Elbamycellarosea gen. et sp. nov. (Juncigenaceae, Torpedosporales) isolated from the Mediterranean Sea

Abstract Elbamycellaroseasp. nov., introduced in the new genus Elbamycella, was collected in the Mediterranean Sea in association with the seagrass Posidoniaoceanica and with the brown alga Padinapavonica. The affiliation of the new taxon to the family Juncigenaceae is supported by both morphology and phylogenetic inference based on a combined nrSSU and nrLSU sequence dataset. Maximum-likelihood and Bayesian phylogeny proved Elbamycellagen. nov. as a distinct genus within Juncigenaceae. The new genus has been compared with closely related genera and is characterised by a unique suite of characters, such as ascospores with polar appendages and peculiar shape and dimension of ascomata and asci.


Introduction
Marine fungi are a considerable part of the huge diversity of microorganisms that inhabit the Oceans (Richards et al. 2012). These organisms, which are distributed worldwide, live on a broad range of biotic and abiotic substrates (e.g. algae, sponges, corals, sediments) (Jones and Pang 2012) and are divided in two major ecological categories, namely obligate and facultative marine fungi. The former grow and reproduce exclusively in the sea, the latter are terrestrial species that can actively grow and reproduce in marine environments. Those fungi whose obligate or facultative marine nature is undefined are called marine-derived (Raghukumar 2017).
The number of marine fungi has been estimated to exceed 10,000 taxa, but the most recent update in marine mycology listed only 1,206 species belonging to Ascomycota, Basidiomycota, Chytridiomycota, and Mucoromycota. Thus fungal diversity is largely undescribed (Pang and Jones 2017).
In an attempt to clarify the phylogeny of the genera Swampomyces Kohlm. & Volkm.-Kohlm. and Torpedospora Meyers, Sakayaroj et al. (2005) recognised a distinct lineage of marine Ascomycota within the class Sordariomycetes that was then named TBM (Torpedospora/Bertia/Melanospora) clade (Schoch et al. 2007). Following a reevaluation of the marine fungi affiliated to the TBM clade, together with the terrestrial genus Falcocladium, new families were introduced to accommodate its four subclades: Juncigenaceae, Etheirophoraceae, Falcocladiaceae, and Torpedosporaceae, all belonging to the order Torpedosporales (Jones et al. 2014;Abdel-Wahab et al. 2018). Based on phylogeny and morphological data, Maharachchikumbura et al. (2015) introduced the order Falcocladiales (Falcocladiaceae) under the class Sordariomycetes.
Recently, during a survey focused on the fungal diversity in the Mediterranean Sea, two unidentified Sordariomycetes were isolated from the seagrass Posidonia oceanica (L.) Delile (Panno et al. 2013) and from the brown alga Padina pavonica (L.) Thivy ). The present paper provides a phylogenetic and morphological study of the two strains that turn out to represent a new genus within the family Juncigenaceae.

Fungal isolates
The fungal isolates investigated in this paper were previously retrieved from P. oceanica (MUT 4937 = CBS 130520) and P. pavonica (MUT 5443) from the coastal waters of Elba island, in the Mediterranean Sea (Panno et al. 2013;Garzoli et al. 2018) (Table 1). The two strains were originally isolated on corn meal agar medium supplemented with sea salts (CMASS; 3.4% w/v sea salt mix, Sigma-Aldrich, Saint Louis, USA, in ddH 2 O) and are preserved at the Mycotheca Universitatis Taurinensis (MUT), Italy, and CBS Collection of the Westerdijk Fungal Biodiversity Institute, the Netherlands.

Morphological analysis
MUT 4937 and MUT 5443 were pre-grown on CMA-sea water (CMASW; 17 g corn meal agar in 1 L of sea water) for one month at 21 °C prior to inoculation in triplicate onto Petri dishes (9 cm Ø) containing CMASS, CMASW, Potato Dextrose Agar (PDA) SS or PDASW. Petri dishes were incubated at 10 °C and 21 °C. The colony growth, together with macroscopic and microscopic traits, were monitored for 28 days.
Reproductive structures were observed and captured using an optical microscope (Leica DM4500B, Leica microsystems GmbH, Germany) equipped with a camera (Leica DFC320, Leica microsystems GmbH, Germany). Macro-and microscopic features were compared with the available description of Juncigenaceae (Kohlmeyer et al. 1997;Abdel-Wahab et al. 2001;Jones et al. 2014;Abdel-Wahab et al. 2018).

DNA extraction, PCR amplification, and data assembling
Genomic DNA was extracted from about 100 mg of mycelium carefully scraped from CMASS plates. Mycelium was transferred to a 2 mL Eppendorf tubes and disrupted in a MM400 tissue lyzer (Retsch GmbH, Haan, Germany). Extraction was accomplished using a NucleoSpin kit (Macherey Nagel GmbH, Duren, DE, USA) following the manufacturer's instructions. The quality and quantity of DNA samples were measured spectrophotometrically with Infinite 200 PRO NanoQuant (TECAN, Switzerland) and stored at −20 °C.
The primer pairs ITS1/ITS4 (White et al. 1990), LROR/LR7 (Vilgalys and Hester 1990), and NS1/NS4 (White et al. 1990) were used to amplify the partial sequences of the internal transcribed spacers including the 5.8S rDNA gene (ITS), partial large ribosomal subunit (nrLSU), and partial small ribosomal subunit (nrSSU), respectively. Ribosomal genes were amplified in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA), as previously described (Bovio et al. 2018). Reaction mixtures consisted of 60-80 ng DNA template, 10× PCR Buffer (15 mM MgCl 2 ,500 mM KCl, 100 mM Tris-HCl, pH 8.3), 200 µM each dNTP, 1 µM each primer, 2.5 U Taq DNA Polymerase (Qiagen, Chatsworth, CA, USA), in 50 µL final volume. Following visualization of the amplicons on a 1.5% agarose gel stained with 5 mL 100 mL −1 ethidium bromide, PCR products were purified and sequenced at Macrogen Europe Laboratory (Madrid, Spain). The resulting ABI chromatograms were processed and assembled to obtain consensus sequences using Sequencer v. 5.0 (GeneCodes Corporation, Ann Arbor, Michigan, USA http://www. genecodes.com). Newly generated sequences were deposited in GenBank (Table 1). Phylogenetic inference was estimated using both Maximum Likehood (ML) and Bayesian Inference (BI). The ML analysis was performed using RAxML v. 8.1.2 (Stamatakis 2014) under GTR + I + G evolutionary model (best model) and 1,000 bootstrap replicates. Support values from bootstrapping runs (MLB) were mapped on the globally best tree using the "-f a" option of RAxML and "-x 12345" as a random seed to invoke the novel rapid bootstrapping algorithm. BI was performed with MrBayes 3.2.2 (Ronquist et al. 2012) with the same substitution model (GTR + I + G). The alignment was run for 10 million generations with two independent runs each containing four Markov Chains Monte Carlo (MCMC) and sampling every 100 iterations. The first quarter of the trees were discarded as "burn-in". A consensus tree was generated using the "sumt" function of MrBayes and Bayesian posterior probabilities (BPP) were calculated. Consensus trees were visualized in FigTree v. 1.4.2 (http:// tree.bio.ed.ac.uk/software/figtree). Members of Xylariales (i.e. Xylaria hypoxylon, Hypoxylon fragiforme, and Daldinia concentrica) were used as outgroup taxa. Due to topological similarity of the two resulting trees, only ML analysis with MLB and BPP values is reported (Fig. 1).

Phylogenetic inference
Preliminary analyses were carried out individually with nrSSU and nrLSU. The topology of the single-locus trees was very similar and the ILD test confirmed the congruence between them (p = 0.001). The combined dataset consisted of an equal number of nrSSU and nrLSU sequences relative to 39 taxa (including MUT 4937 and MUT 5443) that represented 23 genera and 33 species (Table 1). Nine sequences (3 nrSSU, 3 nrLSU, and 3 nrITS) were newly generated while 72 were retrieved from GeneBank. SSU and LSU sequences relative to MUT 4937 and MUT 5443 displayed 100% and 99% similarity ( Etymology. In reference to the geographic isolation site, Elba Island, Tuscany (Italy) Phylogenetic placement. Juncigenaceae, Sordariomycetes, Ascomycota. The genus Elbamycella gen. nov. clusters together with genera Marinokulati, Khaleijomyces, Juncigena, and Fulvocentrum (Fig. 1).
Asexual morph not observed. Colony description. Colonies reaching 21-23 mm diameter on CMASW and 19-29 mm diameter on CMASS in 28 days at 21 °C, plane, thin, mycelium mainly submerged. Colonies pale pink in the centre becoming brown with age, colourless at the margins. Black spots due to ascomata groups in fruiting colonies. Reverse of the same colour of the surface (Fig. 3A, B).
Colonies on PDASW and PDASS reaching 10-14 mm diameter in 28 days at 21 °C, convolute, developing in height with irregular margins, salmon. Reverse of the same colour of the surface (Fig. 3C, D).
At 10 °C colony growth on all media very poor, attaining 5-8 mm diameter in 28 days. Colonies plane to slightly convolute with regular margins, pale pink to cyclamen. Reverse of the same colour of the surface (Fig. 3E-H).

Discussion
The novel genus Elbamycella is introduced in this study and has been compared to the closest genera. Herein, the two strains MUT 4937 and MUT 5443 represented a new species that formed a well-supported cluster phylogenetically distant from the related genera of Juncigenaceae.
From a morphological point of view, the relatedness with the other species belonging to Juncigenaceae is confirmed by i) 3-septate spores (1-4 only in K. marinus), ii) 8-spored asci, and iii) ascomata with an elongated neck (Kohlmeyer et al. 1997;Abdel-Wahab et al. 2001;Abdel-Wahab et al. 2010;Jones et al. 2014;Abdel-Wahab et al. 2018). Elbamycella rosea sp. nov. is furthermore characterised by the presence of polar appendages on the ascospores. Marinokulati chaetosa displays this feature too, although it can be distinguished from E. rosea sp. nov. by additional, equatorially placed appendages. Additonally, in the new species, spores are cylindrical, not fusiform-ellipsoidal as in M. chaetosa (Jones et al. 2014). Khaleijomyces marinus, Juncigena adarca, Fulvocentrum aegyptiacum, F. clavatisporum, and the recently described F. rubrum differ in the shape and dimensions of the ascospores (Jones et al. 2014;Abdel-Wahab et al. 2018;Abdel-Wahab et al. 2019); generally asci and ascomata are larger than those observed in E. rosea sp. nov.
As no sexual form is known for J. fruticosae, the comparison with E. rosea sp. nov. is not possible. However, the similarity or identity to this species is excluded by the phylogenetical distance.
Ecologically, the described Juncigenaceae are species having a marine origin. So far, they have all been retrieved from driftwood in the intertidal of salt marshes (Kohlmeyer et al. 1997;Jones et al. 2014). The new species was found for the first time underwater, in association with the seagrass P. oceanica and the brown alga P. pavonica, two different organisms that were sampled in close proximity. This could be related to a successful spore dispersal; indeed polar appendages are known to facilitate floatation and attachment (Overy et al. 2019).