Three Novel Cheiroid Hyphomycetes in Dictyocheirospora and Dictyosporium (Dictyosporiaceae) from Freshwater Habitats in Guangdong and Guizhou Provinces, China

Over the past two decades, numerous novel species have been identified within Dictyosporiaceae, primarily in Dictyocheirospora and Dictyosporium. A recent monograph has revealed that these two genera exhibit a distinct preference for freshwater habitats, particularly in southern China. However, further investigation into the distribution and diversity of the two genera in Guangdong and Guizhou Provinces remains insufficient. In this study, we conducted an analysis of four intriguing cheiroid hyphomycetes collected from flowing rivers in these two regions. Through morphological and phylogenetic analyses incorporating combined LSU, SSU, ITS, and tef1-α sequence data, we have identified them as a novel species in Dictyocheirospora (Dictyoc. submersa sp. nov.), two novel species in Dictyosporium (Dictyos. guangdongense sp. nov. and Dictyos. variabilisporum sp. nov.), and one previously documented species (Dictyos. digitatum). Specifically, the identification of Dictyos. guangdongense is primarily based on its distinct morphology, characterized by complanate, cheiroid, and brown to dark brown conidia, with a hyaline, short, and atrophied appendage arising from the apical cell of the outer row. In addition, the morphological distinctions between Dictyocheirospora and Dictyosporium are further clarified based on our new data. This study also highlights a few phylogenetic matters regarding Dictyosporiaceae.

During our ongoing study of the taxonomy and diversity of freshwater fungi in southern China and along a north-south gradient [27], four interesting collections that morphologically belong to Dictyosporiaceae were encountered.Their phylogenetic placements were inferred using a combined dataset of the nuclear ribosomal large subunit 28S rRNA gene (LSU), ribosomal small subunit 18S rRNA gene (SSU), nuclear ribosomal internal transcribed spacers (ITSs), and fragments of the translation elongation factor 1-alpha (tef1-α).Three new species are proposed and a new fresh collection is described.

Sampling, Isolation, and Morphological Examination
The specimens were derived from freshwater habitats in Guangdong and Guizhou Provinces, China.The decaying plant materials, including wood, branches, and twigs, submerged in freshwater were collected and placed in zip-lock plastic bags.Essential information such as the collecting site, date, and collector details were recorded.All specimens were transported to the laboratory within three days.If the surfaces of the specimens were heavily covered with mud, they were gently rinsed with tap water.Subsequently, around 4-5 specimens were transferred to a new zip-lock plastic bag or plastic box containing moistened tissue paper/cotton and incubated at room temperature (24-27 • C) for 1-2 weeks.Sampling and specimen incubation followed the method described by Senanayake et al. [28].A stereomicroscope (Chongqing Optec Instrument Co., Ltd., Chongqing, China) was employed to examine the sporodochia developed on the natural substratum.A compound microscope (Nikon Eclipse Ni-U, Tokyo, Japan) equipped with a digital camera (Canon 750D, Tokyo, Japan) was utilized to capture the fungal structures.Single-spore isolations were made from conidium on potato dextrose agar (PDA, Shanghai Bio-way technology Co., Ltd., Shanghai, China) at room temperature.After aseptic transfer, the germinated spores were incubated at room temperature.
Herbarium specimens (dry wood with fungal colonies) were deposited in the herbaria at Zhongkai University of Agriculture and Engineering (MHZU), Guangzhou, China.Living cultures were deposited in the Zhongkai University of Agriculture and Engineering Culture Collection (ZHKUCC), Guangzhou, China.The novel taxa were registered in the databases Facesoffungi (http://www.facesoffungi.org,accessed on 12 February 2024) [29] and Index Fungorum (http://www.indexfungorum.org/names/names.asp,accessed on 12 February 2024).

DNA Extraction, PCR Amplification, and Sequencing
After a month of cultivation, fungal mycelia were carefully scraped from the colonies for subsequent DNA extraction.The entire genomic DNA was extracted from 100-200 mg of axenic mycelia.The cell fragmentation was accomplished using a homogenizer (Allsheng, Hangzhou, China).The Biospin fungi DNA isolation kit (Bioer, Hangzhou, China) was utilized for genomic DNA extraction in accordance with the manufacturer's instructions.In this study, primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4, and EF1-983F/EF1-2218R were used to amplify the LSU, SSU, ITSs, and tef1-α, respectively.The amplifications were carried out in a 25 µL reaction volume containing 9.5 µL of double-distilled sterilized water (ddH 2 O), 12.5 µL of 2 × FastTaq PCR Master Mix (Vazyme Co., Nanjing, China), 1 µL of DNA template, and 1 µL of each forward and reverse primer (10 µM).The PCR thermal cycle program for the amplification of the LSU, SSU, ITSs, and tef1-α was started with an initial denaturation step at 97 • C for 3 min, followed by 38 cycles consisting of denaturation at 94 • C for 30 s, annealing at 53 • C for 50 s, elongation at 72 • C for 1 min, and a final extension step at 72 • C for 10 min [30][31][32].PCR products were checked on 1% agarose electrophoresis gels stained with ethidium bromide (EB).The sequencing reactions were carried out by Tianyi Huiyuan Biotechnology Co., Ltd.(Guangzhou, China).

Phylogenetic Analyses
The newly obtained sequences were analyzed using FinchTV v. 1.4.0.The consensus sequences were generated using BioEdit v. 7.2 [33].The Blast analysis was performed on the NCBI platform (https://blast.ncbi.nlm.nih.gov(accessed on 10 February 2024)) to identify taxonomic matches.The taxa selected for constructing the phylogenetic tree were based on a recent published paper [25] and Blast results obtained from the NCBI.The dataset of each gene region was initially aligned independently using the 'auto' strategy (based on data size) by MAFFT v. 7 [34].Subsequently, manual editing was performed to eliminate uninformative gaps or ambiguous regions in BioEdit v. 7.2 [33].Each dataset (LSU, SSU, ITS, and tef1-α) was concatenated in Mesquite v. 3.81 [35].The fasta file was converted to PHYLIP (for ML) and NEXUS (for BI) format in the Alignment Transformation Environment (ALTER) online program (http://www.sing-group.org/ALTER/(accessed on 10 February 2024)).Phylogenetic analyses were conducted with maximum likelihood (ML) and Bayesian inference (BI) algorithms in the CIPRES Science Gateway (http://www.phylo.org/portal2(accessed on 10 February 2024)) [36].The ML analysis was performed with RAxML-HPC2 v. 8.2.12 on XSEDE with 1000 rapid bootstrap replicates [37].The model selected for ML analysis was GTR + GAMMA.The BI analysis was performed in MrBayes v. 3.2.7a[38] and the best-fitting model was estimated via MrModeltest v. 2.2 [39].
The Markov Chain Monte Carlo (MCMC) was run for 10,000,000 generations, and the trees were sampled every 100th generation.The first 25% of the trees that represented the burn-in phase were discarded, and the remaining 75% of the trees were used for calculating the posterior probabilities (PPs) for the majority-rule consensus tree [40].Phylogenetic trees were visualized using FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 10 February 2024).The editing and typesetting were accomplished using Microsoft Office PowerPoint 2007 (Microsoft Corporation, Redmond, WA, USA).

Taxonomy
Cultural characteristics: Conidia germinating on PDA within 24 h and germ tubes produced from the basal cell.Colonies on PDA reaching 35 mm diam.at room temperature (24-27 °C) in natural light after 20 days, irregular, rough, dry, fluffy, and dense; margins undulate with sparse mycelia, yellow in the middle and white at the margin; and the reverse brown in the middle and pale brown at the margin.Cultural characteristics: Conidia germinating on PDA within 24 h and germ tubes produced from the basal cell.Colonies on PDA reaching 35 mm diam.at room temperature (24-27 • C) in natural light after 20 days, irregular, rough, dry, fluffy, and dense; margins undulate with sparse mycelia, yellow in the middle and white at the margin; and the reverse brown in the middle and pale brown at the margin.
Notes: In the phylogenetic analysis, Dictyocheirospora submersa clustered as a distinct branch within Dictyocheirospora, exhibiting close affinities with Dictyoc.gigantica, Dictyoc.pandanicola, Dictyoc.pseudomusae, and Dictyoc.vinaya (Figure 1).Dictyocheirospora submersa can be easily distinguished from Dictyoc.pseudomusae by the absence of globose to subglobose, hyaline appendages growing from the apical cells or side of the outer rows [10].The conidia of Dictyoc.gigantica differ from those of Dictyoc.submersa by their cylindrical and longer conidia measuring 105-121 × 25-32 µm, whereas Dictyoc.submersa has shorter and mostly clavate, ellipsoidal, or occasionally subcylindrical conidia measuring (20-)35-85 × 12-30 µm [3].Dictyocheirospora submersa is quite similar to Dictyoc.pandanicola and Dictyoc.vinaya in terms of their conidial morphology and dimensions.However, they can be easily distinguished based on the color of their conidia.Dictyocheirospora submersa exhibits pale olivaceous, brown to dark brown conidia, while Dictyoc.vinaya has reddish-brown conidia (from their photo plate), and Dictyoc.pandanicola has pale brown conidia [1,41].In addition, the conidial arms of Dictyoc.submersa are tightly appressed, with minimal divergence observed upon squashing.The conidial arms of Dictyoc.pandanicola and Dictyoc.vinaya, in contrast, easily become divergent, with the conidial arms of Dictyoc.pandanicola even detaching from the conidial body.After conducting a full morphological comparison, we found that Dictyoc.submersa does not correspond to any existing species within the genus.Therefore, it is introduced as a novel species of Dictyocheirospora.
Cultural characteristics: Conidia germinating on PDA within 24 h, and all the cells can produce germ tubes.Colonies on PDA reaching 30 mm diam.at room temperature (24-27 • C) in natural light after 15 days, irregular, rough, dry, fluffy, and dense in the middle; margin sparse and wavy, white and orange-brown; and the reverse orange-brown in the middle and pale orange-brown at the margin.
Cultural characteristics: Conidia germinating on PDA within 36 h and germ tubes produced from the basal cell.Colonies on PDA reaching 50 mm diam.at room temperature (24-27 • C) in natural light after 20 days, irregular, rough, dry, fluffy, and dense; margins undulate with sparse mycelia, white in the middle, with masses of black sporodochia produced at the margin; and the reverse yellowish-brown in the middle and pale yellowishbrown at the margin.
Notes: In the phylogenetic analysis, Dictyosporium guangdongense forms a clade with Dictyos.alatum, Dictyos.appendiculatum, Dictyos.pandanicola, Dictyos.strelitziae, and Dictyos.thailandicum (Figure 1).The distinct and well-defined shape of the appendages in Dictyos.alatum, Dictyos.appendiculatum, Dictyos.strelitziae, and Dictyos.thailandicum can be identified to distinguish them easily from Dictyos.guangdongense [3,9,15,41].In contrast, the appendages of Dictyos.guangdongense are hyaline, short, and atrophied structures arising from the apical cell of the outer row.Dictyosporium pandanicola is quite similar to Dictyos.guangdongense in terms of their conidial morphology and dimensions.However, Dictyos.pandanicola can be distinguished by the absence of any appendages [41].According to a comprehensive morphological comparison of all species mentioned/not mentioned in the referenced key [3,17,18], Dictyos.guangdongense does not match to any known species within the genus.The phylogenetic analyses cannot distinguish between Dictyos.guangdongense and its related species mentioned above due to the limited sequence data available for them, which only include LSU and SSU for Dictyos.appendiculatum, Dictyos.Strelitziae, and Dictyos.thailandicum, as well as LSU, SSU, and ITS for Dictyos.alatum.The tef1-α sequence data are available for Dictyos.pandanicola, but not for Dictyos.guangdongense.

Discussion
Numerous studies in the early 2,000s on lignicolous fungi in freshwater streams revealed Dictyosporium species [46][47][48]; however, the taxa were identified based on morphology, and the names should be questioned.The taxonomy of freshwater fungi and Dictyo- Cultural characteristics: Conidia germinating on PDA within 24 h and germ tubes produced from the basal cell.Colonies on PDA reaching 50 mm diam.at room temperature (24-27 • C) in natural light after 15 days, circular, rough, dry, fluffy, and dense; margin entirely covered with white mycelia; and the reverse pale brown in the middle and white at the margin.
Notes: In the phylogenetic analysis, Dictyosporium variabilisporum clustered as a distinct branch within Dictyosporium (Figure 1).The complanate, cheiroid conidia with apical appendages of Dictyos.variabilisporum correspond to the generic concept of Dictyosporium.However, it is unusual within the genus for the variable color of its conidia that are initially yellowish-brown and become olivaceous-brown.In addition, although several phylogenetically related species, viz., Dictyos.alatum, Dictyos.appendiculatum, Dictyos.bulbosum, Dictyos.krabiense, Dictyos.strelitziae, and Dictyos.thailandicum, have apical appendages that are similar to those of Dictyos.variabilisporum, they can be distinguished based on the morphology or quantity of their appendages [9,15,[41][42][43][44][45].The earlier stage of Dictyos.variabilisporum is also similar to Dictyos.digitatum in having digitate, curved, hyaline, thin-walled terminal cells [10,20,41,42].However, they can be easily distinguished by the conidial morphology, color, and appendages at maturity as shown in Figures 3 and 5.After conducting a full morphological comparison based on the literature and key provided by Goh et al. [3], we found that Dictyos.variabilisporum does not correspond to any existing species within the genus.Therefore, it is introduced as a novel species of Dictyosporium.

Discussion
Numerous studies in the early 2,000s on lignicolous fungi in freshwater streams revealed Dictyosporium species [46][47][48]; however, the taxa were identified based on morphology, and the names should be questioned.The taxonomy of freshwater fungi and Dictyosporaceae has been extensively investigated over the past decades by incorporating barcoding of nuclear ribosomal regions (LSU, SSU, and ITS) and protein-coding genes (tef1-α and rpb2).The unresolved fungal groups and intriguing taxonomic issues have been effectively addressed using the extensive collections obtained from China and Thailand [49][50][51][52][53]. Furthermore, the continuous generation of a wealth of information has resulted in the publication of numerous reviews, books, and monographs aimed at compiling an updated account of freshwater fungi [24,[51][52][53][54][55].A significant milestone has been achieved by Calabon and his co-authors who provide a comprehensive overview of the different facets of freshwater fungal biology [56].
Within Dictyosporaceae, Dictyocheirospora and Dictyosporium are the two most prominent genera that accommodate the majority of freshwater species, with 11 and 9 reported freshwater species, respectively [24,25].A geographical distribution review showed that numerous species within the family were reported from China and Thailand [2].In this study, we describe an additional four hyphomycetes collected from freshwater habitats in China.The findings of our study further validate the role of freshwater habitats as important reservoirs for species of Dictyosporiaceae.
Dictyocheirospora and Dictyosporium are phylogenetically related and share quite similar morphological characteristics.Yang et al. [20] highlighted that Dictyocheirospora can be distinguished from the latter genus by its non-complanate or cylindrical conidia, which are predominantly characterized by closely clustered terminal cells at the apex.Based on this classification scheme, Dictyosporium hydei, Dictyos.indicum, Dictyos.musae, and Dictyos.tetraploides have been transferred to Dictyocheirospora [20].The confirmation of the transfer through sequence data is still pending; however, we concur with this conclusion based on our newly acquired data.The newly discovered species Dictyoc.submersa has non-complanate and subcylindrical conidia with closely clustered terminal cells at the apex (Figure 2), while Dictyos.digitatum, Dictyos.guangdongense, and Dictyos.variabilisporum possess complanate conidia in which the terminal cells are not significantly clustered (Figures 3-5).
The phylogenetic relationships between genera in Dictyosporiaceae have been well investigated using DNA sequence data [1,2,7,12,24,25].However, the interspecific phylogenetic relationships in some cases remain ambiguous due to the limitation of insufficient sequence data included in the phylogenetic tree.In this study, the establishment of Dictyosporium guangdongense as a novel species is primarily based on its morphological characteristics, as the limited molecular data available cannot distinguish between Dictyos.guangdongense and its related species (see notes on Dictyos.guangdongense) (Table S1, Figure 1).In addition, Dictyosporium aquaticum, Dictyos.digitatum, Dictyos.palmae, and Dictyos.stellatum exhibit indistinguishable genetic relationships despite their distinct morphologies (Figure 1) [9,15,42,57].In the phylogenetic analysis, Dictyocheirospora clematidis and Dictyoc.thailandica also cannot be distinguished due to the absence of tef1-α sequence data for Dictyoc.thailandica [25].The protein-coding genes are quite necessary in species identification within this fungal group.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof10040259/s1,Table S1: Taxa used for phylogenetic analyses and their corresponding GenBank accession numbers.Newly generated sequences are indicated in red, ex-type strains are indicated in bold, and missing sequences are indicated with "-".

Figure 1 .
Figure1.The maximum likelihood (ML) tree is constructed using combined LSU, SSU, ITS, and tef1α sequence data.Bootstrap support values with an ML greater than 60% and Bayesian posterior probabilities (PPs) greater than 0.95 are indicated above the nodes as "ML/PP".The tree is rooted to Periconia igniaria (CBS 379.86 and CBS 845.96).Newly generated sequences are highlighted in red, and the type strains are indicated in bold.

DictyosporiumFigure 1 .
Figure1.The maximum likelihood (ML) tree is constructed using combined LSU, SSU, ITS, and tef1-α sequence data.Bootstrap support values with an ML greater than 60% and Bayesian posterior probabilities (PPs) greater than 0.95 are indicated above the nodes as "ML/PP".The tree is rooted to Periconia igniaria (CBS 379.86 and CBS 845.96).Newly generated sequences are highlighted in red, and the type strains are indicated in bold.
Author Contributions: Conceptualization, W.D. and M.D.; writing-original draft preparation, Y.-X.S.; writing-review and editing, W.D., M.D. and S.B.; supervision, W.D. and B.X.; funding acquisition, W.D. and B.X.All authors have read and agreed to the published version of the manuscript.