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

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Abstract
Helicosporous hyphomycetes is a class 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 can be recognized as three novel species, 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.
Keywords: 
Subject: Biology and Life Sciences  -   Biology and Biotechnology

1. Introduction

Based on the type species H. vegetum, Nees [1] established Helicosporium as one of the earliest described genera of helicosporous hyphomycetes. The majority of this group’s species inhabit subtropical to tropical habitats [2,3,4,5,6,7,8,9,10,11]. Typically, they inhabit woody substrates in terrestrial and freshwater environments [3,7,8,9,10,12]. Index Fungorum (accessed on 20 May 2023) [13] currently lists 105 taxa of Helicosporium, of which 75 species have been excluded or transferred to other genera. Most of these taxa were transferred to the genera Helicoma and Neohelicosporium. Currently, there are eighteen accepted species of Helicosporium, twelve of which have molecular data [3,7,8,10,11].
Most species in this genus are reported to have a helicosporous asexual morph. There are three species with reported sexual morph, viz. H. flavum, H. sexuale and H. vegetum [7,8,14]. The sexual morph of Helicosporium is characterized by solitary, yellowish brown, globose to subglobose ascomata, cylindric-clavate, 8-spored bitunicate asci, and hyaline to yellowish brown, fusiform ascospores [7,8,14]. The asexual morph is distinguished by pale yellow to yellow green colonies on the natural woody substratum, erect, setiferous, cylindrical conidiophores with denticulate conidiogenous cells arising laterally from the lower 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 certain potential in the production of 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.

2. Materials and Methods

2.1. 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 (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan).
Single spore isolation was referred from the method described by Chomnunti et al. [17]. Purified cultures were cultured in a 25 ℃ 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.

2.2. 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, 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 to 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).

2.3. 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 edit trees and figures layout 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 in GenBank (Table 1).

2.4. 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 a 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.

3. 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 chiangmaiensis (MFLUCC 10–0125) and A. perpusillum (UAMH 7237). The concatenated LSU, ITS, tef1α, and rpb2 datasets were analyzed using ML and BI methods, yielding similar tree topologies.
The resulting multigene phylogenetic tree confirms that our newly isolated Helicosporium liuzhouense, H. multidentatum and H. nanningense have a distant clade from other taxa within the genus Helicosporium (Figure 1).

4. 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 2). 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.

5. Taxonomy

Helicosporium liuzhouense X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 3.
Index Fungorum number: IF900461
Etymology: The epithet “liuzhouense” named after the city from which the holotype was found.
Holotype: HKAS 125865
Saprobic on decaying wood in a freshwater stream. Sexual morph: Undetermined. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, bright lime green. Mycelium partly immersed, partly superficial, composed of branched, septate, hyphae, pale brown to brown hyphae. Conidiophores macronematous, mononematous, erect, flexuous or straight, unbranched, apical sterile, septate, cylindrical, (102) 110–180 (213) × 4–5 μm ( x ¯   = 145 ×5 μm, n = 25), brown to dark brown, thick-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, discrete, determinate, arising laterally from the lower portion of the conidiophores as tiny tooth-like protrusions, hyaline to pale brown, smooth-walled. Conidia solitary, pleurogenous, helicoid, rounded at tip, 13–15 μm diam., and conidial filament 1–2 μm wide ( x ¯ = 14 × 1.5 μm, n = 25), 90–105 μm long, tightly coiled 2–3 times, becoming loosely coiled in water, indistinctly multi-septate, guttulate, hyaline to pale green, smooth-walled.
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 ℃, and exhibited a pale brown center with brown edges on PDA.
Material examined: CHINA, Guangxi Zhuang Autonomous Region, Liuzhou City, Luzhai County, on decaying wood in a freshwater stream, 4 May 2021, Xing-Juan Xiao & Jian Ma, LZ3 (HKAS 125865, holotype; GZAAS 22–2014, isotype), ex-type living culture CGMCC, GZCC 22–2014. Ibid., LZ3-2 (HKAS 125870, paratype), living culture GZCC 23–0586.
Notes: Phylogenetically, Helicosporium liuzhouense is strongly supported as a sister species to H. multidentatum and H. nanningense (97% ML/ 1.00 PP). Morphologically, Helicosporium liuzhouense is distinguished from H. multidentatum by having conidiogenous cells with tiny tooth-like protrusions, while H. multidentatum has conidiogenous cells with integrated multi-dentate protrusions. Additionally, H. liuzhouense is characterized by shorter conidia (90–105 µm vs. 105–128 µm) and a larger conidial diameter (13–15 µm vs. 12–13 µm) compared to H. multidentatum. H. liuzhouense can be differentiated from H. nanningense by having shorter conidiophores (90–115 µm vs. 100–215 µm) and conidial filaments (82–92 µm vs. 90–105 µm). The PHI test provides strong evidence showing that they are separate species (Figure 2). Although H. liuzhouense and H. viridisporum share similar conidiophores, conidiogenous cells and conidial features[11], the phylogenetic analyses indicating that they are distinct species.
Helicosporium multidentatum X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 4.
Index Fungorum number: IF900460
Etymology: The epithet “multidentatum” referring to the multi-dentate integration protrusions conidiogenous cells.
Holotype: HKAS 125856
Saprobic on decaying wood in a terrestrial habitat. Sexual morph: Undetermined. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, bright lime green. Mycelium partly immersed, partly superficial, composed of branched, septate, pale brown to brown hyphae. Conidiophores macronematous, mononematous, erect, flexuous or straight, unbranched, apical sterile, septate, cylindrical, 130–200 × 4–6 μm ( x ¯ = 165 × 5 μm, n = 20), brown to dark brown, thick-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, discrete, determinate, arising laterally from the lower portion of the conidiophores as integrated multi-dentate protrusions, hyaline to pale brown, smooth-walled. Conidia solitary, pleurogenous, helicoid, rounded at tip, 12–13 μm diam., and conidial filament 1.5–3 μm wide ( x ¯ = 12.5× 2.5 μm, n = 30), 105–130 μm long, tightly coiled 31/4–33/4 times, becoming loosely coiled in water, indistinctly multi-septate, guttulate, hyaline to pale green, smooth-walled.
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 35 mm in 5 weeks at 25 ℃, and exhibited a pale brown center with brown edges on PDA.
Material examined: CHINA, Guangxi Zhuang Autonomous Region, Guilin City, Qixingyan Scenic spot, on decaying wood, 4 May 2021, Xing-Juan Xiao & Jian Ma, QXY8 (HKAS 125856, holotype; GZAAS 22–2013, isotype), ex-type culture CGMCC, GZCC 22–2013. Ibid., QXY8-2 (HKAS 125855, paratype), living culture GZCC 23–0585.
Notes: Morphologically, Helicosporium multidentatum is similar to H. hainanense and H. vesicarium 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 to H. liuzhouense with strong support and is distant from H. hainanense and H. vesicarium (Figure 1).
Helicosporium nanningense X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 5.
Index Fungorum number: IF900556
Etymology: The epithet “nanningense” named after the city from which the holotype was found.
Holotype: HKAS 128858
Saprobic on decaying wood in a terrestrial habitat. Sexual morph: Undetermined. Asexual morph: Hyphomycetous, helicosporous. Colonies on the substratum superficial, effuse, gregarious, bright green. Mycelium partly immersed, partly superficial, branched, septate, pale brown to brown hyphae. Conidiophores macronematous, mononematous, flexuous or straight, unbranched, apical sterile, septate, cylindrical, 90–115 × 4–5 μm ( x ¯ = 102 × 5 μm, n = 20), brown to dark brown, thick-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, discrete, determinate, arising laterally from the lower portion of the conidiophores as tiny tooth-like protrusions, hyaline to pale brown, smooth-walled. Conidia solitary, pleurogenous, helicoid, rounded at tip, 11–14 μm diam., and conidial filament 1.5–2.0 μm wide ( x ¯ = 13 × 1.8 μm, n = 30), 82–92 μm long, tightly coiled 2–31/2 times, becoming loosely coiled in water, indistinctly multi-septate, guttulate, hyaline to pale green, smooth-walled.
Culture characteristics: Conidia germinated on water agar and produced germ tubes within 12 h. The colonies grew on PDA, had a circular shape with a flat surface and undulate edge. They reached a size of 42 mm in 5 weeks at 25 ℃, and exhibited a brown center with nigger-brown edges on PDA.
Material examined: CHINA, Guangxi Medicinal Botanical Garden, Nanning City, on decaying wood in a terrestrial habitat, 4 May 2021, Xing-Juan Xiao & Jian Ma, GXZWY3.2 (HKAS 128858, holotype; GZAAS 23–0591, isotype), ex-type living culture CGMCC, GZCC 23–0587. Ibid., GXZWY3.5 (GZAAS 23–0592, paratype), living culture GZCC 23–0588.
Notes: Phylogenetically, Helicosporium nanningense shares a sister relationship to H. multidentatum with high statistical support (94% ML/ 1.00 PP). Morphologically, H. nanningense differs from H. multidentatum in having different conidiogenous cells (tiny tooth-like protrusions vs. integrated multi-dentate protrusions). Additionally, H. nanningense differs from H. multidentatum in having shorter conidiophores (90–115 µm vs. 130–200 µm) and larger conidia (105–130 µm vs. 82–92 µm). Moreover, H. nanningense is similar to H. viridisporum in conidiophores, conidiogenous cells and conidial features [11], but the phylogenetic analysis result supports they are distinct species.

6. Discussion

Helicosporous fungi are filamentous fungi whose conidia curve through at least 180 degrees in one plane or three-dimensional space as they grow longer [2,3,9,10,11,27,28,29,30] Due to their ability to produce active secondary metabolites with distinct structures, these fungi have attracted scientists’ interest and become a popular study area. Numerous novel helicosporous taxa have recently been discovered in subtropical to tropical terrestrial and freshwater habitats [9,10,11,12,21,22,30,31,32]. In addition, a growing number of active secondary metabolites have been isolated from helicosporous fungi [33,34,35,36].
Helicomyces [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. [49] 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 1). 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. H. sexuale exists 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.
Key to species of Helicosporium
1. Ascomata present 2
1. Ascomata lacking 4
2. Ascomata bright yellow to brown yellow, without setae H. vegetum
2. Ascomata bright yellow to brown yellow, with sparsely setae 3
3. Ascomata globose to subglobose, 185–235 µm high, 116–214 µm diam. H. sexuale
3. Ascomata globose to subglobose, 320–350 µm high, 220–260 µm diam. H. flavum
4. Fresh colonies on decaying woody substrate are bright green 5
4. Fresh colonies on decaying woody substrate are hyaline, gray, yellow, yellow green, or dark brown 7
5. Conidiophores unbranched, with multi-dentate protrusions conidiogenous cells H. multidentatum
5. Conidiophores unbranched, with tiny tooth-like protrusions conidiogenous cells 6
6. Conidiophores < 120 μm long H. nanningense
6. Conidiophores > 120 μm long H. liuzhouense or H. viridisporum
7. Fresh colonies on decaying woody substrate are yellow green 8
7. Fresh colonies on decaying woody substrate are hyaline, gray, yellow, or dark brown 14
8. Conidiophores with bladder-like protrusions conidiogenous cells 9
8. Conidiophores with tiny tooth-like protrusions conidiogenous cells 10
9. Conidiophores < 120 μm long H. vesicarium
9. Conidiophores > 120 μm long H. hainanense
10. Conidia are acropleurogenous H. thailandense
10. Conidia are pleurogenous and apical sterile in conidiophores 11
11. Conidiophores < 180 μm long 12
11. Conidiophores > 180 μm long 13
12. Conidia 10–14 µm diam., 70–90 µm long H. aquaticum
12. Conidia 13–21 µm diam., 100–130 µm long H. setiferum
13. Conidiophores 184–257 µm long H. flavidum
13. Conidiophores 250–425 µm long H. viridiflavum
14. Fresh colonies on decaying woody substrate are yellow 15
14. Fresh colonies on decaying woody substrate are hyaline, gray, or dark brown 16
15. Conidiophores with bladder-like protrusions conidiogenous cells H. flavisporum
15. Conidiophores with tiny tooth-like protrusions conidiogenous cells H. luteosporum
16. Fresh colonies on decaying woody substrate are hyaline, branched conidiophores 200–300 µm long H. albidum
16. Fresh colonies on decaying woody substrate are gray, or dark brown 17
17. Fresh colonies on decaying woody substrate are gray, conidiophores 350–400 µm long, with bladder-like protrusions conidiogenous cells H. neesii
17. Fresh colonies on decaying woody substrate are dark brown 18
18. Conidiophores are unbranched with tooth-like protrusions conidiogenous cells H. murinum
18. Conidiophores are rarely branched 19
19. Tooth- or bladder-like protrusions conidiogenous cells, conidia 6–9 µm diam. H. decumbens
19. Tooth-like protrusions conidiogenous cells, conidia 11.4–19 µm diam. H. melghatianum

Author Contributions

The specimens in this study were collected by X.-J.X., J.M. and Y.-Z.L. Morphological data were collected by X.-J.X. and J.M. Molecular data and phylogenetic analyses were performed by X.-J.X. and J.M. Writing-original were completed by X.-J.X., J.M. Review and editing were done by X.-J.X., J.M., L.-J.Z., N.-G.L., Y.-P.X., Z.-L.L. and Y.-Z.L.

Funding

This work was funded by Guizhou Provincial Key Technology R&D Program (grant No. Qian Ke He Zhi Cheng [2021] Generally 200), Guizhou Province high-level talent innovation and entrepreneurship merit funding project (No. 202104), Youth Science and Technology Talent Development Project from Guizhou Provincial Department of Education (QJHKYZ [2021]263) and the academic emerging project of the Guizhou Institute of Technology (GZLGXM-15).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences generated in this study were submitted to GenBank database.

Acknowledgments

We would like to thank Dr. Shaun Pennycook (Manaaki Whenua Landcare Research, New Zealand) for advising on fungal nomenclature.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The phylogenetic tree generated by maximum likelihood (ML) analysis based on a combined LSU, ITS, tef1α, and rpb2 sequence data. ML and Bayesian posterior probabilities (PP) near the nodes are indicated as ML/PP. The Acanthostigma chiangmaiensis (MFLUCC 10–0125) and A. perpusillum (UAMH 7237) were used as outgroup taxa. Ex-type strains represented in bold. Newly generated sequences are represented in red.
Figure 1. The phylogenetic tree generated by maximum likelihood (ML) analysis based on a combined LSU, ITS, tef1α, and rpb2 sequence data. ML and Bayesian posterior probabilities (PP) near the nodes are indicated as ML/PP. The Acanthostigma chiangmaiensis (MFLUCC 10–0125) and A. perpusillum (UAMH 7237) were used as outgroup taxa. Ex-type strains represented in bold. Newly generated sequences are represented in red.
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Figure 2. Results of the PHI test of Helicosporium liuzhouense, H. multidentatum and H. nanningense with closely related species (combined LSU-ITS-tef1α-rpb2) using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset. The new taxon is in red bold type. Our new species are indicated in red, type strains are marked with “T”.
Figure 2. Results of the PHI test of Helicosporium liuzhouense, H. multidentatum and H. nanningense with closely related species (combined LSU-ITS-tef1α-rpb2) using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset. The new taxon is in red bold type. Our new species are indicated in red, type strains are marked with “T”.
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Figure 3. Helicosporium liuzhouense (HKAS 125865, holotype). (a,b) Colonies on natural substrate. (c–f) Conidiophores. (g–i) Conidiogenous cells. (j) Germinating conidium. (k–p) Conidium. (q–r) Colonies on PDA from above and below. Scale bars: (c–f) 50 μm, (j) 20 μm, (g–i,k–p) 10 μm.
Figure 3. Helicosporium liuzhouense (HKAS 125865, holotype). (a,b) Colonies on natural substrate. (c–f) Conidiophores. (g–i) Conidiogenous cells. (j) Germinating conidium. (k–p) Conidium. (q–r) Colonies on PDA from above and below. Scale bars: (c–f) 50 μm, (j) 20 μm, (g–i,k–p) 10 μm.
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Figure 4. Helicosporium multidentatum (HKAS 125856, holotype). (a,b) Colonies on natural substrate. (c–f) Conidiophores. (g–i) Conidiogenous cells. (j) Germinating conidium. (k–p) Conidium. (q–r) Colonies on PDA from above and below. Scale bars: (c–f) 50 μm, (j–k, n–p) 20 μm, (g–i,l–m) 10 μm.
Figure 4. Helicosporium multidentatum (HKAS 125856, holotype). (a,b) Colonies on natural substrate. (c–f) Conidiophores. (g–i) Conidiogenous cells. (j) Germinating conidium. (k–p) Conidium. (q–r) Colonies on PDA from above and below. Scale bars: (c–f) 50 μm, (j–k, n–p) 20 μm, (g–i,l–m) 10 μm.
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Figure 5. Helicosporium nanningense (HKAS 128858, holotype). (a,b) Colonies on natural substrate. (c–g) Conidiophores. (h–j) Conidiogenous cells. (k–q) Conidium. (r,s) Colonies on PDA from above and below. Scale bars: (c–g) 20 μm, (h–q) 10 μm.
Figure 5. Helicosporium nanningense (HKAS 128858, holotype). (a,b) Colonies on natural substrate. (c–g) Conidiophores. (h–j) Conidiogenous cells. (k–q) Conidium. (r,s) Colonies on PDA from above and below. Scale bars: (c–g) 20 μm, (h–q) 10 μm.
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Table 1. The taxa used in this study, along with their corresponding GenBank accession numbers for DNA sequences, are presented in the following table.
Table 1. The taxa used in this study, along with their corresponding GenBank accession numbers for DNA sequences, are presented in the following table.
Taxon Strain GenBank Accessions
ITS LSU tef1α rpb2
Acanthostigma chiangmaiensis MFLUCC 10-0125T JN865209 JN865197 KF301560 -
Acanthostigma patagonicum BBB MVB 573 JN127358 JN127359 - -
Acanthostigma perpusillum UAMH 7237T AY916492 AY856892 - -
Helicoma vaccinii CBS 216.90 AY916486 AY856879 - -
Helicosporium aquaticum MFLUCC 17-2008T MH558733 MH558859 MH550924 MH551049
Helicosporium flavisporum MFLUCC 17-2020T MH558734 MH558860 MH550925 MH551050
Helicosporium flavum MFLUCC 16-1230T KY873626 KY873621 KY873285 -
Helicosporium hainanense GZCC 22-2006T OP508730 OP508770 OP698081 OP698070
Helicosporium liuzhouense GZCC 22-2014T OQ981394 OQ981402 OQ980476 OQ980474
Helicosporium liuzhouense GZCC 23-0586 OR066416 OR066423 OR058862 OR058855
Helicosporium luteosporum MFLUCC 16-0226T KY321324 KY321327 KY792601 -
Helicosporium luteosporum MFLUCC 16-1233 - KY873624 - -
Helicosporium multidentatum GZCC 22-2013T OQ981395 OQ981403 OQ980477 OQ980475
Helicosporium multidentatum GZCC 23-0585 OR066417 OR066424 OR058863 OR058856
Helicosporium nanningense GZCC 22-2175T OR066418 OR066425 OR058864 OR058857
Helicosporium nanningense GZCC 23-0588 OR066419 OR066426 OR058865 OR058858
Helicosporium setiferum BCC 3332 AY916490 AY856907 - -
Helicosporium setiferum BCC 8125 AY916491 - - -
Helicosporium setiferum MFLUCC 17-1994T MH558735 MH558861 MH550926 MH551051
Helicosporium setiferum MFLUCC 17-2006 MH558736 MH558862 MH550927 MH551052
Helicosporium setiferum MFLUCC 17-2007 MH558737 MH558863 MH550928 MH551053
Helicosporium sexuale GZCC 22-2007 OP508731 OP508771 OP698082 OP698071
Helicosporium sexuale MFLUCC 16-1244T MZ538503 MZ538537 MZ567082 MZ567111
Helicosporium sp. NBRC 9014 AY916489 AY856903 - -
Helicosporium thailandense MFLUCC 18-1407T MT627698 MN913718 MT954371 -
Helicosporium vegetum CBS 254.75 - DQ470982 DQ471105 -
Helicosporium vegetum CBS 269.52 AY916487 AY856893 - -
Helicosporium vegetum CBS 941.72T AY916488 AY856883 - -
Helicosporium vegetum NBRC 30345 - AY856896 - -
Helicosporium vesicarium MFLUCC 17-1795T MH558739 MH558864 MH550930 MH551055
Helicosporium viridiflavum MFLUCC 17-2336T MH558738 - MH550929 MH551054
Helicosporium viridisporum GZCC 22-2008T OP508736 OP508776 OP698087 OP698076
Note: “T” represents the ex-type strain. Newly generated sequences are represented in bold. “-” indicates that no sequence data are available in GenBank.
Table 2. Checklist of accepted Helicosporium species.
Table 2. Checklist of accepted Helicosporium species.
Species Habitats Distribution Molecular data References
H. albidum Terrestrial Belgium, Britain (Birminghan) No Grove 1886
H. aquaticum Freshwater Thailand Yes Lu et al. 2018b
H. decumbens Terrestrial Austria, Brazil No Linder 1929
flavidum Freshwater China No Hsieh 2021
H. flavisporum Freshwater Thailand Yes Lu et al. 2018b
H. flavum Freshwater Thailand Yes Brahamanage et al. (2017)
H. hainanense Terrestrial China Yes Lu et al. 2022
H. liuzhouense Freshwater China Yes This study
H. luteosporum Terrestrial Thailand Yes Lu et al. 2017a
H. melghatianum Terrestrial India No Dharkar et al. 2010
H. murinum Terrestrial Argentina, Austria, Brazil, Canada, China, Cuba, Malaysia, USA No Linder 1929; Goos 1989; Zhao et al. 2007
H. multidentatum Terrestrial China Yes This study
H. nanningense Terrestrial China Yes This study
H. neesii Terrestrial USA No Moore 1957
H. setiferum Freshwater Thailand Yes Lu et al. 2018b
H. sexuale Freshwater, Terrestrial China, Thailand Yes Boonmee et al. 2021; Lu et al. 2022
H. thailandense Freshwater Thailand Yes Dong et al. 2020
H. vegetum Terrestrial Worldwide Yes Boonmee et al. 2014
H. vesicarium Freshwater Thailand Yes Lu et al. 2018b
H. viridiflavum Terrestrial Thailand Yes Lu et al. 2018b
viridisporum Freshwater China Yes Lu et al. 2022
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