﻿Three new species of Neohelicomyces (Tubeufiales, Tubeufiaceae) from freshwater and terrestrial habitats in China

﻿Abstract Neohelicomyces species are a group of helicosporous hyphomycetes with the potential to produce secondary metabolites. During our investigation of helicosporous fungi, six collections were isolated from both terrestrial and freshwater habitats in Guizhou Province, China. Based on multigene phylogenetic analysis (ITS, LSU, tef1α and rpb2), coupled with morphological data, three new Neohelicomyces species, viz. N.guizhouensis, N.helicosporus and N.hydei were established. A list of accepted Neohelicomyces species with molecular data was provided. The strain of Neohelicomycespallidus (UAMH 10535) was synonymised under N.denticulatus based on molecular data.

In this study, six helicosporous taxa were collected from both freshwater and terrestrial habitats in Zunyi City, Qianxinan Buyi and Miao Autonomous Prefecture, Guizhou Province, China.Based on morphological descriptions, illustrations and multi-gene phylogenetic analyses, three novel species are herein introduced, namely Neohelicomyces guizhouensis, N. helicosporus and N. hydei.

Sampling of the collections, macro-and micro-morphological examinations
Specimens were collected from freshwater and terrestrial habitats from August 2021 to March 2022 in Zunyi City and Qianxinan Buyi and Miao Autonomous Prefecture, Guizhou Province, China.Specimens from freshwater habitats were cultured at room temperatures, with moisture maintained for 1-2 weeks.Fungal colonies and micromorphological structures on the surface of the natural substrates were observed using a stereomicroscope (SMZ-168, Nikon, Japan) and photographed using an ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan), equipped with a Canon 90D digital camera.

Isolations and material deposition
Single spore isolations were conducted following the method described by Chomnunti et al. (2014).Subsequently, the germinating spores were aseptically transferred to fresh potato dextrose agar (PDA) plates, following the method outlined in Senanayake et al. (2020).Fungal mycelia were cultured on PDA and incubated at 25 °C for 45 to 50 days.Their colony characteristics, such as shape, colour, size, margin and elevation, were monitored and recorded.
Dried fungal specimens were deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (Herb.HKAS), Kunming, China and the Herbarium of Guizhou Academy of Agriculture Sciences (Herb.GZAAS), Guiyang, China.Cultures were deposited at the Guizhou Culture Collection (GZCC), Guiyang, China.The descriptions of the newly-introduced taxa were uploaded in the Faces of Fungi webpage following the guidelines outlined in Jayasiri et al. (2015).The new species were registered in the MycoBank database (https://www.mycobank.org/).

DNA extraction, PCR amplification and sequencing
Fresh mycelia were scraped with a sterilised toothpick and transferred to a 1.5 ml microcentrifuge tube.Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, China), following the manufacturer's protocol.Primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999) were used to amplify ITS, LSU, tef1α and rpb2 sequence fragments, respectively.The PCR amplification reactions were carried out in a 50 µl reaction volume, including 2 µl DNA, 2 µl of the forward and reverse primer each and 44 µl of 1.1 × T3 Supper PCR Mix (Qingke Biotech, Chongqing, China).The thermal-cycling parameters of the ITS, LSU, tef1α and rpb2 regions were as follows: initial denaturation at 98 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 1 min, elongation at 72 °C for 10 s and final extension at 72 °C for 2 min.The PCR products were detected by 1% agarose gel electrophoresis and the sequencing results were provided by Beijing Qingke Biotechnology Co., Ltd.
Bayesian Inference (BI) was performed using OFPT methods described by Zeng et al. (2023).The aligned Fasta file was converted to a Nexus format file for Bayesian analysis using AliView v. 1.27 (Daniel et al. 2010).The best-fit substitution model of the four gene matrices was selected using MrModelTest 2.3 under the Akaike Information Criterion (AIC) (Nylander et al. 2008).
The multi-gene phylogenetic trees were visualised using FigTree v. 1.4.4 and the final layout of the phylogram was edited using Adobe Illustrator CC 2019v.23.1.0(Adobe Systems, USA).Photo-plates and scale bars were processed using Adobe Photoshop CC 2019 (Adobe Systems, USA) and Tarosoft (R) Image Frame Work programme, respectively.

Phylogenetic results
The phylogenetic positions of our newly-introduced species were determined, based on multi-gene (ITS-LSU-tef1α-rpb2) phylogenetic analysis.The concatenated sequence matrix comprised 3,353 characters (ITS: 1-547, LSU: 548-1,405, tef1α: 1,406-2,308 and rpb2: 2,309-3,353) across 40 ingroup and two outgroup taxa (Helicotubeufia hydei and H. jonesii).Both the ML and BI analyses of the concatenated ITS, LSU, tef1α and rpb2 datasets yielded similar tree topologies.Fig. 1 illustrates the best scoring ML tree, with a final likelihood value of -17,148.363.The decision to introduce new species based on a polyphasic approach follow the guidelines of Chethana et al. (2021).
Culture characteristics.Conidia producing germ tubes on PDA within 9 hours of incubation at 25 °C.Colonies on PDA are circular with flat surface and undulate edge, reaching 40 mm diameter after 45 days of incubation at 25 °C, top view of colony pale pink to brown, reverse brown to dark brown.
Culture characteristics.Conidia producing germ tubes on PDA within 9 hours of incubation at 25 °C.Colonies on PDA are irregular with umbonate surface and filiform edge, reaching 43 mm diameter after 48 days of incubation at 25 °C, top view of colony reddish-brown to black brown, reverse brown to black brown.
Culture characteristics.Conidia producing germ tubes on PDA within 12 hours of incubation at 25 °C.Colonies on PDA are circular with umbonate surface and entire edge, reaching 42 mm in diameter after 50 days of incubation at 25 °C, top view of colony brown to black brown, reverse pale brown to black brown.

Discussion
In this study, six helicosporous taxa were collected for the first time in northern and south-western regions of Guizhou Province, China.Based on multigene (ITS-LSU-tef1α-rpb2) phylogenetic analysis, coupled with morphological descriptions and illustrations, we establish three novel Neohelicomyces species, namely N. guizhouensis, N. helicosporus and N. hydei.
A list of accepted Neohelicomyces species with known sequence data is also provided (Table 2).There are 16 species (including three new species described in the present study) in Neohelicomyces, of which ten were found from freshwater habitats, while the remaining six ones were reported from terrestrial habitats, with 13 species collected from China (Linder 1929;Goos 1989;Tsui et al. 2006;Zhao et al. 2007;Luo et al. 2017;Lu et al. 2018bLu et al. , 2022;;Tibpromma et al. 2018;Crous et al. 2019aCrous et al. , 2019b;;Dong et al. 2020;Hsieh et al. 2021;Yang et al. 2023).Neohelicomyces pallidus is the most widely distributed member of Neohelicomyces genus and has been reported from terrestrial habitats in various regions of the world, including China, Czechia, Italy, Japan, Netherlands and USA (Linder 1929;Goos 1989;Tsui et al. 2006;Zhao et al. 2007;Lu et al. 2018b).Given that most Neohelicomyces species and many helicosporous genera (Berkleasmium, Helicoma, Helicosporium, Helicotubeufia, Neohelicosporium, Parahelicomyces, Pleurohelicosporium, Pseudotubeufia and Tubeufia) in Tubeufiaceae were reported from China, we infer that China is a biodiversity hotspot for helicosporous fungi (Lu et al. 2018b;Hsieh et al. 2021;Ma et al. 2023a).Therefore, we anticipate to discover and classify more helicosporous taxa from different habitats.A plausible explanation for the prevalent number of Neohelicomyces species in China might be attributed to limited sampling in other areas or they probably occur in understudied hosts and substrates.The conidial morphology of most Neohelicomyces species closely resembles those of Helicomyces and the typical helicoid Tubeufia genera (Zhao et al. 2007;Luo et al. 2017;Lu et al. 2018b;Ma et al. 2023b).However, most Neohelicomyces species can easily be distinguished by their longer, hyphae-like and conspicuous conidiophores, when compared to those of Helicomyces and Tubeufia (Morgan 1892;Linder 1929;Rao and Rao 1964;Goos 1985;Zhao et al. 2007;Hyde et al. 2016;Lu et al. 2017bLu et al. , 2018bLu et al. , 2023b;;Kuo and Goh 2018;Tian et al. 2022;Ma et al. 2023b).Only two species, Neohelicomyces longisetosus and N. thailandicus, exhibit morphological variations in conidiophores when compared to other Neohelicomyces species.However, molecular data confirm their taxonomic placement in Neohelicomyces (Dong et al. 2020;Hsieh et al. 2021).For example, Neohelicomyces longisetosus resembles Helicosporium flavum in having shorter, unbranched and less septate conidiophores and terminal, ampulliform conidiogenous cells.Nonetheless, they are delineated, based on their distinct conidial morphology and DNA molecular data (Brahmanage et al. 2017;Hsieh et al. 2021).
Herein, based on multigene phylogenetic analyses, we reclassify Neohelicomyces pallidus (UAMH 10535) under N. denticulatus.Nevertheless, we were unable to compare its morphology as this taxon lacks morphological data (Kodsueb et al. 2006;Tsui and Berbee 2006;Tsui et al. 2006;Lu et al. 2018b).In our phylogenetic analyses, Neohelicomyces pallidus (UAMH 10535) clusters with Neohelicomyces denticulatus (GZCC 19-0444) with 93% ML and 1.00 PP support.In comparison of their sequence data, there were only four nucleotide differences across ITS and one nucleotide difference across LSU (Lu et al. 2018b;Yang et al. 2023).Additionally, our phylogenetic analyses showed that Tubeufia amazonensis (ATCC 42524) shares a sister relationship to Neohelicomyces species, which suggests that this taxon neither belongs to genus Neohelicomyces nor to genus Tubeufia.However, due to the lack of morphological data about Tubeufia amazonensis (ATCC 42524), we were unable to compare its features with other Neohelicomyces and Tubeufia species.Therefore, Tubeufia amazonensis (ATCC 42524) is re-categorised here as a member of Tubeufiaceae (ATCC 42524).Further studies focusing on the re-collections, isolations and morphological examinations of these strains are a prerequisite to having a more stable and resolved taxonomy.

Figure 1 .
Figure1.Phylogenetic tree generated from Maximum Likelihood (ML) analysis, based on the combined ITS, LSU, tef1α and rpb2 sequence data.Bootstrap support values of ML equal to or greater than 75% and Bayesian posterior probabilities (PP) equal to or greater than 0.95 are given near the nodes as ML/PP, respectively.Helicotubeufia hydei(MFLUCC 17-1980)  and H. jonesii (MFLUCC 17-0043) were selected as outgroup taxa.The new species are typed in bold red; " T " denotes ex-type strains.

Table 1 .
Taxa used in this study and their GenBank accession numbers.
Note: " T " indicates ex-type strains.Newly-generated sequences are typed in bold."-" indicates the unavailable data in GenBank.

Table 2 .
Checklist of accepted Neohelicomyces species with molecular data.
Note:The newly-isolated species in this study are typed in bold.