Molecular data reveals a new holomorphic marine fungus, Halobyssothecium estuariae, and the asexual morph of Keissleriella phragmiticola

ABSTRACT This study introduces a novel holomorphic marine fungal species, Halobyssothecium estuariae (Lentitheciaceae, Pleosporales), from dead Phragmites communis. The new species has semi-immersed, subglobose or ellipsoidal, papillate, conical ascomata, clavate to subcylindrical, short pedicellate asci and 3-septate, fusoid to ellipsoidal ascospores with rounded ends, pale brown to dark brown central cells and hyaline end cells. The asexual morph has multiseptate, filiform, intercalary, catenate, branched chlamydospores that resemble Xylomyces. The asexual morph of Keissleriella phragmiticola based on combined LSU, SSU, ITS and TEF1 sequence analyses is reported. The role of molecular identification in delineating cryptic species are also discussed.

The genus Phragmites includes predominant perennial grasses found in marine coastal environments worldwide, throughout temperate and tropical regions and Phragmites australis has been widely studied for marine fungi Wong and Hyde 2002;Van Ryckegem and Verbeken 2005). More than 300 fungi have been reported in association with this plant Hyde 2001, 2002;Calado et al. 2015;Goonasekara et al. 2019), of which 109 species were recorded from intertidal marshes in Hong Kong. Poon and Hyde (1998) described three new species Massarina phragmiticola, Phomatospora phragmiticola and Cytoplacosphaeria phragmiticola from this plant and Wong et al. (1998) introduced the novel genus Phragmitensis typified by P. marina. Karunarathna et al. (2017) introduced a new aquatic genus and species Yunnanensis phragmitis collected on Phragmites australis from Dali Lake, Yunnan Province in China, while Wanasinghe et al. (2018) described Keissleriella phragmiticola on Ph. communis collected from Poole, Dorset, U.K.
The ascomycete Halobyssothecium obiones has a chequered history assigned to various genera and families and is reported from a variety of host plants. Assigned initially as two separate species: Pleospora obiones by Crouan and Crouan (1867) and Leptosphaeria discors by Saccardo and Ellis (1882). Subsequently, they have been assigned to various genera: Metasphaeria (Saccardo 1883), Heptameria (Cooke 1889), Passeriniella (Apinis and Chesters 1964;Hyde and Mouzouras 1988;Khashnobish and Shearer 1996a) and more recently, based on a multi locus phylogenetic study to Halobyssothecium (Dayarathne et al. 2018). Didymosphaeria spartinae is also included in synonymy with H. obiones (Grove 1933). As a consequence, it has also been referred to different families in the Dothideomycetes. Studies of Halobyssothecium obiones/Leptosphaeria discors reveal significant differences in the ascospore dimensions: in most collections, they are 24-38 × 8-14 μm (Dayarathne et al. 2018), while others measure 38-56 × 16-22 μm (Jones 1962;Cavaliere 1968;Webber 1970). Kohlmeyer and Kohlmeyer (1979) provided a description of Leptosphaeria discors based on examination of the type material in Herb Crouan at Concarneau, France and the account of spermagonia for this species by Wagner (1965). Kohlmeyer and Kohlmeyer (1979) concluded that because of these differences in ascospore measurements "it appears to be a new species in need of thorough examination". The results of our study and the above observations suggest that Halobyssothecium obiones is a species complex.
The present study aims to examine if Halobyssothecium obiones is a species complex and introduces a new species Halobyssothecium estuariae with both sexual and asexual morphs that are found on dead culms of Ph. communis. The genus Keissleriella, typified by Keissleriella aesculis, was introduced by Höhnel (1919). It is characterised by ascomata with ostiolar necks filled with black setae and one to multi-septate, hyaline ascospores with a pycnidial coelomycetous asexual morph producing 0-3 septate hyaline conidia (Barr 1990;Tanka et al. 2015). In our ongoing studies, we have found a coelomycetous asexual morph inhabiting Ph. communis and identified as Keissleriella phragmiticola ) based on sequence data. The asexual morph is illustrated and supported by molecular evidence.

Sample collection, isolation and morphological studies
Dead and decaying culms of Phragmites communis and Spartina sp. were collected from Slebech Estuary, Pembrokeshire, UK. and the Ketch Nature Reserve, Hayling Island, UK. Specimens were placed in a Ziplock plastic bags and incubated at room temperature in the laboratory. Specimens were examined under a Leica EZ4 stereo zoom microscope. Hand sections of the ascomata were made and the centrum contents were taken out with the aid of a needle and fixed in sterile distilled water. The microscopic characters were photographed using Carl Zeiss Discovery V8 stereo-microscope fitted with Axiocam and Nikon ECLIPSE TiU upright microscope with DIC objectives connected to Nikon DS-Fi2 digital camera. The morphological measurements were taken by means of Tarosoft (R) Image Frame Work program v. 0.9.7. The pictures in the photo plates were arranged by using Adobe Photoshop CS6 Extended v. 13.0 Isolates were obtained by using a single spore isolation method as described in Choi et al. (1999) using sea salt agar media. The germinating ascospores from ascomycetes and conidia from asexual morphs were transferred to sea salt malt extract agar media (SMEA) plates and incubated at 25°C for 10 to 20 days with regular observations. The herbaria and the axenic type cultures were deposited in Mae Fah Luang University herbarium and Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand. Facesoffungi and Index Fungorum numbers were acquired as elucidated in Jayasiri et al. (2015) and Index Fungorum (2019).

DNA extraction, PCR amplification and sequencing
The hyphal mass from freshly grown colonies were scraped by using a sterile lancet and transferred to a 1.5 ml Eppendorf tube (Christian et al. 1990). Total genomic DNA was isolated by following CTAB methods described by Jeewon et al. (2002) and Suwannarach et al. (2010). Four loci were amplified by employing well-known primer pairs: ITS4 and ITS5 to amplify ITS region and nuclear small subunit rDNA region with NS1 and NS4 (White et al. 1990). Nuclear large subunit rDNA (LSU) was amplified using LR0R and LR5 (Vilgalys and Hester 1990). The translation elongation factor 1-alpha gene (TEF-1α) was amplified using primers EF1-983F and EF1-2218R (Rehner and Buckley 2005).
PCR reactions were carried out using volume of 50 µL composed of 5 µL of Ex Taq buffer, 4 µL of dNTP mixture, 1 µL of each primer, 1 µL (50-100 ng) genomic DNA, 0.3 µL of TaKaRa Ex Taq ™ polymerase and the remaining volume with that of double distilled water. PCR amplification conditions were set as follows; an initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, primer annealing at 54°C for SSU; 52°C for ITS, LSU and TEF1α, primer extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. All PCR products were visualised on a 1.2% agarose gel stained with ethidium bromide and purified by Qiagen purification kit (Qiagen, USA) following the manufacturer's procedure. PCR products of different genes were then sequenced with primers stated above by Biomed company, Beijing, China.

Sequence alignment and phylogenetic analyses
Sequences of different gene regions from both forward and reverse primers were assembled to obtain a consensus sequence with BioEdit v.7.0.5.2 (Hall 1999). Based on the mega BLAST searched in NCBI two strains were assigned to Halobyssothecium and one strain to Keissleriella, Lentitheciaceae. The taxa for the phylogenetic analyses were downloaded from GenBank and those identified from a recent study of Halobyssothecium (Dayarathne et al. 2018). Multiple sequence alignments for different gene regions were generated online at MAFFT server (http://mafft.cbrc.jp/ alignment/server/) (Katoh and Standley 2013) and alignments were manually adjusted using BioEdit, wherever essential. The individual sequence datasets (LSU, SSU, ITS, TEF-1α) were concatenated using BioEdit v.7.0.5.2 (Hall 1999).
For Maximum Likelihood and Bayesian analyses, MrModeltest v. 2.3 (Nylander 2004) was used to determine the best-fit model of nucleotide evolution for the dataset. GTR+I + G model was selected.
RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008;Stamatakis 2014) in the CIPRES Science Gateway platform ) was used to build a maximum likelihood (ML) tree using GTR+I + G model of evolution. Maximum Likelihood bootstrap values greater than 70% were given above each node for ( Figure 1). Bayesian analysis was implemented by using MCMC sampling in MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate Bayesian posterior probabilities by Markov Chain Monte Carlo sampling (MCMC) with two runs and four chains (Rannala and Yang 1996;Zhaxybayeva and Gogarten 2002). A total of 10,000,000 MCMC generations and trees were sampled every 1000th generation, resulting in a total of 10,000 trees. First 1000 trees were discarded as burn-in phase and the remaining 9000 trees were used to calculate posterior probabilities in the majority rule consensus tree. BYPP greater than 0.95 are given above each node (Figure 1).

Culture characteristics
Both sexual and asexual morphs have identical morphological characters in culture. Colonies on MEA, slow growing, reaching 10-25 mm diam., after 10 days of incubation at room temperature, colonies velvety, lobate edges, irregular, surface umbonate. Colony form top pale brown to reddish brown, below reddish brown with white margin.
Phylogenetically, H. obiones and H. estuariae differ by 5.1% nucleotide base pair differences in ITS and 2.9% in TEF gene regions and justifies the introduction of a new species

Notes
Halobyssothecium obiones is frequently reported on Spartina stems, which plays a major role in the breakdown of lignocellulosic secondary walls of plant cells and nutrient recycling (Gessner and Goos 1973;Newell et al. 1995;Barata 2002;Calado et al. 2015Calado et al. , 2019. Our collection of Halobyssothecium obiones on Spartina sp. culms shares similar morphological characters and overlapping measurements with the recently epitypified H. obiones (Dayarathne et al. 2018). However, Halobyssothecium obiones observed in this study has shorter ascospore dimensions in contrast to the previous collections (Jones 1962;Cavaliere 1968;Webber 1970), which suggests that Halobyssothecium is a complex with at least three or more taxa. Hence, further collections and molecular studies may reveal the complexity in Halobyssothecium species.

Culture characteristics
Colonies on MEA, fast growing, reaching 20-35 mm diam., after 10 days of incubation at room temperature, colonies dense, cottony, circular with erose margins, surface raised, colony from above hyaline to cream, below pale yellow without pigmentation.

Notes
The genus Keissleriella comprises 36 species epithets in Index Fungorum 2019. Wanasinghe et al. (2018) described Keissleriella phragmiticola on Phragmites communis from Poole, Dorset, UK. The species is characterised by superficial to semi-immersed erumpent, globose ascomata and ascospores that are narrowly fusiform with 2-3 large guttules in each cell and surrounded by a thick mucilaginous sheath ). Our preliminary blast search analyses based on LSU and ITS sequence data of our taxon indicated 99.8% similarity to Keissleriella phragmiticola (sexual morph). Hence, we consider our taxon as an asexual morph to Keissleriella phragmiticola (sexual morph) and an asexual morph connection is established in this study. Keissleriella phragmiticola (asexual morph) resembles Setoseptoria phragmitis in having subcylindrical hyaline straight to slightly curved ascospores. However, Keissleriella phragmiticola (asexual morph) is distinct from Setoseptoria phragmitis in having larger pycnidia and 5-7-transverse septate ascospores. While Setoseptoria phragmitis is characterised by smaller conidiomata and 1-3-transverse septate ascospores that are shorter in contrast to Keissleriella phragmiticola (Quaedvlieg et al. 2013). The asexual morphs in Keissleriella are distinguishable from asexual morph of Keissleriella phragmiticola in having cylindrical to bone-shaped hyaline conidia with 0-3-transversesepta (Tanaka et al. 2015).