Cryptic marine fungi from the Mediterranean Sea: description of novel taxa.

Marine fungi are a relevant and active component of the microbial communities that inhabit the Ocean. These largely diffused organisms are pivotal to marine food webs due to recycling of recalcitrant substrata. Marine fungi are interesting for both ecological and biotechnological applications. The diversity of marine fungi remains largely undescribed: the number of described marine fungi is lower than the estimated one. This gap depends on the fact that many fungi have been detected by NGS techniques, but they have never been cultivated. Considering the Sea as an extreme environment sensu latu, it is challenging to learn cultivating in vitro these extremophiles organisms, mimicking natural conditions. Furthermore, many marine fungi remain sterile under axenic conditions making their identification particularly difficult. The present work is part of a research program carried out at the Mycotheca Universitatis Taurinensis to study the marine fungal diversity, mainly in the Mediterranean Sea. The main object of the present Thesis was to identify cryptic strains belonging to Lulworthiaceae, Halospheriaceae, Roussoellaceae and Thyridariaceae families and to describe new taxa affiliated to these groups. The 73 fungal isolates investigated in this work were retrieved in the Mediterranean Sea from oil spilled sea water, submerged and driftwood, Posidonia oceanica, Padina pavonica, Flabellia petiolata and Dysidea fragilis. All isolates were inoculated in triplicate onto Petri dishes containing MEA + sea water and incubated at 21 °C. Colony growth, macroscopic and microscopic traits were monitored for 28 days. In attempt to induce sporulation sterile specimens of Quercus ruber cork and Pinus pinaster wood were placed on 3 weeks old fungal colonies and incubated for 4 weeks at 21°C. Following, cork and wood pieces were submerged into tubes containing 20 ml sterile sea water and incubated at 21°C for one month. Morphological features were repeatedly monitored for more than 6 months. Following DNA extraction, the partial sequences of different genetic markers were amplified by PCR: the ITS, the LSU, the SSU, the TEF1-α and the RPB1 and RPB2. Datasets were assembled, multi-locus phylogenetic trees were built and inspected, and isolates that clustered in the same group and deriving from the same substrate underwent mini- and microsatellites analysis to exclude duplicates from further analysis. The strongly supported phylogenetic analyses allowed the description of i) a new putative genus (Parathyridariella gen. nov.) and seven new putative species of Roussoellaceae and Thyridariaceae, namely Parathyridariella dematiacea sp. nov., Parathyridaria thyrrenica sp. nov., P. flabelliae sp. nov., Neoroussoella lignicola sp. nov., Roussoella mediterranea sp. nov., R. margidorensis sp. nov., R. padinae sp. nov.; ii) a new putative genus (Paralulworthia gen. nov.) and two new putative species of Lulworthiaceae, Paralulworthia gigaspora sp. nov. and P. posidoniae sp. nov. As for Halospheriaceae, since neither distinctive morphological characteristics were observed, nor a multi-locus phylogenetic analysis was definite, no conclusion could be drawn, although the strains investigated are clearly representative of one or more new species of Corollospora. In conclusion, this work contributes to fill the void of knowledge on fungal marine biodiversity and once again underlines the importance of applying specific culture methods for extremophile fungi.