F. pyriformis and F. aquatica [44]. Fuscosporella guizhouensis can be distinguished from F. aquatica (17/859 in LSU, 60/524 in ITS) and from F. pyriformis (13/814 in LSU, 58/587 in ITS and 52/1024 in RPB2). Therefore, Fuscosporella guizhouensis is introduced as a new species, and this is the first Fuscosporella species reported from China.   (Figure 1) showed that Mucispora aquatica has a close phylogenetic relationship with M. infundibulata and M. obscuriseptata, but it can be recognized as a distinctly phylogenetic species. Therefore, we introduced Mucispora aquatica as a new species based on morphology and phylogeny.     Culture characteristics: Conidia germinated on WA within 24 h, and germ tubes produced from basal cell. Colonies growing on PDA reached 8−10 mm in diameter after one month at 25 • C, with white and dense mycelium on the surface, the center greyish green, reverse greyish brown and with a dark greyish brown ring in the middle. After one month, the diameter did not increase significantly. Mycelium hyaline to brown, septate, branched, 2−4 µm (x = 3 µm, n = 30) wide, Chlamydospores are apparent, hyaline at the beginning, becoming brown or dark brown, 8−13 × 6−10 (x = 11 × 9 µm, n = 30).
Material examined: CHINA, Guizhou Province, Guiyang City, Wudang District, Xiangsihe scenic spot, undisturbed forests with freshwater habitats, 26 • (Figure 1), Neoascotaiwania guizhouensis was sister to N. terrestris, but they are distinguishable in morphology and phylogeny. Therefore, we introduced Neoascotaiwania guizhouensis as a new species.

Discussion
The phylogenetic analyses based on the combined gene regions (LSU, SSU, ITS, RPB2 and TEF1α) placed three new species, Fuscosporella guizhouensis, Mucispora aquatica and Neoascotaiwania guizhouensis, in Fuscosporellaceae and Savoryellaceae (Savoryellomycetidae, Sordariomycetes) and are described in asexual stages without known sexual morphs. Species in Fuscosporella and Mucispora are reported from freshwater habitats in Thailand

Discussion
The phylogenetic analyses based on the combined gene regions (LSU, SSU, ITS, RPB2 and TEF1α) placed three new species, Fuscosporella guizhouensis, Mucispora aquatica and Neoascotaiwania guizhouensis, in Fuscosporellaceae and Savoryellaceae (Savoryellomycetidae, Sordariomycetes) and are described in asexual stages without known sexual morphs. Species in Fuscosporella and Mucispora are reported from freshwater habitats in Thailand and China [7,11,44,54]; they may be exclusive in freshwater habitats. In this study, we provide the first record of Fuscosporella in China. Neoascotaiwania taxa are widely distributed in France, Spain and Thailand [7,8,14]. Neoascotaiwania guizhouensis, N. fusiformis, and N. limnetica are also found on decaying submerged wood in freshwater habitats [7,8,14,16,55], while N. terrestris was isolated from soil [14], which indicates that they are widely distributed and not limited by the growth environment.
Multi-locus phylogenetic analysis has been crucial for delimiting the novel fungi [61]. The use of multi-gene datasets to infer phylogenetic relationships has dramatically improved the resolution, especially when protein genes are combined with other genes, and the solution substantially increased [62,63]. For Fuscosporella and Mucispora, ITS, LSU and SSU rDNA datasets are available for all the species [7,11,44,54]. However, for the protein genes, only two species had the RPB2 sequence (unverified), and no TEF1α dataset. Therefore, the problem of low similarity occurred after the blastn search without a corresponding sequence in the same genus for alignment. This study provides the RPB2 and TEF1α sequences of Fuscosporella and Mucispora, which make up for the lack of protein genes in these two genera.