Additions to the Genus Helicosporium (Tubeufiaceae, Tubeufiales) from China with an Identification Key for Helicosporium Taxa

Helicosporous hyphomycetes is a group of filamentous fungi that shows promising application prospects in metabolizing bioactive natural compounds. During a study of helicosporous fungi in China, six new helicosporous taxa were collected and isolated from decaying wood in Guangxi Zhuang Autonomous Region, China. Morphological comparisons with multi-gene phylogenetic analyses revealed that these six taxa belong to Helicosporium (Tubeufiaceae, Tubeufiales), and they were recognized as three novel species and were named Helicosporium liuzhouense, H. multidentatum, and H. nanningense. Detailed descriptions and illustrations of the newly discovered taxa and comparisons with similar fungi are provided. In addition, a list and a key to accepted Helicosporium species are provided.

portions of conidiophores resembling tiny tooth-like or bladder-like protrusions, and hyaline to yellow green, pleurogenous helicoid conidia that are smaller than 25 µm diameter with conidial filaments usually not exceeding 4 µm thickness [10,11].
Helicosporium fungi have the potential of producing bioactive secondary metabolites. The antimicrobial activity of Helicosporium was first reported by Hardy and Sivasithamparam [15]. The main antimicrobial constituent, 2-methyl resorcinol, was isolated from Helicosporium sp. KCTC 0635BP by Choi et al. [16]. It was reported to have cytotoxicity against mammalian cells and antimicrobial activity against various types of fungi and bacteria [16].
During a study of helicosporous hyphomycetes in China, six new helicosporous taxa were collected from Guangxi Zhuang Autonomous Region. Three new species, Helicosporium liuzhouense, H. multidentatum, and H. nanningense, were identified based on morphological evidence and phylogenetic analyses of combined LSU, ITS, tef1α, and rpb2 sequence data. The results of the PHI test support the taxonomic classification of these three newly discovered species. The present study provides descriptive and illustrative morphological information as well.

Sample Collection, Specimen Examination, and Isolation
Specimens of decaying wood were randomly sampled from terrestrial and freshwater habitats in Guangxi Zhuang Autonomous Region, China. Freshwater samples were incubated in sterile, moist plastic containers at room temperature for approximately 14 days. After two weeks of collection, fresh specimens were examined and observed using a stereomicroscope (SMZ 745 and SMZ 800N, Tokyo, Nikon, Japan). Morphological characteristics of fresh fungi specimens were recorded with stereomicroscopes fitted with a digital camera. The measurement data for the helicoid conidia includes diameter, thickness, and length. The specific measurement method is shown in Figure 1. Single spore isolation was referred from the method described by Chomnunti et al. [17]. Purified cultures were cultured in a 25 °C incubator. The morphological features of colonies, including color, shape, and colony diameter, were recorded regularly.
The dried specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agriculture Sciences (GZAAS), Guiyang, China. The cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, and the Guizhou Culture Collection (GZCC), Guizhou, China.

DNA Extraction, PCR Amplification, and Sequencing
Using sterile toothpicks, 60-day-old mycelium was scraped from PDA plates and transferred to a 1.5-mL microcentrifuge tube. Using the Ezup fungus genomic DNA extraction kit (Sangon Biotech, Shanghai, China), DNA was extracted and sequenced following the manufacturer's instructions. After obtaining the DNA of the fungal strains, EF1- Single spore isolation was referred from the method described by Chomnunti et al. [17]. Purified cultures were cultured in a 25 • C incubator. The morphological features of colonies, including color, shape, and colony diameter, were recorded regularly.
The dried specimens were deposited in the Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agriculture Sciences (GZAAS), Guiyang, China. The cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, and the Guizhou Culture Collection (GZCC), Guizhou, China.

DNA Extraction, PCR Amplification, and Sequencing
Using sterile toothpicks, 60-day-old mycelium was scraped from PDA plates and transferred to a 1.5-mL microcentrifuge tube. Using the Ezup fungus genomic DNA extraction kit (Sangon Biotech, Shanghai, China), DNA was extracted and sequenced following the manufacturer's instructions. After obtaining the DNA of the fungal strains, EF1-983F/EF1-2218R, FRPB2-5F/FRPB2-7CR, ITS5/ITS4, and LR0R/LR5 were used as primers for amplification [18][19][20]. The amplification reactions of ITS, LSU, tef1α, and rpb2 were carried out according to the methods of Lu et al. [21,22]. After PCR amplification, the products were analyzed using 1% agarose gel electrophoresis. The purification and sequencing of PCR products were completed by Beijing Tsingke Biological Engineering Technology and Services Co., Ltd. (Beijing, China).

Phylogenetic Analyses
BioEdit version 7.0.5.3 was used to inspect the original sequences. The forward and reverse sequences were assembled using SeqMan v. 7.0.0 (DNASTAR, Madison, WI, USA) software and submitted to the GenBank database. Based on recent publications, additional sequences similar to Helicosporium were downloaded from GenBank [7][8][9][10][11]14]. Sequence alignments for each locus were carried using the online multiple alignment program MAFFT version 7, and the alignments were further automatically adjusted using the trimAl tool [23]. The phylogenetic tree was constructed using the methods described by Ma et al. [24], which included Maximum Likelihood (ML) and Bayesian Inference (BI).
The phylogenetic trees were edited using FigTree v1.4.0 software. The edited trees and figure layouts were edited using Adobe PhotoShop CC 2018 and Adobe Illustrator CC 2021 (Adobe Systems, San Jose, CA, USA) software. Sequences generated in this study were uploaded to GenBank (Table 1).

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) Analysis
Three new species, H. liuzhouense, H. multidentatum, and H. nanningense, were analyzed using GCPSR with closely related taxa from combined LSU-ITS-tef1-α-rpb2 gene regions. The pairwise homoplasy index (PHI) test was carried out in SplitsTree4 [25,26]. It indicates that there is no statistically significant evidence for recombination for the selected taxa when the P-value is above 0.05. Both the LogDet transformation and splits decomposition options were used to reveal the relationship among closely related species.

Phylogenetic Results
The partial LSU-ITS-tef1α-rpb2 nucleotide sequences were used to determine the phylogenetic position of our newly isolated taxa. The concatenated sequence matrix consisted of LSU (1-842 bp), ITS (843-1398 bp), tef1α (1399-2310 bp), and rpb2 (2311-3337 bp), totaling 3337 characters for 30 taxa and two outgroups, Acanthostigma chiangmaiense (MFLUCC 10-0125) and A. perpusillum (UAMH 7237). The ML and BI analyses of the concatenated LSU-ITS-tef1α-rpb2 dataset yielded similar tree topologies, and the ML tree is shown in Figure 1. The bootstrap support values of ML equal to or greater than 75%, and PP equal to or greater than 0.95 are given near the nodes as ML/PP, respectively.
The resulting multigene phylogenetic tree confirms that our newly isolated Helicosporium liuzhouense, H. multidentatum, and H. nanningense form a distinct clade from other taxa within the genus Helicosporium ( Figure 2).

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) Analysis
Application of the PHI test to the concatenated tree-locus sequences of LSU-ITS-tef1α-rpb2 revealed the recombination level within phylogenetically related species. No significant recombination events were observed between our species Helicosporium liuzhouense, H. multidentatum, and H. nanningense and closely related species in Helicosporium (Figure 3). The test results show Φ w = 1 for the combined sequence data, Φ w = 1 for LSU dataset, Φ w = 0.80 for ITS dataset, Φ w = 0.09 for tef1α, and Φ w = 0.93 for rpb2 data. Figure 2. The phylogenetic tree generated using Maximum Likelihood (ML) analysis based on combined LSU, ITS, tef1α, and rpb2 sequence data. ML and Bayesian Posterior Probabilities (PP) near the nodes are indicated as ML/PP. The Acanthostigma chiangmaiense (MFLUCC 10-0125) and A. perpusillum (UAMH 7237) were used as outgroup taxa. Ex-type strains are represented in bold. Newly generated sequences are represented in red.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) Analysis
Application of the PHI test to the concatenated tree-locus sequences of LSU-ITS-tef1α-rpb2 revealed the recombination level within phylogenetically related species. No significant recombination events were observed between our species Helicosporium liuzhouense, H. multidentatum, and H. nanningense and closely related species in Helicosporium ( Figure  3). The test results show Φw = 1 for the combined sequence data, Φw = 1 for LSU dataset, Φw = 0.80 for ITS dataset, Φw = 0.09 for tef1α, and Φw = 0.93 for rpb2 data.
Culture characteristics: Conidia germinated on water agar and produced germ tubes within 8 h. The colonies grew on PDA, had a circular shape with a flat surface and undulate edge. They reached a size of 46 mm in 6 weeks at 25 • C and exhibited a pale brown center with brown edges on PDA.    (Figure 3). Although H. liuzhouense and H. viridisporum Y.Z. Lu & J.C. Kang share similar conidiophores, conidiogenous cells, and conidial features [11], the phylogenetic analyses indicate that they are distinct species.
Culture characteristics: Conidia germinated on water agar and produced germ tubes within 8 h. The colonies grew on PDA and had a circular shape with a flat surface and undulate edge. They reached a size of 35  Hyde in having brown to dark brown, unbranched and septate conidiophores with integrated multi-dentate protrusions arising laterally from its lower portion, and hyaline to pale green or yellowish, pleurogenous, helicoid conidia [10,11]. However, H. multidentatum differs from H. hainanense in having longer conidial filaments (105-130 µm vs. 55-60 µm), and from H. vesicarium in having smaller conidial diameter (12-13 µm vs. 13-18 µm) [10,11]. Phylogenetically, H. multidentatum forms a sister clade of H. liuzhouense with strong support and is distant from H. hainanense and H. vesicarium (Figure 2).

Index Fungorum number: IF900556
Etymology: The epithet "nanningense" named after the city in which the holotype was found.
Culture characteristics: Conidia germinated on water agar and produced germ tubes within 12 h. The colonies grew on PDA and had a circular shape with a flat surface and undulate edge. They reached a size of 42 mm in 5 weeks at 25 • C and exhibited a brown center with nigger-brown edges on PDA.
Material  [11], but the phylogenetic analysis result supports that they are distinct species.
Helicomyces Link [37], Helicosporium [1], and Helicoma [38] are the three earliest described helicosporous genera. Based on morphological characteristics, researchers including Linder [2], Moore [39], and Goos [3,[27][28][29] carried out systematic classification studies on these three genera. Tsui et al. [40] conducted a phylogenetic analysis of helicosporous fungi. They discovered that the species of Helicomyces, Helicosporium, and Helicoma did not cluster within their respective genus-level taxonomy units but instead interbred and dispersed within the family Tubeufiaceae. Kuo and Goh [41] also reported the chaotic phylogenetic relationships between these three genera. Lu et al. [10] reevaluated these three genera, redefined their generic concepts based on morphological and phylogenetic evidence, and provided recommendations for classifying and identifying helicosporous fungi. However, some taxa within these genera still require additional morphological and molecular data to resolve their taxonomic issues. For example, Boonmee et al. [7] combined Helicosporium sp. NBRC 9014 (as Tubeufia cerea NBRC 9014 in Tsui et al. [40]) with H. vegetum based on phylogenetic analyses. However, Lu et al. [10] disagreed with this treatment as Helicosporium sp. NBRC 9014 did not cluster with other H. vegetum strains in the multi-gene phylogenetic tree. The taxonomic status of this strain remains unresolved due to insufficient morphological information [10].
Lu et al. [11] highlighted the challenge of taxonomic studies on helicosporous fungi due to their similar morphological characteristics. In this study, three new helicosporous fungi, namely, Helicosporium liuzhouense, H. multidentatum, and H. nanningense, were identified using morphological and phylogenetic analyses, with supporting evidence from the PHI test. Helicosporium liuzhouense and H. nanningense share similarities with H. sexuale and H. viridisporum in terms of conidiophores, conidiogenous cells, and conidial features, respectively, while H. multidentatum is comparable to H. hainanense and H. vesicarium. Notably, they have distinct phylogenetic positions (Figure 2). These findings reinforce the significance of molecular data in precisely distinguishing helicosporous hyphomycetes.
A checklist of accepted Helicosporium species is provided in this study ( Table 2). Nine species are found in freshwater habitats and 11 species in terrestrial habitats, including the newly described species in this study. Helicosporium sexuale occurs in both freshwater and terrestrial habitats. Among them, 18 species are reported only in their helicosporous asexual morph, while three species, viz., H. flavum, H. sexuale, and H. vegetum, have asexual-sexual links that have been confirmed by molecular data. The taxonomic status of 15 species has been determined through phylogenetic analyses, while six species do not have any molecular data and require further research to determine their phylogenetic relationships [42][43][44][45].
A key to the species accepted in Helicosporium is provided as well. Data Availability Statement: All sequences generated in this study were submitted to GenBank database.