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Article

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

by
Xing-Juan Xiao
1,
Jian Ma
1,2,
Li-Juan Zhang
1,
Ning-Guo Liu
1,
Yuan-Pin Xiao
1,
Xing-Guo Tian
1,
Zong-Long Luo
3 and
Yong-Zhong Lu
1,*
1
School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China
2
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
3
College of Agriculture and Biological Science, Dali University, Dali 671003, China
*
Author to whom correspondence should be addressed.
J. Fungi 2023, 9(7), 775; https://doi.org/10.3390/jof9070775
Submission received: 21 June 2023 / Revised: 17 July 2023 / Accepted: 18 July 2023 / Published: 22 July 2023
(This article belongs to the Special Issue Polyphasic Identification of Fungi 3.0)
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Abstract

:
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.

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 Corda and Neohelicosporium Y.Z. Lu, J.C. Kang & K.D. Hyde. Currently, there are eighteen accepted species of Helicosporium, and twelve of which have molecular data [3,7,8,10,11].
All species in this genus are reported to have a helicosporous asexual morph. There are three species with reported sexual morph, viz., H. flavum Brahaman., Y.Z. Lu, Boonmee & K.D. Hyde, H. sexuale Boonmee, Promputtha & K.D. Hyde, and H. vegetum [7,8,14]. The sexual morph of Helicosporium is characterized by solitary, yellowish brown, globose to subglobose ascomata, cylindric-clavate, eight-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 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.

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 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.

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 (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).

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 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).

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 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 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).

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 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.

5. Taxonomy

Helicosporium liuzhouense X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 4.
Index Fungorum number: IF900461
Etymology: The epithet “liuzhouense” named after the city in which the holotype was found.
Holotype: HKAS 125865
Saprobic on decaying wood in a freshwater stream. Sexual morph: Unknown. 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, septate, apical sterile, cylindrical, (102) 110–180 (213) × 4–5 μm ( x ¯   = 145 × 5 μm, n = 25), brown to dark brown, thick-walled, smooth-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, determinate, cylindrical, with denticles, 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 °C 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. Helicosporium 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 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.
Helicosporium multidentatum X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 5.
Index Fungorum number: IF900460
Etymology: The epithet “multidentatum” refers to the multi-dentate integration protrusions conidiogenous cells.
Holotype: HKAS 125856
Saprobic on decaying wood in a terrestrial habitat. Sexual morph: Unknown. 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, septate, apical sterile, cylindrical, 130–200 × 4–6 μm ( x ¯ = 165 × 5 μm, n = 20), brown to dark brown, paler towards the apex, thick-walled, smooth-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, discrete, bladder-like, arising laterally from the lower portion of the conidiophores, 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 and had a circular shape with a flat surface and undulate edge. They reached a size of 35 mm in 5 weeks at 25 °C 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 Y.Z. Lu & J.C. Kang and H. vesicarium Y.Z. Lu, J.C. Kang & K.D. 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).
Helicosporium nanningense X.J. Xiao, J. Ma & Y.Z. Lu, sp. nov., Figure 6.
Index Fungorum number: IF900556
Etymology: The epithet “nanningense” named after the city in which the holotype was found.
Holotype: HKAS 128858
Saprobic on decaying wood in a terrestrial habitat. Sexual morph: Unknown. 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, septate, apical sterile, cylindrical, 90–115 × 4–5 μm ( x ¯ = 102 × 5 μm, n = 20), brown to dark brown, thick-walled, smooth-walled. Conidiogenous cells holoblastic, monoblastic to polyblastic, integrated, determinate, with denticles, 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 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 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 that they are distinct species.

6. Discussion

Helicosporous fungi are filamentous fungi whose conidia curve moves by 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 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.
Key to species of Helicosporium
1. Fresh colonies on decaying woody substrate are bright green····················································································································2
1. Fresh colonies on decaying woody substrate are hyaline, gray, yellow, yellow green, or dark brown··························5
2. Conidiophores unbranched, with multi-dentate protrusions conidiogenous cells····················································H. multidentatum
2. Conidiophores unbranched, with tiny tooth-like protrusions conidiogenous cells··············································································3
3. Conidiophores < 120 μm long··········································································H. nanningense
3. Conidiophores > 120 μm long····················································4
4. Conidiophores (102) 110–180 (213) µm long, 4–5 µm wide, conidia 13–15 µm diam., 90–105 µm long····················································H. liuzhouense
4. Conidiophores 80–206 µm long, 3–7 µm wide, conidia 12–14 µm diam., 75–97 µm long····················································H. viridisporum or H. viridisporum
5. Fresh colonies on decaying woody substrate are yellow green····················································6
5. Fresh colonies on decaying woody substrate are hyaline, gray, yellow, or dark brown····················································14
6. Conidiophores with bladder-like protrusions conidiogenous cells····················································7
6. Conidiophores with tiny tooth-like protrusions conidiogenous cells··························8
7. Conidiophores < 120 μm long··········································································H. vesicarium
7. Conidiophores > 120 μm long··········································································H. hainanense
8. Conidia are acropleurogenous····················································9
8. Conidia are pleurogenous and apical sterile in conidiophores··························10
9. Conidiophores 450–550 µm long··························H. thailandense
9. Conidiophores 184–257 µm long····················································H. flavidum
10. Conidiophores < 180 μm long····················································11
10. Conidiophores > 180 μm long····················································13
11. Conidiophores unbranched, conidia 10–14 µm diam., 70–90 µm long····················································H. aquaticum
11. Conidiophores rarely branched····················································12
12. Conidiophores 125–320 µm long, conidia 13–21 µm diam., 100–130 µm long··························H. setiferum
12. Conidiophores 60–129 µm long, conidia 11–20 µm diam., 68–91 µm long····················································H. sexuale
13. Conidiophores 30–360 µm long, conidia 10–15 μm diam.··························H. vegetum
13. Conidiophores 250–425 µm long, conidia 20–23 μm diam.··························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··························17
15. Conidia are acropleurogenous, conidiophores 36–48 µm long··························H. flavum
15. Conidia are pleurogenous and apical sterile in conidiophores··························16
16. Conidiophores with bladder-like protrusions conidiogenous cells··························H. flavisporum
16. Conidiophores with tiny tooth-like protrusions conidiogenous cells··························H. luteosporum
17. Fresh colonies on decaying woody substrate are hyaline, branched conidiophores 200–300 µm long·································H. albidum
17. Fresh colonies on decaying woody substrate are gray, or dark brown··························18
18. Fresh colonies on decaying woody substrate are gray, conidiophores 350–400 µm long, with bladder-like protrusions conidiogenous cells··················H. neesii
18. Fresh colonies on decaying woody substrate are dark brown····················································19
19. Conidiophores are unbranched with tooth-like protrusions conidiogenous cells····················································H. murinum
19. Conidiophores are rarely branched··············································································20
20. Tooth- or bladder-like protrusions conidiogenous cells, conidia 6–9 µm diam.····················································H. decumbens
20. 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., J.M. and X.-G.T. Writing—original was completed by X.-J.X. and J.M. Review and editing were done by X.-J.X., J.M., L.-J.Z., N.-G.L., Y.-P.X., X.-G.T., Z.-L.L. and Y.-Z.L. All authors have read and agreed to the published version of the manuscript.

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 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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Jof 09 00775 g001 550
Figure 1. The measurement method for helicoid conidia.
Figure 1. The measurement method for helicoid conidia.
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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.
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.
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Jof 09 00775 g003 550
Figure 3. 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. New species are indicated in red, and type strains are marked with “T”.
Figure 3. 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. New species are indicated in red, and type strains are marked with “T”.
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Figure 4. 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) Anverse and reverse colonies on PDA. Scale bars: (c–f) 50 μm, (j) 20 μm, and (g–i,k–p) 10 μm.
Figure 4. 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) Anverse and reverse colonies on PDA. Scale bars: (c–f) 50 μm, (j) 20 μm, and (g–i,k–p) 10 μm.
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Figure 5. 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) Anverse and reverse colonies on PDA. Scale bars: (c–f) 50 μm, (j–k,n–p) 20 μm, and (g–i,l–m) 10 μm.
Figure 5. 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) Anverse and reverse colonies on PDA. Scale bars: (c–f) 50 μm, (j–k,n–p) 20 μm, and (g–i,l–m) 10 μm.
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Figure 6. Helicosporium nanningense (HKAS 128858, holotype). (a,b) Colonies on natural substrate. (c–g) Conidiophores. (h–j) Conidiogenous cells. (k–q) Conidium. (r,s) Anverse and reverse colonies on PDA. Scale bars: (c–g) 20 μm and (h–q) 10 μm.
Figure 6. Helicosporium nanningense (HKAS 128858, holotype). (a,b) Colonies on natural substrate. (c–g) Conidiophores. (h–j) Conidiogenous cells. (k–q) Conidium. (r,s) Anverse and reverse colonies on PDA. Scale bars: (c–g) 20 μm and (h–q) 10 μm.
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Table
Table 1. List of taxa used in this study along with their corresponding GenBank accession for DNA sequences.
Table 1. List of taxa used in this study along with their corresponding GenBank accession for DNA sequences.
TaxonStrainGenBank Accessions
ITSLSUtef1αrpb2
Acanthostigma chiangmaienseMFLUCC 10-0125TJN865209JN865197KF301560-
Acanthostigma patagonicumBBB MVB 573JN127358JN127359--
Acanthostigma perpusillumUAMH 7237TAY916492AY856892--
Helicoma vacciniiCBS 216.90AY916486AY856879--
Helicosporium aquaticumMFLUCC 17-2008TMH558733MH558859MH550924MH551049
Helicosporium flavisporumMFLUCC 17-2020TMH558734MH558860MH550925MH551050
Helicosporium flavumMFLUCC 16-1230TKY873626KY873621KY873285-
Helicosporium hainanenseGZCC 22-2006TOP508730OP508770OP698081OP698070
Helicosporium liuzhouenseGZCC 22-2014TOQ981394OQ981402OQ980476OQ980474
Helicosporium liuzhouenseGZCC 23-0586OR066416OR066423OR058862OR058855
Helicosporium luteosporumMFLUCC 16-0226TKY321324KY321327KY792601-
Helicosporium luteosporumMFLUCC 16-1233-KY873624--
Helicosporium multidentatumGZCC 22-2013TOQ981395OQ981403OQ980477OQ980475
Helicosporium multidentatumGZCC 23-0585OR066417OR066424OR058863OR058856
Helicosporium nanningenseGZCC 22-2175TOR066418OR066425OR058864OR058857
Helicosporium nanningenseGZCC 23-0588OR066419OR066426OR058865OR058858
Helicosporium setiferumBCC 3332AY916490AY856907--
Helicosporium setiferumBCC 8125AY916491---
Helicosporium setiferumMFLUCC 17-1994TMH558735MH558861MH550926MH551051
Helicosporium setiferumMFLUCC 17-2006MH558736MH558862MH550927MH551052
Helicosporium setiferumMFLUCC 17-2007MH558737MH558863MH550928MH551053
Helicosporium sexualeGZCC 22-2007OP508731OP508771OP698082 OP698071
Helicosporium sexualeMFLUCC 16-1244TMZ538503MZ538537MZ567082MZ567111
Helicosporium sp.NBRC 9014AY916489AY856903--
Helicosporium thailandenseMFLUCC 18-1407TMT627698MN913718MT954371-
Helicosporium vegetumCBS 254.75-DQ470982DQ471105-
Helicosporium vegetumCBS 269.52AY916487AY856893--
Helicosporium vegetumCBS 941.72TAY916488AY856883--
Helicosporium vegetumNBRC 30345-AY856896--
Helicosporium vesicariumMFLUCC 17-1795TMH558739MH558864MH550930MH551055
Helicosporium viridiflavumMFLUCC 17-2336TMH558738-MH550929MH551054
Helicosporium viridisporumGZCC 22-2008TOP508736OP508776OP698087 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
Table 2. Checklist of accepted Helicosporium species.
Table 2. Checklist of accepted Helicosporium species.
SpeciesHabitatsDistributionMolecular DataReferences
H. albidumTerrestrialBelgium, Britain (Birminghan)NoGrove 1886 [43]
H. aquaticumFreshwaterThailandYesLu et al. 2018 [10]
H. decumbensTerrestrialAustria, BrazilNoLinder 1929 [2]
H. flavidumFreshwaterChinaNoHsieh 2021 [44]
H. flavisporumFreshwaterThailandYesLu et al. 2018 [10]
H. flavumFreshwaterThailandYesBrahmanage et al. 2017 [14]
H. hainanenseTerrestrialChinaYesLu et al. 2022 [11]
H. liuzhouenseFreshwaterChinaYesThis study
H. luteosporumTerrestrialThailandYesLu et al. 2017 [9]
H. melghatianumTerrestrialIndiaNoDharkar et al. 2010 [42]
H. murinumTerrestrialArgentina, Austria, Brazil, Canada, China, Cuba, Malaysia, USANoLinder 1929 [2]; Goos 1989 [3]; Zhao et al. 2007 [6]
H. multidentatumTerrestrialChinaYesThis study
H. nanningenseTerrestrialChinaYesThis study
H. neesiiTerrestrialUSANoMoore 1957 [39]
H. setiferumFreshwaterThailandYesLu et al. 2018 [10]
H. sexualeFreshwater, TerrestrialChina, ThailandYesBoonmee et al. 2021 [8]; Lu et al. 2022 [11]
H. thailandenseFreshwaterThailandYesDong et al. 2020 [12]
H. vegetumTerrestrialWorldwideYesBoonmee et al. 2014 [7]
H. vesicariumFreshwaterThailandYesLu et al. 2018 [10]
H. viridiflavumTerrestrialThailandYesLu et al. 2018 [10]
H. viridisporumFreshwaterChinaYesLu et al. 2022 [11]
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Xiao, X.-J.; Ma, J.; Zhang, L.-J.; Liu, N.-G.; Xiao, Y.-P.; Tian, X.-G.; Luo, Z.-L.; Lu, Y.-Z. Additions to the Genus Helicosporium (Tubeufiaceae, Tubeufiales) from China with an Identification Key for Helicosporium Taxa. J. Fungi 2023, 9, 775. https://doi.org/10.3390/jof9070775

AMA Style

Xiao X-J, Ma J, Zhang L-J, Liu N-G, Xiao Y-P, Tian X-G, Luo Z-L, Lu Y-Z. Additions to the Genus Helicosporium (Tubeufiaceae, Tubeufiales) from China with an Identification Key for Helicosporium Taxa. Journal of Fungi. 2023; 9(7):775. https://doi.org/10.3390/jof9070775

Chicago/Turabian Style

Xiao, Xing-Juan, Jian Ma, Li-Juan Zhang, Ning-Guo Liu, Yuan-Pin Xiao, Xing-Guo Tian, Zong-Long Luo, and Yong-Zhong Lu. 2023. "Additions to the Genus Helicosporium (Tubeufiaceae, Tubeufiales) from China with an Identification Key for Helicosporium Taxa" Journal of Fungi 9, no. 7: 775. https://doi.org/10.3390/jof9070775

APA Style

Xiao, X. -J., Ma, J., Zhang, L. -J., Liu, N. -G., Xiao, Y. -P., Tian, X. -G., Luo, Z. -L., & Lu, Y. -Z. (2023). Additions to the Genus Helicosporium (Tubeufiaceae, Tubeufiales) from China with an Identification Key for Helicosporium Taxa. Journal of Fungi, 9(7), 775. https://doi.org/10.3390/jof9070775

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