Morphological, molecular and 3D synchrotron X-ray tomographic characterizations of Helicascus satunensis sp. nov., a novel mangrove fungus

Sample collection, isolation, morphological examination, and materials availability

Mature dead Nypa palm fruits were collected from mangroves at Mangrove Forest Resource Development Station 36 in Satun Province, southern Thailand (6°54′14.9616″N and 99°41′17.4912″E). Collecting procedure was made as previously described in Preedanon et al. (2017). The collected Nypa palm fruits were placed in a sealed plastic Ziploc bag and brought back to the laboratory for further examination. The specimens were washed with natural seawater in order to remove sediment and other debris then kept in a moist plastic box and incubated at room temperature (approximately 25–28 °C). The samples were examined directly under a stereo-zoom microscope for the presence of H. satunensis sp. nov. Photographic documentation of the sporulating structures was carried out using the Olympus BX51 and Olympus DP21 software (Olympus, Tokyo). The ascomata specimens were fixed with embedding matrix on stage and cutting sections through a cryostat microtome (MEV; SLEE Mainz, Mainz, Germany). The fresh ascomata of H. satunensis sp. nov. were selected for single spore isolation (Vrijmoed, 2000). Spore suspension was diluted and plated on 1.5% seawater corn meal agar (SCMA) medium with the addition of antibiotics (streptomycin sulfate 0.5 g/L, penicillin G 0.5 g/L). The germinated spores were placed onto freshly SCMA medium and incubated at room temperature (approximately 25–28 °C) (Preedanon et al., 2017). Axenic cultures (BCC 83546, BCC 86189, BCC 86190) were then transferred to 1.5% seawater potato dextrose agar (SPDA). Colony characteristics, growth and sporulation were observed and recorded. The type cultures were deposited at the BIOTEC Culture Collection (BCC), Pathum Thani, Thailand. In addition, dried voucher type specimens (BBH 50658, BBH 50659 and BBH 50660) were deposited at BIOTEC Bangkok Herbarium (BBH), Pathum Thani, Thailand.

Three-dimensional synchrotron X-ray tomography

The microstructure of the fungal fruiting bodies and the outer exocarp of Nypa palm fruit was visualized in three dimensions using synchrotron radiation X-ray tomographic microscopy (SRXTM). Prior to the SRXTM experiment, the fresh fungal fruiting bodies samples were fixed with 3% formaldehyde. For tomographic imaging, each sample was placed in a sample holder attached to a brass stub with glue to stabilize the sample during tomography scanning. The SRXTM examination of the samples was carried out at the X-ray tomographic microscopy beamline (BL1.2W: XTM) at the Siam Photon Source Facility, Synchrotron Light Research Institute. The X-ray beam was generated from a 2.2-Tesla multipole wiggler radiation source optimized with a toroidal focusing mirror and filtered with aluminum foils to achieve an average energy of 10 keV. All X-ray projections were acquired with a pixel size of 3.61 µm using an imaging system consisting of a 2X objective lens-coupled microscope (Optique Peter, Lentilly, France), a YAG-Ce scintillator (CRYTUR, Turnov, Czech Republic) and the PCO.edge 5.5 sCMOS camera (Excelitas PCO GmbH, Kelheim, Germany). To enhance fine details of the entire sample, a tomographic volume was reconstructed from enlarged composite projections obtained from multiple scans. Each scan covered 180°with a step of 0.2°, resulting in a dataset. Subsequently, the X-ray projection datasets underwent pre-processing, which included flat-field correction, beam intensity normalization and image stitching. Tomographic reconstruction was performed using Octopus Reconstruction software (TESCAN, Ghent, Belgium). The resulting computed tomographic slices were analyzed using ImageJ (http://rsbweb.nih.gov/ij/) and Fiji (http://fiji.sc/Fiji), and the 3D visualization of the tomographic volume was displayed using Drishti software (Limaye, 2012).

DNA extraction, PCR amplification and DNA sequencing

Genomic DNA from fungal mycelia was extracted according to the methods of O’Donnell et al. (1997) and Sakayaroj, Pang & Jones (2011). Ribosomal DNA genes (ITS, SSU, LSU) and protein-coding gene sequences (TEF-1α, RPB2) were amplified by polymerase chain reaction (PCR). The ITS rDNA region was amplified with the primer pair ITS4/ITS5 (White et al., 1990), the SSU region with NS1/NS4 (White et al., 1990), the LSU region with LROR/LR5 (Vilgalys & Hester, 1990), the TEF1-α region with EF1-983F/EF1-2218R (Rehner & Buckley, 2005), the RPB2 region with fRPB2-5F/fRPB2-7cR (Liu, Whelen & Hall, 1999) (Table 1). The component of PCR reaction was performed in a total volume of 50 µL, containing 1 µL DNA template (30–50 ng/ µL), 1 µL of each forward and reverse primers (10 µM), 10 µL master mix of Taq DNA polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 37 µL of double-distilled water. The PCR conditions for all the genes used were set up using the T100TM thermal cycler (BIO-RAD Laboratories, Inc., California) (Table 1). The PCR products were subsequently purified and sequenced by Macrogen (Seoul, South Korea).

Phylogenetic analyses

Multiple sequence alignments were analyzed with the closely matched sequences obtained from GenBank (Table 2) according to Jones et al. (2015), Maharachchikumbura et al. (2016) and Hongsanan et al. (2017), Dong et al. (2020), Yang et al. (2023a) and Yang et al. (2023b). The newly generated sequences from this study are listed in Table 2. The nucleotide sequences were assembled and aligned using BioEdit 7.2.5 (Hall, 1999) and Muscle 3.8.31 (Edgar, 2004). Specifically, NCBI blast searches were used to determine sequence similarity to sequences published in the GenBank database. Phylogenetic analyses of the combined SSU, LSU, ITS rDNA, TEF-1α and RPB2 sequences were performed using maximum likelihood (ML) and Bayesian algorithms. Maximum likelihood (ML) analysis was evaluated in RAxMLHPC2 on XSEDE (Stamatakis, 2014) via the CIPRES Science Gateway platform (Miller, Pfeiffer & Schwartz, 2010) under the GTR + GAMMA model with BFGS method to optimize the GTR rate parameters. Finally, Bayesian posterior probabilities of branches were performed using MrBayes 3.2.6 (Ronquist et al., 2012), with the best-fitting model (GTR+I+G) selected by AIC in MrModeltest 2.2 (Nylander, 2004), which was tested with hierarchical likelihood ratios (hLRTs). Three million generations were run in four Markov chains and a sample was drawn every 100 generations with a burn-in value of 3,000 sampled trees. Finally, the consensus tree was displayed using the interactive Tree Of Life (iTOL) (Letunic & Bork, 2021) and adjusted in Adobe Photoshop 2020. All sequences obtained in this study were submitted to GenBank, and the typification were published in the MycoBank database (Crous et al., 2004). The resulting alignments were submitted to TreeBASE (submission numbers: 31389).

Nomenclature

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication (MB 854336) to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE, and CLOCKSS.

Taxonomy

Type: THAILAND, Satun Province, mangrove forests, on a piece of dead palm (Nypa fruticans) fruit, 22 December 2016, S. Preedanon, A. Klaysuban, O. Pracharoen & J. Sakayaroj, BBH 50658, holotypus, cultura dessicata, (holotype designated here) (BIOTEC Bangkok Herbarium, Pathum Thani, Thailand).

Ex-type culture: MCR 00699 (BCC 83546) (BIOTEC Culture Collection, Pathum Thani, Thailand).

Etymology: ‘satunensis’ referring to the collecting location, Satun Province, southern Thailand, where the fungus was collected.

Sexual morph: Ascomata 1,000–2, 400 × 160–280 µm, semi-immersed, lenticular ascomata, 3–4 locules, dark brown to black, carbonaceous, solitary (Fig. 1).

Three-dimensional synchrotron X-ray tomographic analysis reveals that the fungal tissues growing in the outer exocarp of Nypa palm fruits, enclosing 3–4 locules with flattened base, horizontally arranged under the pseudostroma. Cellular, numerous, persistent, hyaline pseudoparaphyses.

Asci 475–642. 5 × 62.5–80 µm, 8-spored, bitunicate asci, cylindrical, thick-walled, short hook pedunculate, with an ocular chamber. Ascospores 22.5–25 × 5–8.75 µm, unequally two-celled, smooth, dark-brown, and slightly constricted at the septum, thick-walled (Fig. 1).

Habitat and distribution: mangrove forests, Satun Province, southern Thailand.

Asexual morph: Undetermined

Culture characteristics: Ascospores germinated on SCMA after 1–2 days, colonial grown on SPDA attaining 2–3 cm in diameter after 60 days incubation at room temperature (approximately 25–28 °C), dense, circular, irregular and grey (7D1) with orange patches (7C5) in the center, white (7A1) at the edge; dark brown at reverse side. Colour codes in the fungal description follow “Methuen Handbook of Colour” (Kornerup & Wanscher, 1978).

Phylogenetic analyses

The phylogenetic relationships of the Pleosporales, Dothideomycetes were reconstructed using the combined five-gene dataset (SSU, LSU, ITS rDNA, TEF-1α, RPB2), with Lophiostoma macrostomum JCM 13544 and Sigarispora arundinis JCM 13550 as the outgroups (Table 2). The alignment of 74 taxa comprised 5,844 base pairs (1,342 for SSU, 1,358 for LSU, 1,192 for ITS, 968 for TEF-1α and 984 for RPB2). Total 3,825 characters were constant; 1,570 characters were parsimony-informative and 449 variable characters were parsimony-uninformative. Phylogenetic analyses showed that our novel species (in bold) belongs to the Morosphaeriaceae. Although the topology of the BI tree and the exhibited ML tree are comparable, the BI tree is not shown. The phylogenetic trees representing the unique position of other species in the marine habitat were deposited in MycoBank. Significant ML bootstrap values (≥50%) and Bayesian posterior probabilities (≥0.95) are indicated in the phylogenetic tree (Fig. 2).

In the multigene phylogenetic analysis, the Morosphaeriaceae are divided into subclades representing the seven accepted genera including Neohelicascus, Aquihelicascus, Helicascus, Morosphaeria, Clypeoloculus, Microvesuvius, and Aquilomyces. Our fungal strains (BCC 83546, BCC 86189 and BCC 86190) are monophyletic and well placed in the Morosphaeriaceae with robust bootstrap and Bayesian supports. They are phylogenetically distinct from the type species H. kanaloanus and form sister subclades with H. nypae and H. mangrovei (Fig. 2). Within the Helicascus subclade, we compared the base substitutions of our new fungus with the type species H. kanaloanus. The result shows the base substitutions at several sites of SSU (960/969 = 99.0% similarity), LSU (803/853 = 94.1% similarity), ITS rDNA (448/714 = 62.7% similarity), TEF-1 α (872/933 = 93.4% similarity) and RPB2 (793/899 = 88.2% similarity) (Table 3).

Taxonomy

Jones et al. (2022) reported 1,900 marine fungi in 769 genera that have evolved for marine life as saprobes, parasites and endophytes. Devadatha et al. (2021) and Zhang et al. (2024) reported that many new taxa have been described from mangrove trees and salt marsh plants. Among these host plants, palms found in mangroves and estuaries, such as Phoenix paludosa, Oncosperma tigillarium, and N. fruticans, harbour a great diversity of fungi (Zhang et al., 2024).

Some of the fungi are found as saprobes on the petioles of N. fruticans: Bacusphaeria nypae, Manglicola guatemaelensis, and Tirisporella baccariana (Jones et al., 1996; Suetrong et al., 2009; Abdel-Wahab et al., 2017). Acuminatispora palmarum, Fasciatispora nypae, Helicascus nypae, Neomorosphaeria mangrovei, Pleurophomopsis nypae, Striatiguttula nypae, and S . phoenicis grow on submerged rachis and petioles of N. fruticans and Ph. paludosa (Hyde et al., 1999; Hyde & Alias, 2000; Loilong et al., 2012; Zhang et al., 2018; Zhang et al., 2019; Zhang et al., 2024). A few fungi, however, were discovered on Nypa fruits: Anthostomella nypae., Fasciatispora spp., and Vaginatispora nypae (Jayasiri et al., 2019; Zhang et al., 2024).

The genus Helicascus is a distinct marine ascomycete characterized by having a pseudostroma composed of host cells enclosed in fungal hyphae, subcylindrical asci, uniseriate, obovoid, dark brown color at maturity, and asymmetrical ascospores (Kohlmeyer, 1969). Recently, three species have been identified in the genus, namely, H. kanaloanus (type species), H. nypae, and H. mangrovei (Kohlmeyer, 1969; Hyde, 1991; Preedanon et al., 2017). We found a new fungus, H. satunensis, that inhabits the brackish waters of Nypa palm fruit in Satun Province, southern Thailand. Helicascus satunensis shares similar ascostromata with H. kanaloanus and H. nypae in having semi-immersed or immersed, carbonaceous, multilocules in the ascostromata arranged under a black pseudoclypeus, while H. mangrovei does not have separate locules in the ascomata (Table 4).

Pseudostroma is a unique taxonomic characteristic of the Morosphaeriaceae at the genus level. Zhang et al. (2015) reported that multilocular pseudostroma are important in delineating species of Helicascus-like species. Members of the Morosphaeriaceae develop somatic hyphae into ascostroma, which subsequently form locules that include the genera Helicascus, Neohelicascus, Aquihelicascus, Morosphaeria, Clypeoloculus, and Aquilomyces (Dong et al., 2020). The coiling and stretching mechanism of the basal endoascus with an ocular chamber are regarded as unique types of asci in the genus Helicascus. Our new fungus H. satunensis shares this type of asci with three other species in the genus (Zhang et al., 2015). All species share the same arrangement of cellular pseudoparaphyses. The ascospores of H. satunensis can be distinguished from those of other species because they are smaller (22.5–25 × 5–8.75 µm) than those of H. kanaloanus (30–55 × 17–25 µm), H. nypae (25–35 × 12–15 µm), and H. mangrovei (40–45 × 18.5–20 µm). Germ pores were not observed in H. satunensis, while they appeared at only one end in H. mangrovei (Preedanon et al., 2017). Moreover, the unequal number of H. satunensis 2-cell cones with constriction of ascospores could be a defined taxonomic marker at the species level in the genus Helicascus.

Molecular phylogeny

Phylogenetic analyses of multigene sequences revealed that Helicascus satunensis forms a well-supported clade within the Morosphaeriaceae, Pleosporales, Dothideomycetes. The Morosphaeriaceae family was established by Suetrong et al. (2009) based on morphological features and strong phylogenetic support. Currently, the family comprises eight genera: Aquihelicascus (Dong et al., 2020), Aquilomyces (Knapp et al., 2015), Clypeoloculus (Tanaka et al., 2015), Helicascus (Kohlmeyer, 1969; Dong et al., 2020), Microvesuvius (Fryar, Réblová & Catcheside, 2023), Morosphaeria (Suetrong et al., 2009), Neohelicascus (Dong et al., 2020), and Neomorosphaeria (Zhang et al., 2024).

Members in the Morosphaeriaceae family are found on submerged dead twigs in freshwater and marine environments. The multigene phylogeny comprising freshwater taxa formed sister clades to the marine fungal lineages. Two new freshwater genera, Aquihelicascus and Neohelicascus, were excluded from the genus Helicascus due to morphological and molecular evidence (Dong et al., 2020). Aquihelicascus was established to accommodate one new combination (A. thalassioideus) and two new species (A. songkhlaensis and A. yunnanensis). Neohelicascus was introduced to accommodate one new species (N. submersus) and seven new combinations (N. elaterascus, N. chiangraiensis, N. unilocularis, N. uniseptatus, N. aegyptiacus, N. gallicus, and N. aquaticus) (Dong et al., 2020). The genera Morosphaeria (M. ramunculicola, M. muthupetensis, M. velatispora) (Suetrong et al., 2009; Devadatha et al., 2018), Neomorosphaeria (Zhang et al., 2024), and Helicascus (H. kanaloanus, H. nypae, H. mangrovei) are predominant saprobic on decaying mangroves and marine substrata, while only Aquilomyces patris is a root endophyte of white poplar (Fryar, Réblová & Catcheside, 2023).

The multigene phylogeny in the present study showed that our new fungus H. satunensis forms a distinct lineage within the genus Helicascus with robust statistical support (100% ML bootstrap and 1.00 Bayesian posterior probability). The DNA sequences of H. satunensis differ from those of H. kanaloanus and other species in terms of the number of nucleotide base substitutions in all the DNA regions, which indicates that these species are different. In conclusion, with its unique morphological and multigene phylogeny, we introduce H. satunensis as a novel mangrove fungus.