Phylogenetic Insights Reveal New Taxa in Thyridariaceae and Massarinaceae

Pleosporales is a highly diverse (and the largest) order in Dothideomycetes, and it is widespread in decaying plants in various environments around the world. During a survey of fungal diversity in Sichuan Province, China, specimens of hyphomycetous and Thyridaria-like fungi were collected from dead branches of pine trees and cherry trees. These taxa were initially identified as members of Massarinaceae and Thyridariaceae through morphological examination. Phylogenetic analyses of the Thyridariaceae, combining ITS, SSU, LSU, RPB2, and TEF1 sequence data, indicated a distinct clade sister to Pseudothyridariella and Thyridariella, distinct from any genus in the family. Thus, a new genus, Vaginospora, is proposed to accommodate the type species Vaginospora sichuanensis, which is characterized by semi-immersed globose to oblong ascomata with an ostiolar neck, cylindrical to clavate asci with an ocular chamber, and hyaline to dark brown, fusiform, 3–5-transversely septate ascospores with an inconspicuous mucilaginous sheath. Based on the morphological comparisons and multi-locus phylogenetic analyses (ITS, SSU, LSU, RPB2, and TEF1) of the Massarinaceae, we have identified three collections belonging to the genus Helminthosporium, leading us to propose H. filamentosa sp. nov., H. pini sp. nov., and H. velutinum as a new host record. According to Phylogenetic analysis, H. pini formed an independent clade sister to H. austriacum and H. yunnanense, and H. filamentosa represents the closest sister clade to H. quercinum. Helminthosporium pini is distinct from H. austriacum by the shorter conidiophores and H. yunnanense by the longer and wider conidia. The H. filamentosa differs from H. quercinum in having longer conidiophores and smaller conidia. This study extends our understanding of diversity within Thyridariaceae and Helminthosporium. Our findings underscore the rich biodiversity and potential for discovering novel fungal taxa within these groups.


Introduction
Pleosporales is the largest order in the Dothideomycetes and is primarily characterized by flask-shaped pseudothecia [1,2].The diversity of species in Pleosporales is high, and most species are saprobes on decaying plant material in freshwater, marine, or terrestrial environments [3][4][5][6].The members of Pleosporales can also be epiphytes, endophytes, or parasites on living leaves or stems, as well as hyperparasites on fungi or insects [7][8][9].
Sichuan Province is located in a subtropical zone, where tropical and temperate flora coexist [29].The diverse climatic environment and complex terrain provide highly favorable conditions for the development of biodiversity.Sichuan Province contains many rare plants and a huge fungal diversity that is waiting to be explored [30][31][32][33][34].We regularly conduct fungal diversity surveys in Sichuan Province and investigate the taxonomy of fungi associated with specific plants, such as gymnosperms and cherries.During the study, a Thyridaria-like fungus and four hyphomycetous fungi were collected from Pinus spp.and Prunus spp.Based on morphological characteristics and multi-locus phylogenetic analysis, we identified these four collections as a new genus, Vaginospora, in the Thyridariaceae, along with two new species and a new host record of Helminthosporium.

Sample Collection, Morphological Examination, and Isolation
We surveyed the fungal diversity of Ascomycota on gymnosperms and cherries in Sichuan Province, China, from April 2023 to April 2024.The specimens were taken into the laboratory in paper envelopes for examination.Microscopic characters were observed and recorded using a Nikon SMZ800N stereo microscope equipped with a Nikon DS-Fi3 camera (Nikon Corporation, Tokyo, Japan) and a Nikon ECLIPSE Ni-U microscope (Nikon Corporation, Tokyo, Japan) fitted with a Nikon DS-Ri2 microscope (Nikon Corporation, Tokyo, Japan) camera.Measurements were conducted using the Nikon NIS-Elements Documentation Imaging Version 5.21.00 (Nikon Corporation, Tokyo, Japan).All photographs were processed using Adobe Photoshop version 22.0 (Adobe Inc., San Jose, SA, USA).Single ascospore or conidium isolation was performed following the method described by Senanayake et al. [35].Germinated ascospore or conidia were individually transferred to potato dextrose agar (PDA) media plates and incubated in the dark at 25 • C. Culture characteristics were examined and recorded regularly after 1-3 weeks.
The holotype specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and all specimens were deposited in the Herbarium of the University of Electronic Science and Technology (HUEST), Chengdu, China.The living ex-type cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and all living cultures were deposited in the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China.The taxonomic descriptions of the new taxa have been deposited in MycoBank.

DNA Extraction, PCR Amplification, and Sequencing
Fungal genomic DNA was extracted from mycelia using the Trelief TM Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, China) according to the manufacturer's protocol.For H. pini specimens, obtaining a culture was not feasible, necessitating the direct extraction of DNA from fruiting structures using the method used by Wanasinghe et al. [36].Five loci-the nuclear ribosomal internal transcribed spacer (ITS: ITS1-5.8S-ITS2), the nuclear ribosomal small subunit rRNA (SSU), the nuclear ribosomal large subunit rRNA (LSU), the partial translation elongation factor 1-alpha (TEF1), and the partial second largest subunit of RNA polymerase II (RPB2)-were amplified by polymerase chain reaction (PCR).The primers used were ITS9mun/ITS4_KYO1 [37,38] for ITS, LR0R/LR5 [39,40] for LSU, PNS1/NS41 [41] for SSU, EF1-728F/EF1-2218R [42,43] for TEF1, and fRPB2-5F/fRPB2-7cR [44,45] for RPB2.The final reaction volume of the PCR reagent was 25 µL, containing 2 µL of the DNA template, 1 µL each of the forward and reverse primers, 8.5 µL of double-distilled water (ddH 2 O), and 12.5 µL of 2× Flash PCR MasterMix (mixture of DNA Polymerase, dNTPs, Mg 2+ , and optimized buffer; CoWin Biosciences, Nanjing, China).The amplification conditions for all five loci consisted of initial denaturation at 94 • C for 3 min, followed by 35 cycles of 30 s at 94 • C, 30 s at 56 • C, and 1 min at 72 • C, and a final extension period of 10 min at 72 • C. The PCR products were visualized by 1% agarose gel electrophoresis.Sanger sequencing was conducted by Tsingke Biological Technology (Beijing, China).Newly generated sequences were deposited in GenBank, and the accession numbers are listed in Tables 1 and 2.

Phylogenetic Analyses
According to the corresponding Sanger sequencing chromatograms, misleading data from the ends of raw sequencing fragments were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0(DNASTAR, Inc., Madison, WI, USA).Barcode sequences of all species (Tables 1 and 2) were downloaded from the NCBI nucleotide database using the R package Analysis of Phylogenetics and Evolution 5.0 (APE, http://ape-package.ird.fr,26 June 2024) [46].
The multiple sequence alignments were conducted using MAFFT version 7.310 [47] with options "--adjustdirection --auto," and the alignment files were further trimmed using trimAl version 1.4 [48] with the option "-gapthreshold 0.5", which only allows 50% of taxa with a gap in each site.The best-fit nucleotide substitution models for each locus were selected using ModelFinder version 2.1.1 [49] under the Corrected Akaike Information Criterion (AICC).All sequence alignments were combined using an in-house Python script.
Maximum Likelihood (ML) and Bayesian analysis (BI) were conducted based on individual and combined datasets.Two phylogenetic trees were constructed by combining the ITS, SSU, LSU, RPB2, and TEF1 gene regions.The first tree represents the phylogenetic analysis of the Massarinaceae, while the second tree represents the phylogenetic analysis of the Thyridariaceae.ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [50], and the topology was evaluated using 1000 ultrafast bootstrap replicates.The BI was conducted using parallel MrBayes version 3.2.7a[51].The ML trees were visualized using ggtree version 2.4.1 [52] and further edited in Adobe Illustrator version 16.0.0.
Material examined: CHINA: Sichuan Province, Chengdu City, Jiudaoguai, 30  C in the dark, colonies from above: irregularly circular, white, uneven entire, raised in center, with denser mycelium at the center; from below: yellowish brown, dark brown in the center, margin undulated.
Notes: Helminthosporium velutinum is the type species of the genus Helminthosporium.It is widely distributed around the world, with 110 known host records, excluding Pinus species [60].Helminthosporium velutinum has been reported from both freshwater and terrestrial environments in China [16,24].Multi-locus analyses of combined ITS, LSU, SSU, RPB2, and TEF1 sequence data showed that our collection (UESTCC 24.0189) nests with H. velutinum strains with 99% ML, 1.00 PP statistical support (Figure 1).In addition, the morphological characteristics examined largely overlapped with H. velutinum [16].Thus, based on morphological comparison and phylogenetic analyses, we report our collections (UESTCC 24.0189) as a new host record of H. velutinum from Pinus sp.
Notes: According to phylogenetic analysis of Thyridariaceae, our collection (UESTCC 24.0191) constitutes a distinct clade, positioned as a sister to Pseudothyridariella and Thyridariella, with 100% ML and 1.00 PP statistical support (Figure 2).However, the sister relationship of Vaginospora to Pseudothyridariella and Thyridariella warrants discussion to clarify the issues of intergeneric affinities.Thyridariella can be distinguished from Vaginospora and Pseudothyridariella by having hyaline ascospores [61,62].Pseudothyridariella produces ascospores slightly constricted at the central septum, whereas Thyridariella and Vaginospora have ascospores constricted at every septum [61,62].Furthermore, the size of ascospores and septa in ascospores of Vaginospora is significantly smaller and fewer than in Thyridariella and Pseudothyridariella [61,62].Therefore, Vaginospora is proposed as a new genus to accommodate Vaginospora sichuanensis and expand the scope of the family Thyridariaceae.
Vaginospora sichuanensis W.H. Tian, Y. Jin & Maharachch., sp.nov.(Figure 6).C in the dark, colonies from above: irregularly circular, white, uneven entire, raised in center, with denser mycelium at the center and becoming sparser at the edge; from below: yellowish brown, brown in the center, margin undulated.

Discussion
Seven hundred and seventy-nine epithets of Helminthosporium have been listed in Index Fungorum (http://www.indexfungorum.org;26 June 2024).Konta et al. compiled a list of 216 identified and accepted species of Helminthosporium worldwide based on species records from Index Fungorum [15].However, most species are characterized solely based on morphology, and only 37 species have sequence data.The lack of extensive molecular data is primarily due to most species being reported and identified before sequencing technologies.This results in the possibility that many species within the Helminthosporium may be conspecific or belong to different genera [18,19,[21][22][23].Therefore, it is essential to re-examine the type specimens of formerly described Helminthosporium-like species to address this matter.Simultaneously, it is necessary to collect fresh specimens, sequence them, and combine multi-locus phylogenetic analysis with morphological examination to designate epi-types or neotypes, which is crucial for clarifying the taxonomic status of many doubtful species.
In recent studies, species of Pseudothyridariella have been grouped into two distinct clades based on phylogenetic analysis [70,71].The genus Pseudothyridariella was proposed by Mapook et al. [62], with P. chromolaenae as the type species, and Thyridariella mahakoshae was transferred to the Pseudothyridariella genus as P. mahakoshae.However, not many sequence data and species were available for constructing phylogeny at that time.More data are emerging showing that this species is clearly unrelated to the genus Pseudothyridariella, which supports our study [70,71].Morphologically, P. mahakoshae also differs from P. chromolaenae in that the former has brown or olive-brown to dark brown ascospores with 5-8 transverse septa, whereas the latter has hyaline ascospores with 3-6 transverse septate [61,62].When the genus Thyridariella was introduced, it was mentioned that one of its characteristic features was the production of hyaline muriform ascospores [61].Pseudothyridariella idesiae is not comparable here because it is known only for its asexual morph [71].Our newly introduced genus, Vaginospora sichuanensis, has muriform ascospores that are mostly 3-transversely septate, which are morphologically and phylogenetically distinct from P. mahakoshae.Therefore, more evidence should be collected, and the taxonomic status of P. mahakoshae should be revised in future studies.
The multi-locus phylogenetic analysis in this study revealed that our newly collected taxa of Vaginospora represent a new genus as it constitutes a strongly supported monophyletic clade (Figure 2).Molecular evidence further indicates that Vaginospora belongs to the family Thyridariaceae.Our collection (UESTCC 24.0191) was morphologically similar to the type genus Thyridaria of Thyridariaceae, with immersed or semi-immersed ascomata, composed of textura angularis cells in the peridium, bitunicate, cylindrical to subclavate asci that are apically rounded with an ocular chamber, and fusiform, septate ascospores [12].They differ in ascospore morphology, with our collection having 1-5 septa and being highly constricted at the septa, 1-2 longitudinal septa at maturity, and a sheath around the ascospores, which is not found in Thyridaria [12].Morphological character comparison is a traditional classification method that cannot reflect phylogenetic relationships; it could be informative at the generic level in Thyridariaceae.For example, Parathyridaria was introduced into the family for its characteristic features of discoid ostiolar apices always present and pale to greyish-brown ascospores [12], Thyridariella is distinguished from other genera by its hyaline ascospores [61]; a typical feature of Pseudothyridariella is that the ascospores are constricted only at the central septum [62].Thus, within Thyridariaceae, ascospores morphology is diverse, and when establishing a new genus, it can be combined with molecular data to provide further evidence and support.

Figure 1 .
Figure 1.The phylogram of the family Massarinaceae from ML analysis is based on the concatenated dataset of ITS-LSU-SSU-RPB2-TEF1.The tree is rooted with Periconia pseudodigitata (CBS 139699).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 1 .
Figure 1.The phylogram of the family Massarinaceae from ML analysis is based on the concatenated dataset of ITS-LSU-SSU-RPB2-TEF1.The tree is rooted with Periconia pseudodigitata (CBS 139699).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 2 .
Figure 2. The phylogram of the family Thyridariaceae from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2-SSU-TEF1.The tree is rooted with Torula herbarum (CBS 595.96).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Figure 2 .
Figure 2. The phylogram of the family Thyridariaceae from ML analysis is based on the concatenated dataset of ITS-LSU-RPB2-SSU-TEF1.The tree is rooted with Torula herbarum (CBS 595.96).Support values of ML-UFBoot ≥ 95 and Bayesian posterior probabilities ≥ 0.95 were displayed at the nodes as ML/PP.Support values below 95 and 0.95 are indicated by a hyphen (-).Newly collected taxa are shown in red.Strains from type materials are in bold.

Author Contributions:
Conceptualization, W.-H.T., Y.J. and S.S.N.M.; methodology, W.-H.T.; data sources, W.-H.T. and Y.J.; formal analysis, W.-H.T., Y.-C.L. and Y.J.; visualization, W.-H.T., Y.J. and Y.-C.L.; writing-original draft preparation, W.-H.T.; writing-review and editing, S.S.N.M., T.K.F. and W.-H.T.; supervision, S.S.N.M.; funding acquisition, X.-Y.G. and S.S.N.M.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the 2023 Xinjiang Production andConstruction Corps Agricultural Key Core Technology Research Project, grant number NYHXGG2023AA301; 2023 Seventh Division Huyanghe City "Taking Up the Challenge and Assuming Leadership" Project, grant number QS2023011; 2023 13th Division Xinxing City Science and Technology Plan Project, grant number 2023B4; Talent Introduction and Cultivation Project, University of Electronic Science and Technology of China, grant number A1098531023601245.We extend our appreciation to the Researchers Supporting Project at King Saud University, Riyadh, Saudi Arabia, for funding this research project, fund number RSP2024R487.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Species details and their GenBank accession numbers used in phylogenetic analyses of Massarinaceae.

Table 2 .
Species details and their GenBank accession numbers used in phylogenetic analyses of Thyridariaceae.Type isolates are in bold, and newly generated sequences are in red.