Corollospora mediterranea: A Novel Species Complex in the Mediterranean Sea

The genus Corollospora, typified by the arenicolous fungus Corollospora maritima, consists of twenty-five cosmopolitan species that live and reproduce exclusively in marine environments. Species of this genus are known to produce bioactive compounds and can be potentially exploited as bioremediators of oil spill contaminated beaches; hence their biotechnological importance. In this paper, nine fungal strains isolated in the Mediterranean Sea, from the seagrass Posidonia oceanica (L.) Delile, from driftwood and seawater contaminated by an oil spill, were investigated. The strains, previously identified as Corollospora sp., were examined by deep multi-loci phylogenetic and morphological analyses. Maximum-likelihood and Bayesian phylogeny based on seven genetic markers led to the introduction of a new species complex within the genus Corollospora: Corollospora mediterranea species complex (CMSC). The Mediterranean Sea, once again, proves an extraordinary reservoir of novel fungal species with a still undiscovered biotechnological potential.


Introduction
The last decades have seen an increasing interest in marine fungi due to the need to broaden our knowledge on aquatic biodiversity and to exploit these organisms as a source of novel bioactive molecules. Although more than 1800 species inhabiting the oceans have been described so far, most of the fungal diversity, estimated to exceed 10,000 taxa [1], is yet to be unveiled. Several marine habitats and substrates, both biotic and abiotic, are still being explored worldwide, leading to the discovery of new marine fungal lineages. Among Sordariomycetes, one of the ascomycetous classes mostly detected in the sea, the family Halosphaeriaceae (order Microascales) usually dominates this habitat, with 65 genera and 166 species occurring on driftwood, algae, and seagrasses worldwide [2]. While most of the genera of this family are represented by one or two species, the genus Corollospora, typified by Corollospora maritima Werderm, includes 25 arenicolous species typically found in beach sand, sea-foam, shell fragments, and algal thalli [3][4][5][6]. Besides their ability to rapidly degrade cellulose [7], species affiliated with this genus are known to produce bioactive metabolites [8,9] and can be potentially exploited as bioremediators of oil spill contaminated beaches [10]. For instance, the phthalide derivative corollosporine isolated from C. maritima demonstrated a strong antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli [11], whereas pulchellalactam produced by C. pulchella, along with the antimicrobial dioxopiperazines melinacidins II-IV and gancidin W [12], showed inhibitory activity against the tyrosine phosphatase CD45 in lymphocytes [13]. Weak antibacterial activity was also observed in fractions of C. lacera mycelial extract [14].
In general, this genus includes morphologically diverse species whose most distinctive features are the ascospore apical primary and equatorial secondary appendages, respectively formed from the epispore and by the fragmentation of the exospore layer [15][16][17][18]. Indeed, in reference to the ascospore appendage ontogeny, a revision of the genus was done by scanning and transmission electron microscope investigations [16]. In almost twenty years, other species of Corollospora were morphologically described [3,[19][20][21][22][23][24]. With the upcoming molecular techniques, Campbell and collaborators [15] performed phylogenetic analysis of Corollospora spp. (and related taxa) based on 28S rDNA sequences and confirmed the monophyletic nature of the genus. However, the authors concluded that more genetic markers, including protein-coding genes, were necessary to resolve relationships among the species of Corollospora [15]. New sequence data, including 18S rDNA, ITS, and RPB1, relative to Corollospora spp., were then generated in the framework of the Fungal Barcoding Consortium and AFTOL project (Assembly Fungal Tree Of Life) [25,26].
Recently, nine unidentified strains belonging to the genus Corollospora were isolated in surveys aimed to investigate the underwater fungal diversity of the Mediterranean Sea: 4 isolates were retrieved from the seagrass Posidonia oceanica [27], 4 from seawater contaminated by oil spill [28], and 1 from submerged wood [29]. It is not uncommon, as it is in this case, to come across marine fungi that neither sporulate nor develop reproductive structures in axenic culture, leaving traditional morphology-based identification impossible. Consequently, the identification of these sterile mycelia must rely on molecular data [30][31][32][33].
With this study, we wish to provide an accurate phylogenetic placement of the Mediterranean strains by applying a combined multi-locus molecular phylogeny. Besides, the paper gives morphological insights into the strains that turned out to represent new species.

Fungal Isolates
The isolates analyzed in this study were previously retrieved from the Mediterranean Sea. In detail, 4 isolates derived from a site chronically contaminated by an oil spill in Sicily (Gela, Italy) [28] 1 from driftwood sampled in the seawater off Porto Badisco (Apulia, Italy) [29] and 4 from leaves of P. oceanica collected in good health condition in Tuscany (Elba island, Italy) [27] (Table 1). Originally, the strains were isolated on Corn Meal Agar medium supplemented with sea salts (CMASS; 3.5% w/v sea salt mix, Sigma-Aldrich, SL, USA, in ddH 2 O) and are preserved at the Mycotheca Universitatis Taurinensis (MUT), Italy.

Morphological Analysis
All isolates were pre-grown on Malt Extract Agar-sea water (MEASW; 20 g malt extract, 20 g glucose, 2 g peptone, 20 g agar in 1 L of seawater) for one month at 21 • C prior to inoculation in triplicate onto new MEASW Petri dishes (9 cm Ø). Petri dishes were incubated at 15 and/or 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.
In an attempt to induce sporulation, sterile pieces of Quercus ruber cork and Pinus pinaster wood (species autochthonous to the Mediterranean area) were placed on 3-week old fungal colonies [34]. Petri dishes were further incubated for 4 weeks at 21 • C. Following, cork and wood specimens were transferred to 50 mL tubes containing 20 mL of sterile seawater. Samples were incubated at 21 • C for at least three months.
Morphological structures were observed, and images captured using an optical microscope (Leica DM4500B, Leica microsystems GmbH, Germany) equipped with a camera (Leica DFC320, Leica microsystems GmbH, Germany).

DNA Extraction, PCR Amplification, and Data Assembling
Approximately 100 mg of fresh mycelium were carefully scraped from MEASW plates, transferred to a 2 mL Eppendorf tube, and disrupted by the mean of an 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, Switzerland); DNA samples were stored at −20 • C.

Sequence Alignment and Phylogenetic Analysis
A dataset consisting of nrSSU, nrITS, nrLSU, and RPB1 was assembled based on BLASTn results and of the most recent phylogenetic studies focused on Halosphaeriaceae and Corollospora [30,41]. Reference sequences were retrieved 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. Since no incongruence was observed among single-loci phylogenetic trees, alignments were concatenated into a single data matrix with SequenceMatrix [42]. The best evolutionary model under the Akaike Information Criterion (AIC) was determined with jModelTest 2 [43]. Phylogenetic inference was estimated using Maximum Likelihood (ML) and Bayesian Inference (BI) criteria. The ML analysis was generated using RAxML v. 8.1.2 [44] under GTR + I + G evolutionary model and 1000 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 [45] 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 (BPP) were calculated. Consensus trees were visualized in FigTree v.  Following, a new phylogenetic analysis was conducted only on the strains investigated, whose relationship was unclear. To this aim, TEF-1α, β-TUB, and RPB2 sequences were added to the restricted dataset. Alignments and multi-loci phylogeny were conducted as described above. Sequence alignments and phylogenetic trees were deposited in TreeBASE (http://www.treebase.org, submission number S27921 and S27923, acessed on 12 May 2021).
The dataset combining nrSSU, nrITS, nrLSU and RPB1 had an aligned length of 2431 characters, of which 1530 were conserved, 263 were parsimony-uninformative and 638 par-    (Figure 4), mycelium white, grey, grey-green, sometimes with pinkish shades; dense and feltrose, occasionally umbonate in the middle, with radial grooves; reverse from light brown to dark green, occasionally with concentric rings, lighter to the edges. Neither soluble pigments nor exudates were observed (Figure 3).

Discussion
The description of the strains investigated in this study was particularly tricky and complicated since neither asexual nor sexual reproductive structures developed in axenic conditions. As a consequence, it was not possible to describe the range of diagnostic traits amongst these newly identified lineages.
To better characterize these fungi, we tried to mimic the saline environment by using a culture medium supplemented with seawater, since it is well known that only this method supports a measurable growth of vegetative mycelium [33]. The family Halospaheriaceae, to whom the genus Corollospora belongs, includes the largest number of marine lignicolous species. It was thus realistic to induce sporulation by placing wood and cork specimens on the colony surface prior to transfer them into seawater. Despite wood colonization, sporulation did not occur, whereas chlamydospores were abundantly produced. Likewise, strains of Corollospora sp. isolated from intertidal decayed wood of mangrove trees and driftwood in Saudi Arabia did not develop reproductive structures but only chlamydospores that were much bigger and not comparable to those observed in this study [30]. It must be considered that strictly vegetative growth with no sporulation is a common feature of marine fungi [46][47][48] that are likely to rely on their dispersion to hyphal fragments and/or chlamydospores. Most probably, a necessary requisite for fungi to develop reproductive structures is the occurrence of those environmental conditions these organisms are adapted to (e.g., high salinity, low temperature, high pressure, wet-dry cycles, etc.).
The phylogenetic analysis based on ribosomal genes (nrITS, nrLSU, nrSSU) and RPB1, shows a clear distance between the strains under investigation and the other species of Corollospora, Corollospora maritima being the closest one. This new and strongly supported clade may include one or more novel species. Hypothetically, the tree highlights the presence of three clusters that include:  (Figure 1). Bearing in mind the conclusion drawn by Campbell et al. [15] that indicated the need of sequencing more genetic markers to clarify the relations among the species of the genus Corollospora, a dataset focused on this new group, together with C. maritima, was built with the addition of three more protein-coding genes, namely RPB2, TEF-1α and β-TUB ( Figure 2). From one side, this strongly supported tree points out once more the distance from C. maritima, strengthening the idea that we are dealing with a new species. It is undeniably complicated to define a fungal species, and Corollospora mediterranea was here established following the recommendations outlined by Jeewon and Hyde [49], who, dealing with the issue of the species boundaries and identification of new taxa, pointed out a number of key elements to follow. Notably, all the ITS sequences (including 5.8S) analyzed are longer than the minimum 450 base pairs required and display a percentage of identity with the closest relative C. maritima < 95%. In addition, as recommended, the strongly supported phylogenetic tree includes the minimum number (4)(5) of closely related taxa of the same genus (Figure 1). A thorough inspection of the tree shown in Figure 2  On the other hand, the lack of distinct micromorphological traits (neither sexual nor asexual morph with differentiated conidiogenesis detected) and the huge variability observed among the colonies (in terms of growth rate, texture, surface and reverse color) lead us to introduce the C. mediterranea species complex (CMSC), since we could not discern the species boundaries with certainty ( Figures 5 and 6). Indeed, the term "species complex" comes to help taxonomists when: (i) a group of organisms may represent more than one species; (ii) morphological features are overlapping due to extreme variability; (iii) the species may be somehow related although no certain assumption can be assessed. Furthermore, no clear correlation between colony features, growth rates, source of isolation, and/or phylogenetic position was noticed. In general, researchers introduce a species complex when facing a problematic topic. This is the case for example of Fusarium oxysporum species complex (FOSC) [50,51], Fusarium solani species complex (FSSC) [52], Wallemia sebi species complex (WSSC) [53], Colletotrichum acutatum species complex (CASC) [54] and many others. Most of these cases were resolved with a revision of the species complex, where individual species were identified based on a multiloci phylogeny. However, in our study, this approach did not lead to a sharp resolution of the complex. The genome sequencing of all the strains investigated would be an option to sort out this intriguing issue. This approach would possibly reveal those genetic regions that may allow an easy distinction of the species within the complex [55]. On the other hand, the same goal could be achieved by the analysis of secondary metabolites [53]. An investigation of this sort is important also from another point of view: it is now recognized that marine fungi are a reservoir of novel active metabolites that can be harnessed for pharmaceutical, nutraceutical, cosmetic, and environmental purposes.
Kirk and Gordon [10] demonstrated that a number of strains of C. maritima, C. lacera, and C. intermedia were capable of using hexadecane as the sole carbon source, and assumed that the genus Corollospora could find utility in petroleum degradation. Supporting this idea is the isolation of 4 strains of C. mediterranea (MUT 1938, MUT 1950, MUT 1954, and MUT 1961) from a site chronically contaminated by an oil spill in the Mediterranean Sea [28]. The aforementioned findings indicate the great versatility and adaptability of these fungi to hydrocarbons-contaminated environments, with the consequent possibility of being used as bioremediators. In addition, knowing that the species of Corollospora belong to a family of lignicolous fungi and that are rich in cellulase and lignin-degrading enzymes [7], their retrieval from leaves of the seagrass P. oceanica (MUT 5040, MUT 5048, MUT 5049, and MUT 5082) and from driftwood (MUT 1587) does not come as a surprise. It is, therefore, reasonable to assume a role of C. mediterranea in degrading and recycling organic matter, making them available for other organisms.

Conclusions
In conclusion, the retrieval of C. mediterranea from different substrates, localities and in different sampling campaigns indicates its constant presence in (at least) the Mediterranean Sea and points out how the marine environments is still largely uninvestigated from a mycological point of view. Therefore, it is more and more glaring that the Oceans are a huge reservoir of unidentified microorganisms with a valuable biotechnological potential not yet disclosed. In the next future, we would aim at resolving the species complex, by applying more approaches to induce sexual and/or asexual sporulation. In parallel, all the strains studied in this work, will be investigated for the production of new and powerful bioactive molecules.
Funding: This research was funded by the University of Torino (ex 60%).

Informed Consent Statement: Not applicable.
Data Availability Statement: Sequence data are available at Genbank (NCBI) under the accession numbers reported in the manuscript.