Insights on Lulworthiales Inhabiting the Mediterranean Sea and Description of Three Novel Species of the Genus Paralulworthia

The order Lulworthiales, with its sole family Lulworthiaceae, consists of strictly marine genera found on a wide range of substrates such as seagrasses, seaweeds, and seafoam. Twenty-one unidentified Lulworthiales were isolated in previous surveys aimed at broadening our understanding of the biodiversity hosted in the Mediterranean Sea. Here, these organisms, mostly found in association with Posidonia oceanica and with submerged woods, were examined using thorough multi-locus phylogenetic analyses and morphological observations. Maximum-likelihood and Bayesian phylogeny based on nrITS, nrSSU, nrLSU, and four protein-coding genes led to the introduction of three novel species of the genus Paralulworthia: P. candida, P. elbensis, and P. mediterranea. Once again, the marine environment is a confirmed huge reservoir of novel fungal lineages with an under-investigated biotechnological potential waiting to be explored.


Introduction
The increasing interest in marine fungi continues to widen our knowledge of marine biodiversity. So far, more than 1900 species inhabiting the oceans have been described (www.marinefungi.org); however, most of the fungal diversity, estimated to exceed 10,000 taxa [1], is yet to be uncovered. Marine habitats and substrates, both biotic and abiotic, are continuously being explored worldwide, leading to the discovery of new marine fungal lineages.

Morphological Analysis
The strains were grown on Malt Extract Agar seawater (MEASW; 20 g malt extract, 20 g glucose, 2 g peptone, 20 g agar-Sigma-Aldrich, Saint Louis, MO, USA-in 1 L of seawater) for one month at 21 • C prior to inoculation in triplicate onto new MEASW Petri dishes (9 cm Ø). Plates were incubated at 15 and 21 • C. The colony growth was monitored periodically for 28 days, while macroscopic and microscopic features were assessed at the end of the incubation period.
Efforts to induce sporulation were carried out by applying sterile pieces of Quercus ruber cork and Pinus pinaster wood (species autochthonous to the Mediterranean area) on three-week-old fungal colonies [18]. Plates were further incubated for four weeks at 21 • C. Cork and wood specimens were transferred to 50 mL tubes containing 20 mL of sterile seawater. Samples were incubated at 21 • C for a minimum of three months up to nine months.
Morphological structures were observed, and images captured using an optical microscope (Leica DM4500B, Leica microsystems GmbH, Wetzlar, Germany) equipped with a camera (Leica DFC320, Leica microsystems GmbH, Wetzlar, Germany).

DNA Extraction, PCR Amplification, and Data Assembling
Fresh mycelium carefully scraped from MEASW plates was transferred to a 2 mL Epperndorf tube and disrupted by a MM400 tissue lyzer (Retsch GmbH, Haan, Germany). Genomic DNA was extracted following the manufacturer's instructions of a NucleoSpin kit (Macherey Nagel GmbH, Duren, DE, USA). The quality and quantity of DNA were measured spectrophotometrically (Infinite 200 PRO NanoQuant; Tecan, Männedorf, Switzerland), and DNA samples were stored at −20 • C.

Sequence Alignment and Phylogenetic Analysis
A dataset consisting of nrSSU, nrITS, and nrLSU was assembled on the basis of BLASTn results and of the available phylogenetic studies focused on Lulworthiales, Lulworthiaceae, and Lulworthia [5][6][7]9,11,25,26]. Reference sequences were obtained from GenBank (Table 1). Sequences were aligned using MUSCLE (default conditions for gap openings and gap extension penalties), implemented in MEGA X (Molecular Evolutionary Genetics Analysis), visually inspected, and manually trimmed to delimit and discard ambiguously aligned regions. Alignments were concatenated into a single data matrix with Sequence-Matrix [27] since no incongruence was observed among single-loci phylogenetic trees. The best evolutionary model under the Akaike Information Criterion (AIC) was determined with jModelTest 2 [28]. Phylogenetic inference was estimated using Maximum Likehood (ML) and Bayesian Inference (BI) criteria. The ML analysis was generated using RAxML v. 8.1.2 [29] under GTR + I + G evolutionary model and 1000 bootstrap replicates. Support values from bootstrapping runs (BS) were mapped on the global 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 [30] 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 25% of generated trees were discarded as "burn-in". A consensus tree was generated using the "sumt" function of MrBayes and Bayesian posterior probabilities (BYPP) were calculated. Consensus trees were visualized in FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree). Three species of Pleosporales, namely, Bimuria novae-zelandiae, Letendraea helminthicola, and Setosphaeria monoceras, were used as an outgroup, as indicated in previous studies [26]. Due to a topological similarity of the two resulting trees, only Bayesian analysis with BS and BYPP values was reported ( Figure 1).
Following, a new phylogenetic analysis was conducted, focusing only on the under investigation, whose relationships were unclear. To this aim, TEF-1α, β-TUB, RPB1, and RPB2 sequences were added to the restricted dataset (Table S1). Alignments and multi-loci phylogeny were conducted as described above. Lulworthia cf. purpurea, Halazoon melhae, Lulworthia medusa, and Cirrenalia fusca were used as the outgroup.

Discussion
The morphological description of the strains object of this study was complicated by the absence of reproductive structures in pure cultures, thus making the description of diagnostic features amongst the newly recognized lineages impossible. For the same reason, the morphological comparison between the strains in analysis and the recently accepted species was unfeasible.
For a better characterization of these fungi, we used a culture medium supplemented with seawater to mimic their natural environment. Indeed, it is known that only the addition of seawater supports a measurable growth of vegetative mycelium [16,31]. Placing wood and cork specimens on the colonies' surface, followed by their transfer into seawater to induce sporulation, was not successful. In fact, despite wood colonization, only chlamydospores were produced. Strictly vegetative growth is now a recognized

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Sexual morph not observed. Asexual morph with differentiated conidiogenesis not observed.

Discussion
The morphological description of the strains object of this study was complicated by the absence of reproductive structures in pure cultures, thus making the description of diagnostic features amongst the newly recognized lineages impossible. For the same reason, the morphological comparison between the strains in analysis and the recently accepted species was unfeasible.
For a better characterization of these fungi, we used a culture medium supplemented with seawater to mimic their natural environment. Indeed, it is known that only the addition of seawater supports a measurable growth of vegetative mycelium [16,31]. Placing wood and cork specimens on the colonies' surface, followed by their transfer into seawater to induce sporulation, was not successful. In fact, despite wood colonization, only chlamydospores were produced. Strictly vegetative growth is now a recognized feature of a relatively high percentage of marine fungi isolated from different substrates [31][32][33][34][35][36]. This may be due to the lack of appropriate environmental conditions these organisms are adapted to (e.g., high salinity, low temperature, high hydrostatic pressure, etc.) or simply to the fact that the dispersal of sterile marine fungi relies on hyphal fragments and/or resistance structures. In addition, it must be considered that, in fungi, sexual reproduction is controlled by the mating-type (MAT1) locus and that, contrary to homothallic self-fertile filamentous ascomycetes, mating in heterothallic self-sterile species is possible only between strains morphologically indistinguishable with a different idiomorph at the MAT1 locus [37][38][39]. As a consequence of fungal sterility, the identification of the 21 Lulworthiales was achieved with the help of molecular and phylogenetic data. Notwithstanding, the lack of sporulation in the strains identified as P. gigaspora and P. posidoniae puzzled us since sexual structures had previously been observed and described [8]. A possible reason for this behavior may be found in the strain-specific behavior of a homothallic species: homothallism, may, in fact, be a necessary but not sufficient condition for self-fertility to occur. Strains may be more or less sensitive to a range of conditions such as light, temperature, or salinity. Alternatively, an idiomorph may be eliminated via homologous recombination, as demonstrated in Chromocrea spinulosa-which exhibits both homothallic and heterothallic behaviour [37]-or, as seen in some species such as Thielaviopsis cerberus [40], it may display a unidirectional mating-type switching.
The inspection of the phylogenetic tree, based on the three ribosomal genes (nrITS, nrLSU, and nrSSU), highlights the presence of five hypothetical clades ( Figure 1). In detail, three strains grouped with P. posidoniae, three clustered with P. halima, and seven seemed affiliated with P. gigaspora. The remaining formed two well-supported clusters that did not encompass any known fungus, indicating the presence of new lineages. To clarify and be certain of the relations among the species, a new dataset focusing on the five clades and their closest relatives (L. cf. purpurea, H. mehlae, L. medusa, and H. fuscus) was built with the addition of four protein-coding genes, namely, TEF-1α, RPB1, RPB2, and βTUB ( Figure S1). Given the presence of intron regions, which can evolve at a faster rate compared to ribosomal regions, protein-coding genes are more informative and can be employed to improve phylogenetic accuracy, providing a clear species-level identification [23,41]. Indeed, the phylogenetic tree constructed upon seven markers points out the presence of two groups: (a) the cluster that includes the P. gigaspora, P. halima, P. posidoniae, and the newly found P. mediterranea clades; and (b) the cluster that consists of two additional new species, P. candida and P. elbensis. Besides the ultimate scope of our investigation, all the newly generated protein-coding sequences greatly enrich the public databases, thus increasing the availability of molecular data for researchers dealing with this group of fungi.
One could argue and contest the fact the introduction of novel species is based on molecular and phylogenetic data only. However, we followed the key recommendations outlined by Jeewon and Hyde [42]. As indicated by the authors, all the ITS sequences (including 5.8S) analyzed are longer than the minimum requirement of 450 base pairs; the tree is based on genes with strong phylogenetic signals and is statistically supported and includes the minimum number of closely related taxa of the same genus ( Figure 1). Finally, reliable statistical support for each new clade (at least 60% BS or 0.9 BYPP) confirms taxa distinctiveness (Figure 1 and Figure S1).
The order Lulworthiales, with its sole family Lulworthiaceae, was erected to accommodate the genera Lulworthia and Lindra, once considered part of Halosphaeriales (fam. Halosphaeriaceae) [6,7], and was then moved to the new subclass Lulworthiomycetidae [5]. The polyphyletic nature of these two genera initially confused taxonomists, although nowadays, following a number of revisions [7,8,25,26], it is broadly accepted and is once more demonstrated in our investigation (Figure 1).
Lulworthiaceae are found in cold, temperate, and tropical waters in association with woods, seaweeds, seagrasses, and seafoam [4,8,9]. Likewise Goncalves et al. [9], the strains of P. halima, produced only chlamydospores and derived from submerged woods, indicating a preference of this species for such a substrate. Two strains of P. gigaspora were isolated from an oil spill, while the rest were associated with the seagrass P. oceanica. Members of Lulworthiaceae are known saprobes (http://www.funguild.org) and cellulases producers [10]. Considering the substrates of isolation and the production of lignocellulosic enzymes, we can hypothesize a lignicolous nature of the newly identified species. The retrieval of two strains of P. gigaspora from an oil spill reinforces the idea that these organisms can break down complex lignocellulose and recalcitrant compounds, thus contributing to the recycling of nutrients and possibly degrading contaminants such as polycyclic aromatic hydrocarbons (PAH). Interestingly, Paço and collaborators demonstrated the ability of a strain of Zalerion maritimum to degrade polyethylene [43], suggesting a key role of Lulworthiaceae in offering a solution to microplastic pollution. Further experiments will be necessary to assess the full degradative potential of these organisms that could be harnessed for bioremediation purposes.

Conclusions
In conclusion, the retrieval of fungi affiliated with Lulworthiales-together with the introduction of the novel species Paralulworthia candida, Paralulworthia elbensis, and Paralulworthia mediterranea-greatly contributes to improving our knowledge on this strictly marine order and to step-by-step unveiling the fungal communities hosted in the Mediterranean Sea.
Due to the extraordinary biotechnological potential demonstrated by marine fungi, a few strains described in this paper are currently being investigated for the production of novel bioactive molecules. However, we must bear in mind that the applicative value of these organisms depends on their identification at the species level, safe long-term preservation, and on the accessibility guaranteed by the public collections of biological resources.
Author Contributions: Conceptualization, A.P., E.B. and V.P.; methodology, A.P., G.C.V., E.B. and V.P.; software, A.P.; validation, A.P., I.P. and V.P.; formal analysis, A.P., E.B. and V.P.; investigation, A.P., E.B. and V.P.; resources, V.P. and G.C.V.; data curation, A.P., I.P. and V.P.; writing-original draft preparation, A.P. and V.P.; writing-review and editing, A.P., V.P. and G.C.V.; visualization, A.P. and V.P.; supervision, V.P. and G.C.V.; project administration, A.P., V.P. and G.C.V.; funding acquisition, G.C.V. All authors have read and agreed to the published version of the manuscript. lignocellulosic enzymes, we can hypothesize a lignicolous nature of the newly identified species. The retrieval of two strains of P. gigaspora from an oil spill reinforces the idea that these organisms can break down complex lignocellulose and recalcitrant compounds, thus contributing to the recycling of nutrients and possibly degrading contaminants such as polycyclic aromatic hydrocarbons (PAH). Interestingly, Paço and collaborators demonstrated the ability of a strain of Zalerion maritimum to degrade polyethylene [43], suggesting a key role of Lulworthiaceae in offering a solution to microplastic pollution. Further experiments will be necessary to assess the full degradative potential of these organisms that could be harnessed for bioremediation purposes.

Conclusions
In conclusion, the retrieval of fungi affiliated with Lulworthiales-together with the introduction of the novel species Paralulworthia candida, Paralulworthia elbensis, and Paralulworthia mediterranea-greatly contributes to improving our knowledge on this strictly marine order and to step-by-step unveiling the fungal communities hosted in the Mediterranean Sea.
Due to the extraordinary biotechnological potential demonstrated by marine fungi, a few strains described in this paper are currently being investigated for the production of novel bioactive molecules. However, we must bear in mind that the applicative value of these organisms depends on their identification at the species level, safe long-term preservation, and on the accessibility guaranteed by the public collections of biological resources.