First record of the fungal genus Neodevriesia Quaedvl. & Crous (Ascomycota, Dothideomycetes, Neodevriesiaceae) isolated from scleractinian corals of Perhentian Islands, Malaysia

Abstract Fungal species members of the genus Neodevriesia have been known to occur in marine environments. This report documents the first record of the fungal genus Neodevriesia isolated from scleractinian corals. Three isolated strains were identified from a phylogenetic tree that was constructed, based on the nuclear ribosomal internal transcribed spacer and partial large subunit (ITS + LSU) DNA sequences. Isolates were closely related to both Neodevriesiashakazului (Crous) Crous and Neodevriesiaqueenslandica (Crous, R.G. Shivas & McTaggart) Crous, but formed a distinct clade with strong support that implies a potentially genetic variant of a known species or even a novel species. These findings contribute to the fungal diversity checklist in Malaysia and knowledge about marine fungi associated with scleractinian corals.

More than 20 species from the genus Neodevriesia have been described from a broad range of habitats across wide geographic regions. For example, the extremophilic fungi Neodevriesia bulbillosa Egidi & Zucconi was isolated from limestone in the Malloracan mountain range (Ruibal et al. 2005). The mycoparasitic Neodevriesia coryneliae Crous & A.R. Wood was found growing on the ascomata of Corynelia uberata Fr., which is a common pathogenic fungi of Podocarpus spp., known commercially as podo or East African yellow-wood . Despite being first and primarily described as a terrestrial species, subsequent discoveries have since expanded the distribution of Neodevriesia to include the marine environment. Wang et al. (2018) (Crous et al. 2020). In an effort to examine marine fungi, we investigated fungi associated with scleractinian corals across coral reefs surrounding the Perhentian Islands, Malaysia. Herein, we report the first record of the fungal genus Neodevriesia isolated from several scleractinian corals of Malaysia, based on preliminary identification of their morphology and DNA sequences.

Materials and Methods
Samples of scleractinian coral colonies comprising Acropora sp., Porites sp., Tubastraea sp. were collected on 14 May 2017, from the coral reefs of Perhentian Islands, located at the east coast of Terengganu, Peninsular Malaysia. Sampling sites were Rawa Island (5°57'38.28"N, 102°40'54.59"E), D'Lagoon of Perhentian Besar ( 5°55'56.34"N, 102°43'23.21"E) and Terumbu Tiga of Perhentian Kecil (5°54'2.76"N, 102°46'25.69"E) (Fig.  1). Scleractinian coral samples were collected by SCUBA diving and initially identified underwater using the Coral Finder Guidebook 3.0 (Kelley and Pears 2016). The scleractinian corals were photographed underwater using an Olympus TG-5 digital camera for subsequent identification, based on Coral of the World (Vol. 1-3) (Veron and Stafford-Smith 2000). Scleractinian coral fragments (4 ~ 6 cm) were cut and placed into separately labelled plastic bags with seawater. Once out of the water, coral samples were divided into two sets of labelled 50 ml Falcon tubes; one set containing absolute ethanol for preservation and the other set in natural seawater for isolation of coral-associated fungi. The coral samples were kept cold on ice until transported back to the laboratory and stored in 8 C.
Isolation of coral-associated fungi was conducted following modifed methods of Li et al. (2014). Coral fragments were dipped in 90% ethanol for 3 mins for surface sterilisation and rinsed with sterilised artificial seawater. The coral fragments were ground and mixed with sterilised artificial seawater. The slurry was transferred into a Falcon tube and vortexed for 5 minutes. Ten-fold serial dilutions (n = 3) were made from the slurry. One hundred μl of each dilution was spread on to Petri dishes containing Corn Meal Agar (CMA) prepared with 70% seawater containing Streptomycin (n = 3). The plates were incubated at 26 C for 5 days until the manifestation of mycelium. The fungal strains were then isolated and subcultured on Czapek-Dox Agar (CDA). CDA was used for its sodium nitrate as the sole source of nitrogen that promotes growth. The pure fungal isolates were kept at 8 C for long-term storage.
Pure fungal isolates were identified, based on the morphology of the spores and hyphae. Pure isolates were cultured under continuous normal light on CDA for three weeks at 26°C. Mycelial plugs (5 mm diameter) were cut from colony margins and placed in 9-cm-diameter Petri dishes (n = 3). CDA plugs (1 cm × 1 cm) were placed in clean Petri dishes, each agar First record of the fungal genus Neodevriesia Quaedvl. &amp; Crous (Ascomycota, ... plug was embedded with conidia and a coverslip was placed over each plug according to Riddell (1950). After 7 days, the micrographs were taken under a Nikon Eclipse Light microscope.
DNA sequence reads and chromatograms of the fungal isolates were inspected using Sequence Scanner v.1.0 (Applied Biosystem), edited using BioEdit ver. 7.2 (Hall 1999) and aligned with Clustal-X 2.0 (Thompson et al. 1994, Larkin et al. 2007) to assemble the contiguous sequence for each fungal isolate. BLASTn (Altschul et al. 1990) was used to search for both homologous ITS and LSU sequences in GenBank. Fifty-nine reference sequences  Iterations were performed at 10,000,000 generations per run, sampling frequency of 1,000 and PP was estimated with 25% burn-in and bootstrap support was calculated, based on 1,000 iterations.
The phylogenetic tree analysis of ITS + LSU (1397 bp) confirmed that the strains PERF0613, PERF1511 and PERF1811 belong to the genus Neodevriesia. The strains were closely related to Neodevriesia shakazului and Neodevriesia queenslandica (PP = 1.00, BS = 100) (Fig. 3). Although the marine fungal strains formed a sister group to N. shakazului, they formed a distinct clade (PP = 1.00, BS = 100) with a long branch length (2.83% sequence divergence) (Suppl. material 2) Light micrographs of colonies and fungal morphologies of Neodevriesia strain PERF1811. A Fungal colonies appearance cultured in CDA; B Septate hyphae (arrowed); C Conidiophore (arrowed) with conidia at the tip; D Conidia in a simple chain (arrowed).
First record of the fungal genus Neodevriesia Quaedvl. &amp; Crous (Ascomycota, ... Such phylogenetic affinity of the marine fungal strains were unexpected, since N. shakazului (Crous et al. 2011) and N. queenslandica (Crous et al. 2012) are terrestrial species which was first isolated and described from plants Aloe sp. and Scaevola taccada, respectively. In terms of morphology, they are indistinguishable from the strains in the present study. However, the molecular data seem to suggest they may potentially be a novel species. These Neodevriesia strains are the first to be isolated from the marine environment, particularly from scleractinian corals.
Despite the fungi having been long-deemed as parasitic, studies have shown that fungi are integral members of the microbiome, which allow for hard corals to thrive in oligotrophic water (Amend et al. 2011, Bourne et al. 2016, Wegley et al. 2007). Although information is still scarce and evidence to implicate the functional role of Neodevriesia in coral microbiome remains inconclusive, a more general functional role of nitrogen cycling has been proposed to justify the presence of fungi in hard corals (Rädecker et al. 2015). Fungal functional genes involved in nitrogen cycling were detected in various metagenomic studies on corals (Wegley et al. 2007, Kimes et al. 2010, Zhang et al. 2015, Zhang et al. 2021 Although the three strains are genetically distinct from the other two known species (i.e. Neodevriesia shakazului and Neodevriesia queenslandica), it is still premature to formally describe them as a new species due to the insufficient number of fruiting bodies obtained. Hence, what we are reporting here is preliminary and, thus, requires future work that Consensus tree (BI, ML) based on BI topology of Neodevriesia with closely related genera using concatenated ITS and LSU sequences. Only clades with PP > 0.50 and BS > 50% are indicated at the nodes. 'Triangle' at the node indicated support of 1.00/100. Tree was rooted with Rachlcladosporium spp. from the family Cladosporiaceae. Families are indicated with coloured blocks to the right of the tree. Isolation code numbers are indicated behind the species names. '*' indicated the TYPE and Ex-TYPE specimens. Fungal strains from this study are indicated in orange.
includes some measurements of additional morphological characters for the full description. Additional genetic markers, such as RNA polymerase II gene (RPB2), actin (Act) and calmodulin (Cal), will also be used to further confirm them as a novel species. It is also worthwhile to further investigate and gain insights into the ecological roles of this group of fungi in the marine environment. Nevertheless, there is no doubt that this report documents the first record of genera Neodevriesia found from scleractinian corals. These findings will contribute to the fungal diversity checklist of Malaysia (Lee et al. 2012).

Author contributions
Li Chuen Lee (LCL), Mohammed Rizman-Idid (MRI) and Haifeng Gu (HFG) collected the corals. LCL analysed the data and wrote the manuscript. LCL, MRI and Siti Aisyah Alias (SAS) designed the methodology. LCL isolated the fungi and their DNA. SAS verified the morphology of the fungi. LCL, MRI, SAS, HFG and Kishneth Palaniveloo (KP) conceived and coordinated the study. All authors contributed critically to the drafts and gave final approval for publication.

Conflicts of interest
The authors declare that they have no conflict of interest.