Additions to Bambusicolous Fungi of Savoryellaceae from Southwest China

Asexual and sexual morphs of saprobic bambusicolous fungi were collected from freshwater and terrestrial habitats in Sichuan Province, China. Taxonomic identification of these fungi was carried out on the basis of morphological comparison, culture characteristics, and molecular phylogeny. Multi-gene phylogeny based on combined SSU, ITS, LSU, rpb2, and tef1α sequence data was performed to determine their phylogenetic placement, and the result showed that these fungi belong to Savoryellaceae. Morphologically, four asexual morphs are similar to Canalisporium and Dematiosporium, while a sexual morph well-fits to Savoryella. Three new species, Canalisporium sichuanense, Dematiosporium bambusicola, and Savoryella bambusicola are identified and described. Two new records, C. dehongense and D. aquaticum, were recovered from the bamboo hosts in terrestrial and freshwater habitats, respectively. In addition, the nomenclatural confusion of C. dehongense and C. thailandense is discussed.

Members of Savoryellaceae are often found in freshwater habitats [17,23,26]. For example, the genus Canalisporium is found in freshwater habitats and typified by C. caribense [27].

Isolation and Morphological Examination
Fungi associated with decaying bamboo were collected from Dujiangyan and Qionglai in Sichuan Province, China in 2021. Specimens were placed in envelopes or plastic bags and taken to the laboratory. Morphological characteristics were observed using a Nikon ECLIPSE E200 stereo microscope and photographed by a Nikon ECLIPSE Ni-U compound microscope fitted with a Nikon DS-Ri2 digital camera as per the guidelines provided in Senanayake et al. [31]. Single-spore isolations were used to obtain pure cultures following the methods described by Senanayake et al. [31]. Measurements were made with the Tarosoft (R) Image Framework program v. 0.9.7, following Liu et al. [32]. Photo plates representing fungal structures were processed in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA). Herbarium specimens (dry branches with fungal material) were deposited in the herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS), Kunming, China and the herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China. The isolates obtained in this study were deposited in China General Microbiological Culture Collection Center (CGMCC) in Beijing, China and the University of Electronic Science and Technology Culture Collection (UESTCC) in Chengdu, China. Names of the new taxa were registered in MycoBank [33].

Phylogenetic Analyses
The BLAST searches were performed for finding similar sequences that match our data. A concatenated dataset of the SSU, ITS, LSU, rpb2, and tef1α sequences were used for phylogenetic analyses with the inclusion of reference taxa from GenBank (Table 1). The sequences were aligned by using the online multiple alignment program MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/ (accessed on 27 May 2022)) [38], and the alignment was manually optimized in BioEdit v.7.0.9 [39]. The five-gene dataset was concatenated by Mesquite v. 3.11 (http://www.mesquiteproject.org/ (accessed on 27 May 2022)) for multi-gene phylogenetic analyses. Maximum likelihood (ML) and Bayesian inference (BI) were carried out as detailed in Dissanayake et al. [40]. Maximum likelihood (ML) analysis was performed using RAxML-HPC v.8 tool via the CIPRES Science Gateway V3.3 (https://www.phylo.org/portal2/home.action, accessed on 27 May 2022) with rapid bootstrap analysis, and a general time-reversible model (GTR) was applied with a discrete GAMMA distribution. The Bayesian inference (BI) analyses were performed by using PAUP v.4.0b10 [41] and MrBayes v. 3.1.2 [42,43]. The best model for different genes partition in the concatenated dataset was determined by MrModeltest 2.3. [44], and posterior probabilities were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v.3.1.2 [42,43]. The final alignment and phylogram were submitted to Tree-BASE (https://www.treebase.org/, accessed on 5 May 2023, submission ID: 30350). The phylogenetic trees were visualized by Treeview v. 1.6.6 [45]. Table 1. Taxa used in the phylogenetic analyses and their GenBank accession numbers. Newly generated sequences are indicated with * and the ex-type strains are in bold. "N/A" sequence is unavailable.

Phylogenetic Analyses
To determine the phylogenetic placement of the new collections in this study, the combined SSU, ITS, LSU, rpb2, and tef1α data set comprised 55 sequences with Pleurotheciella aquatica (MFLUCC 17-0464) and P. erumpens (CBS 142447) as the outgroup taxa. The concatenated matrix comprised a total of 4870 characters (SSU: 1081 bp; ITS: 841 bp; LSU: 921 bp; rpb2: 1056 bp; tef1α: 971 bp) including gaps. Maximum likelihood (ML) and Bayesian inference (BI) analyses were resulting in generally congruent topologies. The best-scoring ML tree (Figure 1) was selected to represent the relationships among taxa, in which a final likelihood value of -37,950.559206 is presented. The evolutionary models for Bayesian analysis were selected for each locus, and the best-fit model GTR+I+G for ITS, LSU, rpb2, and tef1α, SYM+I+G for SSU, respectively. Six simultaneous Markov chains were run for 165,000 generations, and trees were sampled every 1000 generations and 165 trees were obtained. The first 33 trees representing the burn-in phase of the analyses were discarded, while the remaining 132 trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01).
J. Fungi 2023, 9, x FOR PEER REVIEW 6 of 21 Figure 1. RAxML tree generated from combined SSU, ITS, LSU, rpb2, and tef1α sequence data of Savoryellaceae. Bootstrap values for ML equal to or greater than 75% are placed above the branches. Branches with Bayesian posterior probabilities (BYPP) from MCMC analysis equal to or greater than 0.95 were in bold. The ex-type strains were indicated in bold, and newly generated sequences were indicated in red. "T" represents the type species of each genus.

Figure 1.
RAxML tree generated from combined SSU, ITS, LSU, rpb2, and tef1α sequence data of Savoryellaceae. Bootstrap values for ML equal to or greater than 75% are placed above the branches. Branches with Bayesian posterior probabilities (BYPP) from MCMC analysis equal to or greater than 0.95 were in bold. The ex-type strains were indicated in bold, and newly generated sequences were indicated in red. "T" represents the type species of each genus.
Culture characteristics: Colonies on PDA reaching 20-30 mm after 5 months at 25 • C, circular, raised to umbonate, rough surface, dense, entire at the edge, brown, dry, reverse dark brown to black. Mycelium subhyaline to pale brown, 1.5-2.5 µm wide in culture. Notes: Canalisporium dehongense was introduced by Hyde et al. [46] from a submerged wood in Yunnan Province, China and has been considered as a later synonym of C. thailandense [47]. However, Goh and Kuo [48] treated Canalisporium dehongense and C. thailandense as distinct species, and this is followed in this study. The new collection clustered with Canalisporium dehongense in the single-and multi-gene phylogeny and share the similar conidiogenous cells and conidial morphology. Therefore, we identify our collection as C. dehongense, and an additional bamboo host record is provided herein.     Figure 6 MycoBank: MB 555673 Saprobic on dead bamboo in freshwater habitat. Sexual morph: Undetermined. Asexual morph: hyphomycetous. Colonies on natural substratum, effuse, scattered to gregarious, dark brown to black, glistening, superficial. Mycelium immersed in natural substrate, unbranched, septate, hyaline to subhyaline hyphae. Conidiophores reduced to conidiogenous cells, or rarely 20-30 × 3.5-4.5 μm, micronematous, mononematous, cylindrical, unbranched. Conidiogenous cells not observed. Conidia 21-28 μm diam (x̅ = 25 μm, n = 50), solitary, acrogenous, globose to subglobose, brown to dark brown, dictyospores, slightly constricted at the septa, with a pore in each cell.
Culture characteristics: Colonies on PDA reaching 10-15 mm after 7 months at 25 °C, circular, with dense mycelium on the surface, dark grayish of the inner ring, and brown.
Culture characteristics: Colonies on PDA reaching 10-15 mm after 7 months at 25 • C, circular, with dense mycelium on the surface, dark grayish of the inner ring, and brown. Notes: Luo et al. [26] introduced a monotypic genus Dematiosporium, which was collected from decaying submerged wood in Erhai lake, Dali City, Yunnan Province, China. Subsequently, Réblová et al. [17] recollected and described it in France, where it occurs on decaying submerged wood of Alnus glutinosa and other unidentified substrates. Species of Dematiosporium are often found on decaying wood in aquatic environments and it is characterized by cylindrical, unbranched, aseptate conidiophores and globose to subglobose, dictyospores conidia with a pore in each cell [17,26]. Our new collection was found from decaying bamboo in freshwater environments. We identify it as D. aquaticum based on the morphological characters and phylogenetic analyses. The SSU, ITS, rpb2, and tef1α regions were attempted, and we were able to obtain the LSU sequences. This is the first report of D. aquaticum from bamboo in a freshwater habitat. We describe D. aquaticum in detail and supplement the description of the conidiophore that was missing from the previous description.
Subsequently, Réblová et al. [17] recollected and described it in France, where it occurs on decaying submerged wood of Alnus glutinosa and other unidentified substrates. Species of Dematiosporium are often found on decaying wood in aquatic environments and it is characterized by cylindrical, unbranched, aseptate conidiophores and globose to subglobose, dictyospores conidia with a pore in each cell [17,26]. Our new collection was found from decaying bamboo in freshwater environments. We identify it as D. aquaticum based on the morphological characters and phylogenetic analyses. The SSU, ITS, rpb2, and tef1α regions were attempted, and we were able to obtain the LSU sequences. This is the first report of D. aquaticum from bamboo in a freshwater habitat. We describe D. aquaticum in detail and supplement the description of the conidiophore that was missing from the previous description.  Asexual morph: hyphomycetous. Colonies on natural substratum, effuse, scattered to gregarious, dark brown to black, glistening, superficial. Mycelium immersed in natural substrate, composed of hyaline, septate, branched, smooth hyphae. Conidiophores 6.5-11.5 × 2-4.5 µm (x = 9 × 3 µm, n = 10), micronematous, mononematous, cylindrical, straight or slightly flexuous, unbranched, aseptate. Conidiogenous cells holoblastic, monoblastic, cuneiform, integrated, terminal, determinate, hyaline. Conidia 35-60 × 27-41 µm (x = 46 × 34 µm, n = 50), solitary, acrogenous, ellipsoidal to subglobose, dark brown to black, smooth-walled, dictyospores, with a pore in each cell (Figure 7j), broadly rounded at apex, subtruncate at the base.   Notes: The phylogenetic analyses showed that three isolates of Dematiosporium bambusicola formed a monophyletic clade in Savoryellaceae and are sister to D. aquaticum with absolute statistical support (100% ML/1.00 BYPP). Dematiosporium Bambusicola resembles D. aquaticum in forming dematiaceous, dictyospores conidia with a pore [26]. However, D. bambusicola has larger conidia than that of D. aquaticus (35-60 × 27-41 µm vs. 21-28 µm diam) [26]. The conidia of D. bambusicola are ellipsoidal to subglobose and dark brown to nearly black, while D. aquaticus has globose to subglobose conidia and brown to dark brown [26]. In addition, D. bambusicola differs from the latter in having smaller conidiophores (6.5-11.5 × 2-4.5 µm vs. 20-30 × 3.5-4.5 µm) [26]. We hereby introduce the new species based on the distinctiveness of morphology and multi-gene phylogeny.

Discussion
Tibpromma et al. [20] introduced Canalisporium krabiense and C. thailandense from Thailand. These two species have different conidiogenous cells, but were found to have almost identical ITS and LSU sequences and were phylogenetically clustered together (Figure 1). Koukol and Delgado [47] speculated that DNA cross-contamination happened
Culture characteristics: Colonies on PDA reaching 10-15 mm after 2 months at 25 • C, circular, with dense mycelium on the surface, light gray of the inner ring, and light yellow of the outer ring; in reverse black.
of this family are more likely to be found from monocotyledons and favorable to hard tissue substrates in freshwater habitats.
Asexual morphs have been found in all six genera in Savoryellaceae, of which Bactrodesmium and Dematiosporium are represented only by asexual morphs; members of Canalisporium mostly are asexual morphs with only C. grenadoideum having a holomorph; while Savoryella is commonly known as a sexual morph with only S. nypae and S. sarushimana represented by a trichocladium-like asexual morph [19]; Ascotaiwania and Neoascotaiwania both have holomorphs, but different in asexual morphs (monodictys-like, monotosporella-like, and trichocladium-like vs. bactrodesmium-like) [53,62,[64][65][66]. In this study, one sexual morph of Savoryella and four asexual morphs of Canalisporium and Dematiosporium are isolated and identified, which contributed to the taxonomy of Savoryellaceae and the diversity of bambusicolous fungi. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The sequences and alignments data were submitted to GenBank and TreeBASE, respectively.