Synhelminthosporium gen. et sp. nov. and Two New Species of Helminthosporium (Massarinaceae, Pleosporales) from Sichuan Province, China

Helminthosporium is a polyphyletic genus in Massarinaceae (Pleosporales). Species of Helminthosporium are characterized by having septate and erect conidiophores, acro-pleurogenous and distoseptate conidia with a ring-shaped scar at the base. During a survey of fungal diversity in Sichuan Province, China, six Helminthosporium-like isolates were collected from dead branches of unknown trees. Five barcodes, including ITS (ITS1-5.8S-ITS2), SSU, LSU, TEF1, and RPB2 were amplified and sequenced. Morphological examination and multi-locus phylogenetic analyses revealed two new Helminthosporium species (H. chengduense sp. nov., and H. chinense sp. nov.), a new genus (Synhelminthosporium gen. nov.) with a type species Synhelminthosporium synnematoferum sp. nov., and two known species (Helminthosporium submersum and H. velutinum) within Massarinaceae. The new genus Synhelminthosporium differs from the phylogenetically closest genus Helminthosporium by producing synnematous conidiophores. This work expands our understanding of the diversity of Helminthosporium-like taxa in Sichuan Province, China.


Introduction
Fungi consist of a highly diverse lineage of eukaryotes with a huge estimated number of between 2.2 and 3.8 million species [1]. Investigating fungal diversity is vital in Assembling the Fungal Tree Of Life (AFToL) [2], which significantly enhances our understanding of the history of life and also strengthens our ability to explore and use fungal resources [3].
The genus Helminthosporium, typified by H. velutinum, is characterized by producing macronematous, cylindrical, rather straight, septate, erect conidiophores with tretic conidiogenous cells and clavate or obclavate, distoseptate conidia with a flat, ringed pore at the base [8,13]. Conidia are produced mainly laterally from tretic conidiogenous cells and the production of a terminal conidium usually determines the end of conidiophore growth. Most Helminthosporium species were introduced based on their asexual morph and only six species, viz., H. tiliae, H. microsorum, H. oligosporum, H. massarinum, H. quercicola, and H. quercinum, were characterized based on both morphs [8,11]. Splanchnonema kalakadense was described as the sexual morph of H. velutinum, but this was only based on pure culture without sequence data [14]. Tanaka et al. [11] first connected the Massarina-like sexual morph and asexual morph of H. massarinum, which was confirmed based on pure culture and sequence data. Voglmayr and Jaklitsch [8] experimentally confirmed three Splanchnonema-like sexual morphs of Helminthosporium species based on pure culture, sequence data, and herbarium studies, which extends the knowledge of sexual morphs of Helminthosporium.
Sichuan Province, located in southwestern China, along the Yangtze River, has enormous fungal diversity [15][16][17][18]. We regularly conduct fungal diversity surveys in Sichuan Province. During the study of preliminary morphological examination and BLASTn analysis of ITS sequences (the ribosomal internal transcribed spacer), a total of six Helminthosporium-like isolates were obtained from July to September 2021. Based on the multi-locus phylogenetic analysis and morphological examination, two known Helminthosporium species including a new habitat record, two new Helminthosporium species, and a new genus Synhelminthosporium with the type species, S. synnematoferum sp. nov. are introduced. This study broadens our understanding of the diversity of Helminthosporium-like taxa.

Sample Collection, Isolation, and Morphological Examination
A survey of the diversity of ascomycetous fungi in Sichuan Province, China, was conducted between July and September 2021. Dead branches were collected from three locations in Sichuan Province (Yunqiao Wetland, Chengdu City; Baiyungou, Chongzhou City; Huilonggou, Pengzhou City). The specimens were taken to the laboratory in paper envelopes for examination. The morphological observation was consistently carried out from material on natural substrates. Tiny pieces of mycelium were mounted in a drop of sterilized water using syringe needles. Microscopic characters were observed and recorded using a Nikon SMZ800N stereo microscope equipped with a Nikon DS-Fi3 microscope camera and a Nikon ECLIPSE Ni-U microscope fitted with a Nikon DS-Ri2 microscope camera, respectively. Measurements were conducted using the Nikon NIS-Elements Documentation Imaging Software Version 5.21.00. All photos were processed using Adobe Photoshop software version 22.0. Isolates were obtained by picking up pieces of mycelium into sterilized water, spreading the suspension onto the surface of potato dextrose agar (PDA) plates, and incubated for 24 h at 25 • C. Germinated conidia were individually transferred to PDA plates and incubated under the dark at 25 • C. Culture characteristics were examined and recorded after one week and later at regular intervals.
The specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, or the Herbarium of University of Electronic Science and Technology (HUEST), Chengdu, China. The living cultures 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) in Chengdu, China.

DNA Extraction, PCR Amplification, and Sequencing
Fungal genomic DNA was extracted from mycelia using the Trelief TM Plant Genomic DNA extraction Kit (TSINGKE Biotech, Shanghai, China) according to the manufacturer protocol. The DNA was stored at −20 • C for long-term storage. Five barcodes including the nuclear ribosomal internal transcribed spacer region (ITS: ITS1-5.8S-ITS2), the partial nuclear ribosomal small subunit rRNA gene (SSU), the partial nuclear ribosomal large subunit rRNA gene (LSU), the partial translation elongation factor 1-alpha gene (TEF1) and the partial second-largest subunit of RNA polymerase II gene (RPB2) were amplified by polymerase chain reaction (PCR). The corresponding primer pairs and PCR processes are listed in Table 1. The final PCR reaction volume was 25 µL containing 2 µL of DNA template, 1 µL each of the forward and reverse primer (10 µM), 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, Taizhou, China). The PCR products were visualized in 1% agarose gel electrophoresis. Sanger sequencing was conducted by Tsingke Biological Technology (Beijing, China).

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 Helminthosporium species currently available in GenBank, representative strains from other genera in Massarinaceae, and the outgroup taxon Periconia pseudodigitata (CBS 139699) were downloaded from the NCBI nucleotide database using the function read.GenBank integrated within the R package Analysis of Phylogenetics and Evolution (APE) [29].
The multiple sequence alignment was conducted using MAFFT version 7.310 [30] with options "-adjustdirectionaccurately-auto", and the alignment results were further trimmed using trimAl version 1.4 [31] 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 alignment dataset were selected using PartitionFinder version 2.1.1 [32] under the Corrected Akaike Information Criterion (AICC).
Maximum Likelihood (ML) and Bayesian analysis were conducted based on individual and combined datasets. Five alignment datasets of SSU, ITS, LSU, TEF1, and RPB2 were concatenated using an in-house python script for multi-locus phylogenetic analysis. ML phylogenetic trees were obtained using the IQ-TREE version 2.0.3 [33], and the topology was evaluated using 1000 ultrafast bootstrap replicates. The Bayesian analysis was conducted using parallel MrBayes version 3.2.7a [34]. Two different runs with 20 million generations and four chains were executed, and the initial 25% of sample trees were treated as burn-in. Tracer version 1.7.1 [35] was used to confirm that the MCMC runs reached convergence with all ESS values above 200. Then, the ML tree was annotated by TreeAnnotator version 2.6.6 implemented in BEAST version 2.6.6 [36] based on MrBayes MCMC trees with no discard of burn-in, and no posterior probability limit. The ML trees were visualized using ggtree [37] and further edited in Adobe Illustrator version 16.0.0.
The best-scoring ML consensus tree (lnL = −21,360.862) with ultrafast bootstrap values from ML analyses and posterior probabilities from MrBayes analysis at the node is shown in Figure 1.

MycoBank: MB 844417
Etymology: The name refers to China, the country where the fungus was collected. Saprobic on decaying wood in damp environment. Sexual morph: Unknown. Asexual morph: Colony on natural substrate effuse, black, hairy. Mycelium mostly immersed, towards the surface forming stroma-like aggregations of light to brown pseudoparenchymatous cells. Conidiophores 214-461 µm long (x = 326, n = 40), 8-16 µm wide (x = 11, n = 38) at the base, tapering to 6-10 µm (x = 8, n = 38) at the apex, arising solitarily or in fascicles from the stroma cells, erect, simple, straight or flexuous, thick-walled, subcylindrical, smooth, pale to dark brown, with well-defined small pores at the apex and rarely laterally beneath the upper 1-5 septa. Conidiogenous cells mono-to poly-tretic, cylindrical, integrated, terminal and intercalary, brown, secession schizo-lytic. Notes: The phylogenetic tree shows that the isolate UESTCC 22.0026 clusters with the ex-type strain (ZM 20380) of H. nanjingensis, which was introduced by Wang et al. [41] from dead branches of an unidentified tree in Nanjing City, Jiangsu Province, China. Our collection (HKAS 124017) shares similar morphological characteristics in the shape and color of conidiophores and conidia with the holotype (HSAUP 02 0198) [41] of H. nanjingensis on natural substrate. However, it differs from H. nanjingensis by having significantly shorter conidia (42-109 µm vs. 64.5-170.5 µm) and smaller number of disto-septa (4-10 vs. 6-17) [41]. The BLASTn analysis of ITS of our isolate UESTCC 22.0026 showed 98% identity (446/453 bp, no gap) with ex-type strain (ZM 20380) of H. nanjingensis. Helminthosporium nanjingensis produced yellow-green pigment on PDA media [41], but the isolate UESTCC 22.0026 does not produce pigment on PDA. Only the ITS sequence is available for H. nanjingensis. Therefore, we cannot compare the sequence difference of other barcodes. Thus, considering the difference in morphology and the ability to produce pigments, we describe the isolate    Culture characteristics: Colony on PDA 19 mm diam after 2 weeks in an incubator under dark conditions at 20 • C, white, irregular circular, surface velvety, with denser mycelium at the center and becoming sparser towards the edge, with unclear margin; reverse pale green at the center, with unclear white margin.
Notes: The phylogenetic tree showed that our isolate (UESTCC 22.0021) from decaying wood in a damp environment clustered with the ex-type strain (MFLUCC 16-1360) of H. submersum, which was introduced by Zhao et al. [42] from submerged wood in freshwater. Morphologically, our collection fits well with H. submersum by having effuse, velvety, dark brown or black colonies, mono-nematous, straight or flexuous, unbranched, pale to dark brown conidiophores, and similar size (49-86 × 14-25 µm vs. 41-55 × 14.5-18.5 µm) conidia [42]. Based on the overlapping morphological characteristics and the multi-locus phylogenetic tree, we identify our isolate as H. submersum. This is the first report of H. submersum isolated from decaying wood in terrestrial habitats.
Helminthosporium velutinum Link [as 'Helmisporium'], Mag. Gesell. naturf. Freunde, Berlin 3(1-2): 10, Table 1 Culture characteristics: Colony on PDA 19 mm after 2 weeks in an incubator under dark conditions at 20 • C, creamy white, irregular circular, surface velvety, with denser mycelium at the center and becoming sparser towards the edge, with clear margin; reverse white, pale green in the center, with clear margin.
Notes: The phylogenetic tree showed that the isolate HUEST 22.0022 clustered with the isolates of H. velutinum. Helminthosporium velutinum, the type of the genus, is a wellknown and most commonly recorded species [8]. It has been recorded mainly from woody substrates, and it is known for more than 100 host records [43]. Zhu et al. [12] first reported H. velutinum from a freshwater habitat in China, which is a less common habitat for this species. Our collection (HUEST 22.0022) displays similar morphological characteristics with the type of H. velutinum in the shape and color of colonies, conidiophores, conidiogenous cells, and conidia on the natural substrate [8]. We identified the isolate UESTCC 22.0022 as H. velutinum, a new record from terrestrial habitats in china considering similar morphological characteristics.   (c) Conidiophores with apical and lateral conidia; (d,e,j) Conidiophores with stroma cells; (f) Conidiophore apex with an apical conidium; (g) Conidiophore with a lateral conidium; (h,i) Front view and back view of culture on PDA after 1 week; (k-z) Conidia. Scale bars: (d-g,j,k,s) = 20 µm. Scale bar k applies to (l-r); (s) applies to (t-z).  The genus Synhelminthosporium is introduced based on the new species S. synnematoferum. Both BLASTn analysis results of five barcode sequences (SSU, ITS, LSU, TEF1, and RPB2) and multi-locus phylogenetic analyses showed that Synhelminthosporium is distinct and phylogenetically close to Helminthosporium. Synhelminthosporium differs from Helminthosporium by having synnematous conidiophores, which is a character only presented in Helminthosporiella within Massarinaceae [7,44]. Phylogenetic analysis shows that Synhelminthosporium is different from Helminthosporiella. In addition, Helminthosporiella shows catenate conidia, but this character is absent in Synhelminthosporium. Based on distinguishing morphological characteristics and multi-locus phylogenetic analyses, we introduce a new genus Synhelminthosporium to accommodate the new species S. synnematoferum in Massarinaceae.

Discussion
To date, there are 775 epithets of Helminthosporium (http://www.indexfungorum.org; accessed on 10 April 2022), whereas many of them are not congeneric with the generic type, and were reclassified into other groups in subsequent studies [46][47][48]. For instance, H. cynodontis was reassigned to the genus Bipolaris (Pleosporaceae, Pleosporales) due to the production of sympodial conidiogenous cells and by having darkly pigmented conidiogenous loci [46]. Recently, Konta et al. [7] accepted 216 Helminthosporium species, however many species are identified only based on morphological studies, and only 25 species have sequence data. The lack of a large amount of molecular data is mainly because most species were introduced before the advent of Sanger sequencing. Considering that numerous Helminthosporium species were characterized only based on morphological studies, it is likely that some of them belong to the same species or even to different genera. During phylogenetic analysis, abnormal long branches were observed in four . These species were introduced and characterized before the 1950s [49][50][51][52], whereas the sequence data related to them were submitted to GenBank by Vu et al. [53]. BLASTn analyses of these sequences showed that the top hits of ITS and LSU sequences for H. anomalum (CBS 161.27) belong to Bipolaris, ITS and LSU sequences for H. asterinum (CBS 203.35) belong to Kirschsteiniothelia, ITS and LSU sequences for H. decacuminatum (CBS 185.47) and H. gibberosporum (CBS 200.32) belong to Curvularia. The present study introduces two new Helminthosporium species and a new genus Synhelminthosporium based on multi-locus phylogenetic analysis and morphological studies. This phylogeny needs to be expanded by re-examining type materials of old described Helminthosporium-like species without molecular data, collecting new fresh specimens, sequencing, and using multi-locus analysis to establish epi-types or neotypes as necessary. Our new species can be distinguished from all other Helminthosporium species by morphological features and multi-locus phylogenetic analysis, and thus we are confident that the newly introduced species are distinct.
Recent studies have no universally accepted standard in selecting barcodes for phylogenetic analysis. Boonmee et al. [54] introduced H. chiangraiense using ITS and LSU. Crous et al. [45] introduced H. erythrinicola and H. syzygii using ITS, LSU, and RPB2. Zhu et al. [12] introduced H. aquaticum using SSU, ITS, and LSU barcodes. Voglmayr and Jaklitsch [8] pointed out that only ITS and/or LSU sequences can be problematic in resolving the phylogeny of Massarinaceae. Other barcodes RPB2 and TEF1 were proposed in multi-gene phylogenetic analyses of Massarineae, as these barcodes usually significantly increase the phylogenetic resolution [8,11]. However, the majority of Helminthosporium species do not have RPB2 and TEF1 barcodes (19 species have SSU sequence data; 25 species have SSU sequence data; 23 species have LSU sequence data; 17 species have RPB2 sequence data and 15 species have TEF1 sequence data). The present study conducted both individual and combined phylogenetic analyses. ITS, RPB2, and TEF1 barcodes offered more parsimony informative sites than SSU and LSU. In addition, more powerful resolution in delineating species and higher bootstrap support values for most clades were observed in single-gene ML trees ( Figures S1 and S2), indicating that these barcodes are better in resolving genera in Massarineae than the other two barcodes.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8070712/s1, Figure S1: Single-gene trees. Novel isolates are indicated in dark blue. Type isolates are in bold. The ML ultrafast bootstrap values/Bayesian PP greater than 95%/0.95 are indicated at the respective nodes. The tree is rooted with Periconia pseudodigitata (CBS 139699) (Periconiaceae, Pleosporales). Figure S2: Comparisons of the single-gene ML trees and the multi-locus ML tree in delineating species. (a) The proportion of highly supported internal nodes, bootstrap values ≥ 95%. Protein-coding barcodes TEF1 and RPB2 offer higher proportions of highly supported internal nodes than three nuclear ribosomal regions (b) Pairwise tree distances. The smaller the value, the higher the similarity in topology. Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.