Morphological and Phylogenetic Analyses Reveal Five New Species in Chaetosphaeriaceae

Chaetosphaeriaceae is a genera-rich and highly diverse group of fungi with a worldwide distribution in terrestrial and aquatic habitats. Eight fresh collections of Chaetosphaeriaceae were obtained during investigations of hyaline-spored hyphomycetes in China and Thailand. Based on morphological characteristics and phylogenetic analysis of a combined LSU and ITS sequence dataset, Chaetosphaeria obovoidea, Codinaea aseptata, Codinaeella hyalina, Dictyochaeta guizhouensis and Paragaeumannomyces guttulatus were introduced as new species, Codinaea terminalis was reported as new host record, and Codinaea dwaya and Phialosporostilbe scutiformis were introduced as new collections. Phylogenetic analysis in this study revealed that Chaetosphaeria was polyphyletic. Detailed descriptions and illustrations of new taxa and identified species are provided, as well as an updated phylogenetic tree to confirm the placements of these eight new collections.


Collection, Isolation and Conservation
Samples of submerged decaying twigs and dead bamboo culms were collected from Chiang Rai Province, Thailand, and Guizhou Province, China. They were packed into plastic bags for transportation to the laboratory, and associated metadata (i.e., date, locality and host) were noted. Fungal colonies on the host surface were examined and observed using a stereomicroscope (Leica EZ4 Microsystems (Schweiz) AG, Singapore). Photomicrographs of micro-morphological characteristics were documented using a Nikon DS-Ri2 digital camera fitted to a Nikon ECLIPSE Ni compound microscope (Nikon, Japan). Measurements were made using the Tarosoft (R) Image Frame Work, and these images used for figures were processed and combined with Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA).
Single-spore isolations were made on water agar (WA) and potato dextrose agar (PDA), and germinating conidia were transferred to fresh malt extract agar (MEA) and PDA following the method of Chomnunti et al. [39]. Dried materials were deposited in the herbaria of Mae Fah Luang University (Herb. MFLU), Chiang Rai, Thailand, Herbarium of Cryptogams, Kunming Institute of Botany, Academia Sinica (HKAS), Kunming, China, and Herbarium of Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China. Cultures were deposited at Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand, and Guizhou Culture Collection, China (GZCC). Faces of Fungi and Index Fungorum numbers were registered according to the guidelines in Jayasiri et al. [40] and Index Fungorum (2022).

DNA Extraction, PCR Amplification and Sequencing
Pure cultures were grown on MEA/PDA media at 25 • C for one month. Fresh fungal mycelia were scraped off from the surface of the cultures and transferred to 1.5 mL microcentrifuge tubes. Meanwhile, fungal genomic DNA was extracted with the Biospin Fungus Genomic DNA Extraction Kit (Biospin Fungus Genomic DNA Extraction Kit, BioFlux ® , Shanghai, China) following the manufacturer's instructions. LR0R and LR5 (Vilgalys and Hester 1990) and ITS5 and ITS4 (White et al. 1990) primers were used to amplify the large subunit of the ribosomal DNA (LSU) and the internal transcribed spacer (ITS) gene regions. The amplification reactions were performed in a 50 µL reaction volume, which contained 2 µL of DNA template, 2 µL of each forward and reverse primer (10 µM), 25 µL of 2× Taq PCR Master Mix with blue dye (Sangon Biotech, Shanghai, China) and 19 µL of distilled-deionized water. The following thermo-cycling parameters were used for the LSU and ITS region: initial denaturation at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 45 s, annealing at 56 • C for 50 s, elongation at 72 • C for 1 min and a final extension period for 10 min at 72 • C. The quality of the PCR products was checked on a 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were performed at Sangon Biotech (Shanghai, China) using the same primers.

Alignments and Phylogenetic Analysis
Original sequences were verified using BioEdit v. 7.1.3.0 [41], and were assembled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA). Consensus sequences were submitted to the NCBI GenBank (Table 1). The new sequences were subjected to BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 May 2022) for preliminary determination of the possible species identification range in the GenBank database. Sequences of the ITS and LSU gene were analyzed to assess relationships among species of Chaetosphaeria, Codinaea, Codinaeella, Dictyochaeta, Paragaeumannomyces, Phialosporostilbe and relevant fungi in Chaetosphaeriaceae [6,9,10,12,14,16,20,29,36,42]. Accession numbers for sequences were retrieved from GenBank database and related publication from previous studies (Tables 2 and S1). The alignments for sequences of each locus were performed with the online multiple alignment program MAFFT version 7 (https://mafft.cbrc. jp/alignment/server/, accessed on 3 May 2022) and then manually verified in BioEdit 7.1.3.0 [43]. The maximum likelihood (ML) and Bayesian inference (BI) analyses inferred phylogenetic relationships, based on the concatenated sequence matrix of a combined LSU and ITS dataset.   The website tool "ALTER" (http://www.singgroup. org/ALTER/, accessed on 3 May 2022) was used to convert the aligned fasta file for RAxML analysis [44]. Subsequently, 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 3 May 2022) with rapid bootstrap analysis [45,46]. Eventually, 1000 non-parametric bootstrap iterations were run with the GTRGAMMA model, and the final tree was selected amongst suboptimal trees from each run by comparing likelihood scores under the GTR-Gamma substitution model.
The aligned fasta file was converted to the nexus file format for BI analysis using AliView. BI analysis was performed using MrBayes 3.2.7a via CIPRES [45]. The best-fit model of DNA evolution was estimated using MrModeltest v. 2.2 [47]. The Bayesian Markov chain Monte Carlo (BMCMC) sampling method in MrBayes v.3.2.7a determined the posterior probabilities (PPs) [48]. Four simultaneous Markov chains were run for 1 million generations, with trees sampled every 100 generations, resulting in 10,000 trees. The first 2000 trees representing the burn-in phase of the analyses were discarded, and the remaining 8000 trees were used for calculating posterior probabilities (PPs) in the majorityrule consensus tree [49].
Phylogenetic trees were printed with FigTree v. 1.4.0 [50], and the layout was established using Adobe Illustrator CS6 (Adobe Systems, San Jose, CA, USA).  Figure 1.

Phylogenetic Analysis
This phylogenetic study confirmed that the family Chaetosphaeriaceae was a robust clade (100% ML/1.00 PP). There are eight new collections, five of which are in Chaetosphaeria, Codinaeella, Dictyochaeta, Paragaeumannomyces and Phialosporostilbe genera, respectively, while the other three belong to the genus Codinaea, under Chaetosphaeriaceae ( Figure 1).
The phylogenetic tree shows that our three new Codinaea collections of Codinaea aseptata sp. nov., and two known species, Co. dwaya and Co. terminalis, clustered with other 16 Codinaea taxa in a monophyletic clade with highly support (90% ML/1.00 PP, Figure 1). CT = 6.118919, GT = 1.000000 and Tree-Length = 11.191081; distribution shape parameter α = 0.304587. Bayesian posterior probabilities (PPs) from MCMC were evaluated with a final average standard deviation of split frequencies of 0.009954. Maximum likelihood and Bayesian analyses were conducted, resulting in generally congruent topologies, and the ML analysis result with a final likelihood value of −27,871.044543 is presented in Figure 1.  This phylogenetic study confirmed that the family Chaetosphaeriaceae was a robust clade (100% ML/1.00 PP). There are eight new collections, five of which are in Chaetosphaeria, Codinaeella, Dictyochaeta, Paragaeumannomyces and Phialosporostilbe genera, respectively, while the other three belong to the genus Codinaea, under Chaetosphaeriaceae (Figure 1).
The phylogenetic tree shows that our three new Codinaea collections of Codinaea aseptata sp. nov., and two known species, Co. dwaya and Co. terminalis, clustered with other 16 Codinaea taxa in a monophyletic clade with highly support (90% ML/1.00 PP, Figure 1).
Eight "Codinaea"-like taxa comprised a new species of Codinaeella hyalina and eight previously identified species, representing the Codinaeella clade, which was phylogenetically well-supported by the generic placement (100% ML/1.00 PP, Figure 1). It is consistent with the studies of Réblová et al. [16]. The phylogenetic tree generated from ML analysis that is based on a concatenated LSU-ITS dataset for the Chaetosphaeriaceae family. Bootstrap support values for ML equal to or greater than 65% and Bayesian posterior probabilities (PPs) equal to or greater than 0.95 were indicated above or below the nodes as ML/PP. Leptosporella arengae (MFLUCC 15-0330) and L. bambusae (MFLUCC 12-0846) were selected as the outgroup taxa. The newly obtained sequences are indicated in blue.
Eight "Codinaea"-like taxa comprised a new species of Codinaeella hyalina and eight previously identified species, representing the Codinaeella clade, which was phylogenetically well-supported by the generic placement (100% ML/1.00 PP, Figure 1). It is consistent with the studies of Réblová et al. [16].
One of the eight fresh collections was identified as a new species in Dictyochaeta species, namely D. guizhouensis. It clustered together with the other six known species and one unidentified Dictyochaeta sp. (CBS 138684) with good support (97% ML/1.00 PP, Figure 1). All species of Dictyochaeta formed into a single monophyletic clade and fit well with the narrow genetic concept of conidia without setulae [14]. Furthermore, the phylogenetic placement of the Dictyochaeta clade and the phylogenetic relationship of taxa within Dictyochaeta presented similar results to those obtained by Réblová et al. [14].
Culture characteristics: Conidia germinating on PDA within 15 h and hyaline germ tube produced from the base of conidia. Colonies growing on PDA at 25 • C reach 17 mm in three weeks, circular, unbonate, entire, with filamentous, dense, aerial mycelium on the surface, white at the center, pale grey at the edge from above; yellowish to greyish brown to pale brown in reverse from the center to the margin of the colony, and do not produce pigmentation in culture.
Culture characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from the base and the upper part. Colonies growing on PDA, reaching 19 mm in diam. in 10 days at 25 • C, circular, flat, entirely to slightly filamentous, section to fan shape at the surface, taupe, becoming white towards the edge from above; yellowish-brown mycelium in the middle and pale-yellow mycelium in the outer ring in reverse, and does not produce pigmentation in culture.
Codinaea aseptata J.Y. Zhang and Y.Z. Lu, sp. nov. (Figure 3).   Notes: Four morphotypes (C1-C4) of Codinaea were provided by Réblová et al. [16]. The morphological characteristics of our new isolate match well with the generic concept of Codinaea and fit well with the morphotype C1. Codinaea aseptata is most similar to Co. terminalis due to having setae in fascicles with conidiophore, phialidic and terminal conidiogenous cells and aseptate, falcate conidia with setulae at both ends [9]. However, our new isolate differs from Co. terminalis in its obviously longer and rarely fertilizable setae. BLAST results of ITS and LSU sequence data are Codinaea acacia OTU5 (96.09% similarity) and Co. paniculate MFLU 34876 (99.43 similarity), respectively. The phylogenetic tree (Figure 1) showed that our new isolate of Cod. aseptata formed an individual lineage in the Codinaea clade, but without statistical support shown in phylogenetic tree (44% ML/-). This may be due to the consideration that many close phylogenetic relatives of our new collection have not yet been discovered. Hence, Codinaea aseptata was introduced as a new species based on its distinct morphology and phylogenetic evidence. Notes: Following BLASTn searches of NCBI GenBank, the closest matches of the LSU and ITS sequences of our new isolate is Codinaea dwaya (strain CBS 261.77; LSU, OL654135, 99.88% shared identity; ITS, OL654078, 97.79% shared identity). Our new collection fits well with the description of species of Co. dwaya in mononematous conidiophores, integrated, terminal, cylindrical, phialidic conidiogenous cells and hyaline, spherical, aseptate conidia with setulae [16,[53][54][55]. Phylogenetically, our isolate grouped together with Co. dwaya (CBS 261.77) with high support value (100% ML/1.00 PP, Figure 1). Thus, we identified this isolate as a new collection of Co. dwaya from a freshwater habitat.    Notes: Codinaea terminalis was introduced by Lin et al. [9], isolated from decaying leaves in China. The morphology of our isolate shares similar characters with the ex-type strain Codinaea terminalis (GZCC 18-0085). However, there are some differences in the size of the microstructure such as obviously longer setulae (9.6-11.5 μm vs. 4-9.5 μm). Our new isolate clustered among two Dictyochaeta terminalis strains (GZCC 18-0085 and GZCC19-0525) with strong statistical support (99% ML/1.00 PP, Figure 1). Thus, we identified this isolate as a new host record of Co. terminalis on dead bamboo culms in China.
Culture characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from conidia. Colonies growing on PDA, reaching 19 mm in diam. in 10 days at 25 • C, circular, flat with a protuberance in the center, white, entire margin with thin mycelia; pale yellow to white from center to edge in reverse and not producing pigmentation in culture.
Material examined: CHINA, Guizhou province, Zunyi city, Chishui county, Hushi town, Chishui Alsophila Natural Reserve (28 •  Notes: Codinaea terminalis was introduced by Lin et al. [9], isolated from decaying leaves in China. The morphology of our isolate shares similar characters with the ex-type strain Codinaea terminalis (GZCC 18-0085). However, there are some differences in the size of the microstructure such as obviously longer setulae (9.6-11.5 µm vs. 4-9.5 µm). Our new isolate clustered among two Dictyochaeta terminalis strains (GZCC 18-0085 and GZCC19-0525) with strong statistical support (99% ML/1.00 PP, Figure 1). Thus, we identified this isolate as a new host record of Co. terminalis on dead bamboo culms in China.
Culture characteristics: Conidia germinating on PDA within 12 h. and germ tubes produced from conidia. Colonies growing on PDA, slow growth, reaching 20 mm in diam. in 20 days at 25 • C, circular, flat, with dense, white mycelium on the surface with undulate margin, from below olivaceous brown at the center, yellow at the edge and does not produce pigmentation in culture.
Cultural characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from conidia. Colonies grew on PDA medium at 25 • C, circular, with an entire margin, umbonate, with dense, white mycelium in the middle and yellowish-brown mycelium in the outer ring on the surface; in contrast, they are cream yellow in the middle, while yellowish-brown in the center and the outer ring.
Notes: In a BLASTn search in GenBank, the closest match to the LSU and ITS sequences of our new isolate (GZCC 22-0083) exhibited 100% similarity across 100% of the query sequence and 98.49% similarity across 90% of the query sequence to Dictyochaeta sp. (strain CBS 138684), respectively. The phylogenetic tree depicted that our new collection is closely related to unidentified taxon Dictyochaeta sp. (CBS 138684) with 97% ML/1.00 PP support ( Figure 1). Dictyochaeta sp. (CBS 138684) was described by Réblová et al. [14] with its sexual and asexual morphs. However, there was insufficient evidence of morphological characteristics due to a sample ageing; therefore, Dictyochaeta sp. (CBS 138684) was treated as an unidentified species [14]. Our new isolate formed an asexual morph in the natural substrate and is characterized by a lack of setae, microconidia and polyphialidic conidiohenous cells with conspicuous collarette, which can be differentiated from Dictyochaeta sp. (CBS 138684). Given the remarkable differences in morphology between our new collection (HKAS 123753) and Dictyochaeta sp. (CBS 138684), we maintain Dictyochaeta sp. as an unidentified species and introduce our new collection as a novel species in Dictyochaeta.
Culture characteristics: conidia germinating on PDA within 15 h and the hyaline germ tubes germinates from a point of the conidia. Colonies on MEA at 26 • C, slow growth, reach 5 mm in diam. in 30 days, circular, flat, with entire margin, taupe brown from above, while dark brown in reverse, and do not produce pigmentation in culture. myces (P. panamensis) by Réblová et al. [29] based on generic concept and phylogenetic analysis. Morphologically, our new species P. guttulatus formed an asexual morph in the natural substrate and is similar to P. panamensis in having a "Craspedodidymum"-like asexual morph. However, Paragaeumannomyces guttulatus differs from P. panamensis in having bigger conidiogenous cells and smaller and rounder conidia with longer appendages [4,6]. Thus, we introduce P. guttulatus as a new species based on morphological and phylogenetic analyses.    (Figure 1). Chaetosphaeria panamensis was introduced by Huhndorf and Fernández [4] with sexual and asexual morphs. Later, Chaetosphaeria panamensis was transferred to the Paragaeumannomyces (P. panamensis) by Réblová et al. [29] based on generic concept and phylogenetic analysis. Morphologically, our new species P. guttulatus formed an asexual morph in the natural substrate and is similar to P. panamensis in having a "Craspedodidymum"-like asexual morph. However, Paragaeumannomyces guttulatus differs from P. panamensis in having bigger conidiogenous cells and smaller and rounder conidia with longer appendages [4,6]. Thus, we introduce P. guttulatus as a new species based on morphological and phylogenetic analyses.
Cultural characteristics: Conidia germinating on PDA within 15 h and germ tubes produced from the corner of conidia. Colonies on PDA medium, circular, reaching 13 mm in diam. in 10 days at 25 • C, entirely to slightly filamentous, with dense, yellowish-brown mycelium in the middle and thin, slightly yellow mycelium in the edge; yellowish in reverse and not producing pigmentation in culture.
GenBank accession numbers: (LSU) ON678145, (ITS) ON678180. Notes: Phialosporostilbe scutiformis was introduced by Yang et al. [36] based on two specimens collected from freshwater habitats in China and Thailand. A BLASTn search of the ITS and LSU sequence data indicated that our new isolate is closely related to P. scutiformis (MFLUCC 17-0227) with 100% similarity. The morphology of our isolate and the paratype specimen of P. scutiformis (HKAS 102205) are indistinguishable, except that the big guttule in conidia was not observed in our collection. This may be due to the different observation periods. Phylogenetic analysis confirmed that our isolate MFLUCC 22-0053 grouped with Phialosporostilbe scutiformis strains (MFLUCC 17-0227 and MFLUCC 18-1288) with strong bootstrap support (99% ML/1.00 PP, Figure 1). Therefore, we identified this isolate as a new collection of P. scutiformis based on identical morphology and phylogenetic analysis.

Discussion
Most Chaetosphaeriaceous species are saprobic on wood or decaying plants from terrestrial and freshwater habitats, sometimes occurring on soil, while some are fungicolous taxa [1,2,6,11,12,14,20,29,[56][57][58]. Chaetosphaeria mangrovei was only one species reported from the marine habitat [20]. In this study, five new species among eight newly collections, namely Chaetosphaeria obovoidea, Codinaea aseptata, Codinaeella hyalina, Dictyochaeta guizhouensis and Paragaeumannomyces guttulatus, were documented from China. This increased the number of Chaetosphaeriaceous species and exhibited high diversity, and undiscovered in China. Additionally, three previously known species including a new host record and two new collections were described and identified based on morphological comparison and phylogenetic analysis of a combined LSU and ITS sequence dataset.