Rambellisea gigliensis and Rambellisea halocynthiae, gen. et spp. nov. (Lulworthiaceae) from the Marine Tunicate Halocynthia papillosa

In this study, 15 Lulworthiales strains isolated from the marine tunicate Halocynthia papillosa collected in the central Tyrrhenian Sea were characterized using a polyphasic approach (morpho-physiological, molecular, and phylogenetic analyses). Based on multi-locus phylogenetic inference and morphological characters, a new genus, Rambellisea, and two new species, R. halocynthiae and R. gigliensis (Lulworthiales), were proposed. Multi-locus phylogenetic analyses using the nuclear ribosomal regions of DNA (nrITS1-nr5.8S-nrITS2, nrLSU, and nrSSU) sequence data strongly supported the new taxa. Phylogenetic inference, estimated using Maximum Likelihood and Bayesian Inference, clearly indicates that Rambellisea gen. nov. forms a distinct clade within the order Lulworthiales. Moreover, the two new species were separated into distinct subclades, solidly supported by the analyses. This is the first report of Lulworthiales species isolated from animals.

The total number of fungi from marine environments, reported on the "Marine Fungi" specialized website [15], accounts for 1947 species (30 October 2023).However, it has been estimated that this is only a small fraction (<0.2%) of the total marine fungal diversity [11].To date, up to 90% of marine described species belong to the phyla Ascomycota and Basidiomycota [15].With more than 940 species and 385 genera, Ascomycota is the most common taxon, and the greatest number of species are found in the classes Dothideomycetes, Eurotiomycetes, and Sordariomycetes [16].Some orders of Sordariomycetes are exclusively or preferentially marine: Koralionastetales and Lulworthiales host only marine species [17], whereas the Halosphaeriaceae family includes both freshwater and marine species, even though the marine ones are the most numerous [18,19].
The order Lulworthiales, with the single family Lulworthiaceae, was established by Kohlmeyer et al. [20] based on phylogenetic analyses and morphological characters to ac-commodate the genera Lulworthia and Lindra, previously included in the order Halosphaeriales.Over the past few years, several new marine fungi have been described in the family (Lulworthia atlantica, and L. fundyensis), including the recently established genus Paralulworthia, in 2020.This genus was established to accommodate five new species that were discovered in Posidonia oceanica (P.gigaspora, P. posidoniae, P. candida, P. elbensis, and P. mediterranea) [21][22][23][24][25].The species P. candida, P. elbensis, and P. mediterranea, which do not produce reproductive structures, were established solely based on phylogenetic analyses [23].It should be noted that several marine fungal strains exhibit only mycelia sterilia, and they could be identified exclusively by molecular approach [5,26,27].This approach, although significantly different from the traditional taxonomy based on reproductive characterization, is now widely accepted by the scientific community [28].In the last few years, several new species and genera were established in the absence of sexual or asexual structures [29][30][31][32].
During a survey carried out in the central Tyrrhenian Sea to study epizoic fungi, some new strains belonging to Lulworthiales were isolated from the tunicate Halocynthia papillosa.Halocynthia papillosa is a common ascidian species inhabiting the Mediterranean Sea [47,48], and it presents a tunic composed of cellulose, acid mucopolysaccharides, proteins, and sulfated glycans [49].Some of these compounds, such as cellulose, are extremely rare biomolecules in animals [50].
In this study, 15 Lulworthiales strains isolated from H. papillosa were characterized using a polyphasic approach (morpho-physiological, molecular, and phylogenetic analyses).Based on multi-locus phylogenetic inference and morphological characters, a new genus, Rambellisea, and two new species, Rambellisea halocynthiae and Rambellisea gigliensis, are here proposed.

Fungal Isolation
Five specimens of H. papillosa were collected near the "Punta Gabbianara" cape (42 • 21 ′ 50 ′′ N-10 • 55 ′ 24 ′′ E), Giglio Island (Tuscan Archipelago, North Tyrrhenian Sea) at 23-28 m depth in March 2022.The samples were placed in sterile containers and maintained at 4 • C. Isolations were carried out within 24 h as follows: Samples were washed in sterilized artificial seawater (SW; Sea Salts, 35 g dissolved in 1 L, Sigma-Aldrich, St. Louis, MO, USA) to eliminate debris and any potential transient propagules.For each animal, the tunic (T) was separated from the inner tissues (I) to evaluate mycobiota differences related to animal districts.For fungal isolation, the following two different techniques were used: (i) Direct plating: tunic was cut into pieces of about 1 cm 3 and directly plated (5 pieces for each plate) onto Petri dishes (90 mm) containing Malt Extract Agar seawater (MEAsw; 50 g MEA-Sigma-Aldrich dissolved in 1 L of seawater) and Corn Meal Agar seawater (CMAsw; 17 g CMA-Fluka analytical, Buchs, Switzerland, dissolved in 1 L of seawater).(ii) Homogenization: 5 g of each district (T, I) was homogenized in 10 mL of sterile seawater using a sterile device (ULTRA-TURRAX, IKA, Staufen, Germany).A total of 500 µL of each suspension was plated onto Petri dishes (90 mm) containing MEAsw and CMAsw.
To avoid bacterial growth, all media were supplemented with antibiotics (Streptomycin Sulfate, 0.2 g/L; Penicillin G 0.07 g/L; Chloramphenicol, 0.05 g/L).All plates were incubated at 25 • C in the dark and checked daily for four weeks.Strains were isolated in axenic culture on CMAsw and cryogenically maintained at −40 • C in the culture collection of microorganisms of the "Laboratory of Ecology of Marine Fungi" (DEB, University of Tuscia, Viterbo, Italy).Samples of each species were also preserved at the Mycotheca Universitatis Taurinensis (MUT) culture collection.

Morphology and Growth Studies on Different Media
Morphological analyses were carried out on plates utilizing different cultural media: Potato Dextrose Agar seawater (PDAsw; 39 g PDA-Sigma-Aldrich dissolved in 1 L of filtered seawater), Malt Extract Agar seawater (MEAsw), Corn Meal Agar seawater (CMAsw), and Oatmeal Agar seawater (OAsw; 30 g oatmeal powder, 20 g agar dissolved in 1 L of seawater).
The plates (5 cm or 9 cm Ø) were inoculated with a single agar disc (2 mm 2 ) cut from the actively growing margin of 14 d strain cultures on PDAsw and incubated at 25 • C in sealed plastic boxes.These were humidified by a small beaker of distilled water to prevent evaporation and salt precipitation.Growth was monitored for 28 days, and the macroscopic and microscopic features were annotated.
To promote reproduction, fungal strains were inoculated on different natural substrata, such as bark (Quercus cerris), wood (Pinus pinaster), and tunic of H. papillosa (substrate of isolation).All substrata were sterilized, cut into small pieces (3 × 1 cm), and transferred to the surface of PDAsw well-developed colonies (21 days old).The plates were incubated for 4 weeks at 25 • C to allow natural substrata colonization.Following that, some of the inoculated fragments were transferred into tubes containing 20 mL of sterile seawater to simulate natural conditions, while others were transferred to moist chambers and further incubated for 4 months.All inoculated fragments were checked regularly.
The strains' growth preference in relation to salinity was also investigated: each strain was inoculated, as mentioned above, on PDA plates (5 cm Ø) supplemented with different amounts of NaCl (0, 30, 50, 70, 80, and 100‰).The growth diameter was measured daily for 21 days.All experiments were carried out in triplicate.

DNA Extraction, PCR Amplification, and Data Assembling
Genomic DNA was extracted from fresh mycelium (about 100 mg) using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's directions.The extracted DNA was spectrophotometrically quantified (Qubit, Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 • C.

Sequence Alignment and Phylogenetic Analyses
For the phylogenetic analyses, a concatenated dataset of nrSSU, nrITS, and nrLSU sequences (Table 2) based on BLASTn results including the most representative species of the Lulworthiales genera according to the literature was used [21][22][23][24][25].The single gene sequence datasets were aligned with the Clustal X 2.1 software [53] using the default parameters for gap opening and gap extension.Alignments were checked and edited using BioEdit Alignment Editor 7.2.5 [54] and manually adjusted in MEGA 10.2.6 when necessary.Positions where one or more species had a long mutation, as well as ambiguously aligned regions, were excluded from the subsequent phylogenetic analyses.The datasets were concatenated with MEGA X. Phylogenetic inference was estimated using Maximum Likelihood (ML) and Bayesian Inference (BI).
Maximum Likelihood analyses including 1000 bootstrap (BS) replicates were run using the IQ-TREE web server under different models for each dataset in the concatenated matrix [55].ModelFinder on the IQ-TREE web server was used to determine the best nucleotide substitution model for each partition.TNe+G4 is the best-fit model for nrLSU, nr5.8S, and nrITS2, TIM2e+G4 for nrITS1, and TN+F+G4 for nrSSU [56].The best scoring tree, with final likelihood values of −19719.747,was visualized using FigTree v.1.4(http://tree.bio.ed.ac.uk/software/figtree/, accessed on 17 September 2023).The Bayesian Inference was performed with Mr Bayes 3.2.7 [57] under different models for each partition of the matrix as evaluated by jModelTest 2 [58] using Bayesian Information Criterion (TPM2+I+G for nrSSU part1 and TrN+G for nrSSUpart2; TIM1ef+G for nrITS1; TrNef+G for nr5.8S;TrN+G for nrITS2 and nrLSU).Substitution rates, gamma distribution shape parameter, and proportion of invariable sites were reported for each partition in Supplementary Materials (Table S1).The alignment was run for 1 million generations in two independent runs, each with four Markov Chains Monte Carlo (MCMC) and sampling every 100 iterations.As a "burn-in" measure, the first 25% of generated trees were discarded.MrBayes' "sumt" function was used to generate a consensus tree, and Bayesian posterior probabilities (BYPP) were calculated.
The phylogenetic analysis appeared to support the conclusion that the fifteen strains isolated from H. papillosa belong to two novel species within a new genus in the Lulworthiaceae family (Figure 1).
The new genus Rambellisea is herein proposed, with the description of the following two new species: Rambellisea halocynthiae sp.nov.and Rambellisea gigliensis sp.nov.
Nucleotide divergence between the novel species and the closest was annotated for each locus when it occurred and reported as Supplementary Materials (Tables S2-S4).
Diagnosis.Differs from the genus Paralulworthia to which it appears phylogenetically most closely related in the absence of sexual features and conidiogenous structures.
Colony description.Colonies on PDAsw, reaching 13.5 mm in diameter after 28 days at 25 °C, plane centrally umbonate, surface velutinous to feltrose, smoke-grey to pale brown with a light brown marginal area; aerial mycelium sparse, whitish to light brown, mainly in the central area; margins regular, moderately deep, reverse brown.Soluble pigment is yellowish to orange or absent; no exudates were observed (Figure 3).Colonies on MEAsw (Figure S2), reaching 22.5 mm in diameter after 28 days at 25 °C, are morphologically similar to PDAsw.Colonies on CMAsw (Figure S2) reaching 47.3 mm in diameter after 28 days at 25 °C, plane slightly umbonate, surface velutinous, olive-grey to pale brown with a large submerged peripheric area up to 10 mm, aerial mycelium, whitish to light brown, mainly in the central area, reverse brown.Soluble pigment and exudates not produced.S2-S4).
Colony description.Colonies on PDAsw, reaching 13.5 mm in diameter after 28 days at 25 • C, plane centrally umbonate, surface velutinous to feltrose, smoke-grey to pale brown with a light brown marginal area; aerial mycelium sparse, whitish to light brown, mainly in the central area; margins regular, moderately deep, reverse brown.Soluble pigment is yellowish to orange or absent; no exudates were observed (Figure 3).Colonies on MEAsw (Figure S2), reaching 22.5 mm in diameter after 28 days at 25 • C, are morphologically similar to PDAsw.Colonies on CMAsw (Figure S2) reaching 47.3 mm in diameter after 28 days at 25 • C, plane slightly umbonate, surface velutinous, olive-grey to pale brown with a large submerged peripheric area up to 10 mm, aerial mycelium, whitish to light brown, mainly in the central area, reverse brown.Soluble pigment and exudates not produced.
R. halocynthiae isolates can be collected from the tunic and the internal tissues of H. papillosa and can be cultured on media with and without sea salt; the best growth was observed at the sea salinity on Corn Meal Agar (CMAsw).

Discussion
Fungi are key players in terrestrial and marine environments and represent a substantial proportion of the microbial diversity on Earth [15].Even if the role of marine fungi in several basic ecosystem functions, such as their contribution to aquatic carbon pump efficiency or regulation of phytoplankton composition, is largely recognized, the diversity of marine fungi seems to be largely unexplored.It was estimated that up to 90% of marine species have not been described yet [14].Considering this gap, the exploration of habitats and substrates that have never been studied by mycologists appears to be an essential issue to enhance our knowledge of marine fungal biodiversity.Indeed, the new taxa proposed in this study were isolated from H. papillosa, a substratum that has never been previously studied from a mycological point of view.
The fifteen new isolates, obtained from the external tunic and internal tissues of the studied tunicate, developed only sterile mycelia.According to the literature, all strains were cultivated on different substrates, including artificial media (PDAsw, MEAsw, CMAsw, and OAsw), and natural matrices (bark, wood, and tunic of H. papillosa).To promote reproduction and the possible development of reproductive structures, the inoculated matrices were placed in both humid chambers and submerged in seawater during incubation [22,[59][60][61].Fungi development occurred in all studied conditions; nevertheless, sexual reproductive structures or asexual conidia have never been observed.Asexual chlamydospores were observed in 28-day-old cultures of R. gigliensis in all studied conditions, while R. halocynthiae produced vegetative mycelium only.Mycelia sterilia are not unusual among marine fungi [62,63], according to Damare and co-workers [64], it is possible that many marine fungi have evolved hyphal fragmentation as the preferential dispersion system.This would explain the broad presence of the toruloid mycelium observed in R. gigliensis; similar mycelia were reported for other Lulworthiales too [24].
Considering the absence of reproductive structures, except for the mentioned chlamydospores (propagules primarily devoted to perennation, not dissemination) in R. gigliensis, a molecular taxonomical approach was carried out for the taxonomical characterization of the identified strains.A preliminary analysis of the universal barcode for fungi (nrITS region) revealed similarity values inferior to 88% with all sequences deposited in the NCBI nucleotide database.This low identity clearly indicates that these strains were new taxa.Nevertheless, the ITS analyses indicated that all strains belonged to the order Lulwor-thiales, and the multi-locus molecular analyses, based on ribosomal genes (nrLSU, nrITS, and nrSSU), were performed to infer their phylogeny according to recent literature [21][22][23][24][25].The phylogenetic tree clearly showed that our strains formed a well-supported clade that did not encompass any known fungus, indicating the presence of a new lineage inside the family Lulworthiaceae (Figure 1).
The order Lulworthiales includes only strictly marine species [20], commonly found in association with wood, seagrass, and algae.To the best of our knowledge, members of the order Lulworthiales have not been isolated from ascidians yet or from other marine animals [65][66][67][68].The newly studied strains are epizoic, facultative halophytes.They can grow in media devoid of seawater, even if the optimal growth was observed at Mediterranean Sea salinity (38‰).

Conclusions
The present paper provides a morphological and phylogenetic study of fifteen strains obtained from the marine tunicate Halocynthia papillosa collected in the central Tyrrhenian Sea; this tunicate has never been studied for its mycobiota.The strains form a novel lineage within the family Lulworthiaceae.In light of this, the new genus Rambellisea has been established, including the two new species, Rambellisea halocynthiae sp.nov.and Rambellisea gigliensis sp.nov.The identification of fungi belonging to Lulworthiales significantly contributes to the advancement of knowledge about this order of marine species, confirming that the marine ecosystem constitutes an extensive repository of biodiversity, largely unexplored, in particular for its microbial components.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/jof10020127/s1,Table S1: Substitution rates, gamma distribution shape parameter and proportion of invariable sites for each partition, Table S2: The variable sites detected in the nrITS region among Rambellisea halocynthiae, R. gigliensis and its neighbor species belonging to the genera Paralulworthia, Table S3: The variable sites detected in the nrLSU region among Rambellisea halocynthiae, R. gigliensis and its neighbor species belonging to the genera Paralulworthia, Table S4: The variable sites detected in the nrSSU region among Rambellisea halocynthiae, R. gigliensis and its neighbor species belonging to the genera Paralulworthia, Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.All sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/nuccore,accessed on 1 August 2023) and alignments were deposited at TreeBase (https://www.treebase.org/treebase-web/search/studySearch.html,accessed on 8 October 2023).

J 16 Figure 1 .
Figure 1.Phylogenetic inference based on combined nrITS, nrSSU, and nrLSU sequence dataset inferred using the Maximum Likelihood method.The tree is rooted to species of Pleosporales (Bimuria novae-zelandiae, Setosphaeria monoceras, and Letendraea helminthicola).Branch numbers indicate BS and BYPP values.Bar = expected changes per site (0.04).The strains resulting from the current study are in bold and the strains of each new species are distinguished by various colors.

3. 2 Figure 1 .
Figure 1.Phylogenetic inference based on combined nrITS, nrSSU, and nrLSU sequence dataset inferred using the Maximum Likelihood method.The tree is rooted to species of Pleosporales (Bimuria novae-zelandiae, Setosphaeria monoceras, and Letendraea helminthicola).Branch numbers indicate BS and BYPP values.Bar = expected changes per site (0.04).The strains resulting from the current study are in bold and the strains of each new species are distinguished by various colors.

Table 1 .
Details of PCR programs for the different markers used.

Table 2 .
Taxa used for the phylogenetic analyses and GenBank accession number.Newly generated sequences are indicated in bold.