Deciphering changes in the symbiotic microbe community within Corals during bleaching events.

El Niño events have a significant impact on global climate, most notably causing warming events, which affect the stability of marine ecosystems. Widespread bleaching of coral reefs, resulting in high levels of Coral mortality due to heat stress, is now recognized as a global threat to coral. 2015–2017 were the three warmest years in the instrumental record period since 1880 and record high temperatures triggered a pan-tropical Coral bleaching episode. The world’s largest Coral Reef ecosystem, the Great Barrier Reef, experienced the highest temperatures ever recorded and lost nearly 30% of coral cover. Coral is closely associated with a complex group of microorganisms, including Symbiodiniaceae (symbiotic Dinoflagellates), Fungi, Bacteria, Archaea, Endolithic Algae, and Viruses, in a relationship known as Coral  Symbiosis. Coral microbes play an important role in nutrient cycling and antimicrobial protection in Coral Reefs. Therefore, it is important to investigate the effects of bleaching events on the function of Coral microbial communities. Recently, metagenomics has been used to investigate the taxonomic diversity and metabolic capabilities of Coral-associated microbes under thermal stress or bleaching. These studies suggested that microbes can undergo major shifts, from symbionts to opportunistic microbes or potential disease-causing Bacteria, during heat stress or bleaching. In addition to this, the metabolism of the microbial community can shift from autotrophy to heterotrophy, which involves sulphur and nitrogen metabolism, fatty acid and lipid utilisation, and secondary metabolism.

In a paper published in the journal Frontiers in Microbiology on 20 March 2020, Fulin Sun of the State Key Laboratory of Tropical Oceanography and the Daya Bay Marine Biology Research Station of the South China Sea Institute of Oceanology of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory, Hongqiang Yang, also of the State Key Laboratory of Tropical Oceanography and the Key Laboratory of Ocean and Marginal Sea Geology at the South China Sea Institute of Oceanology, and the Nansha Marine Ecological and Environmental Research Station of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory,  and Guan Wang and Qi Shi, again of the Key Laboratory of Ocean and Marginal Sea Geology at the South China Sea Institute of Oceanology of the Chinese Academy of Sciences, and the Southern Marine Science and Engineering Guangdong Laboratory, present the results of a study of the microbiome (micro-organism community) of Corals from Xiane Reef in the Nansha Islands of the South China Sea (part of the disputed Spratly Island group), during the 2016 bleaching event.

Traditionally, research into coral bleaching has mainly focused on studying photosynthetic symbiotic Dinoflagellate Algae known as Symbiodiniaceae. Physiological damage and expulsion of Algal symbionts are thought to be the result of the host immune response triggered by reactive oxygen species produced by Coral hosts, Algal symbionts, or both. Research on Symbiodiniaceae has been focused on changes in diversity and density of Symbiodiniaceae, Photosystem II damage in symbiotic Dinoflagellates, thermal tolerance of Symbiodiniaceae, and functional changes in Symbiodiniaceae. However, few studies have reported the functional response of Symbiodiniaceae to bleaching.

Although previous studies had not focused on Eukaryotes, they have hinted that Eukaryotes are the most abundant component of Coral symbionts. Microeukaryotes have been most widely associated with Coral diseases and mortality. To date, studies on other Microeukaryotes associated with Coral have mainly focused on several key populations, including Fungi, Endolithic Microalgae and Protists. The potential diversity of Coral related Fungi suggests a broader role beyond pathogenicity. Metagenomic analysis has revealed that Endolithic Algae can play a key role in the microbial community by driving important chemical processes. When Zooxanthellae are absent, other Microeukaryotes can provide nutrients that increase Coral survival during periods of acute stress. Despite the fact that other Eukaryotes are ubiquitous in Corals, little is known about their diversity and ecological function during Coral bleaching events.

Until now, fundamental gaps have existed in our understanding of the Coral microbiome and its functional contribution to Coral. In 2016, a bleaching event also affected a large area of Coral in the South China Sea, where mass Coral bleaching had not previously been recorded. In Sun et al.'s study, four different Coral species were studied to provide an overview of the metagenomic (DNA) and metatranscriptome (RNA) response of Coral symbionts (Prokaryotes, Symbiodiniaceae, other Endolithic Eukaryotes and the Coral itself) to bleaching and to highlight differences in their functional performance. Coral species were collected at the same location to eliminate any potential external environmental influences. In order to study the different components of Coral symbionts separately, each symbiotic component was separated based on the National Center for Biotechnology Information non-redundant protein database. Combined with Kyoto Encyclopedia of Genes and Genomes database annotation, the corresponding functions of DNA and RNA in different components were explored.

Coral samples were collected at Xiane reef, Nansha Islands, South China Sea, in June 2016. Four Coral species (with unbleached coral and bleached coral being collected from the same Coral colony), Acropora tenuis, Goniastrea minuta, Pocillopora verrucosa, and Pocillopora meandrina, were sampled. Water temperatures in the sampling area ranged from 30.5 to 31° C. Three replicate samples of unbleached and bleached parts of Coral (including tissue, mucus and skeleton) were collected using a hammer and chisel. Once collected, the Coral samples (1 cm x 1 cm) were washed with sterile seawater three times to remove any surface attachments. Each sample was divided into two parts and placed in sterile centrifuge tubes with Sample Protector (Takara, Japan) for DNA and RNA extraction. DNA and RNA isolation of replicate samples was mixed and conducted using DNeasy and RNeasy plant mini kits (Qiagen, Germany) following the manufacturer’s instructions. Coral tissues were removed with an airbrush for the identification of each species. All Coral samples were identified according to their ecological and morphological characteristics.

Location map of sampling sites in the South China Sea during the 2016 El Niño period. Sun et al. (2020).

At the DNA level, the abundance of unbleached Coral genomes accounted for more than 80% of the total number of sequences, but the DNA content had low abundance in bleached Acropora tenuis (48.55%) and Goniastrea minuta (7.90%) Corals. In comparison with unbleached Corals, Bacterial abundance increased sharply in bleached Acropora tenuis (32.53%) and Goniastrea minuta (78.63%) Corals, as did the abundance of Eukaryotes in bleached Acropora tenuis. At the RNA level, bleached Acropora tenuis Coral had a reduced abundance of Symbiodiniaceae compared with unbleached Acropora tenuis Coral. In contrast, bleached Acropora tenuis Coral had a higher abundance of Prokaryotes and Eukaryotes than unbleached Acropora tenuis. Unlike Acropora tenuis Corals, the abundance of Eukaryotes was higher in bleached Pocillopora verrucosa and Pocillopora meandrina in comparison to unbleached Corals. Symbiodiniaceae abundance was significantly lower in bleached Pocillopora verrucosa and Pocillopora meandrina Corals compared with unbleached bleached Pocillopora verrucosa and Pocillopora meandrina Corals.

The field sampling process of Acropora tenuis. (A) Acropora tenuis is in the process of bleaching during the sampling time, the lower left part of its growth base is bleached Favites sp.; (B) This is the red frame part of the (A), Acropora tenuis in the process of bleaching; (C) This is the yellow frame of the part (B); the upper blue frame of part (C) is the collection part of the bleached Coral sample, and the lower part of the green frame is the collection part of unbleached Coral samples. Sun et al. (2020).

The results showed that there were distinct differences in the composition of symbionts among different Corals at RNA and DNA levels. For Acropora tenuis and Goniastrea minuta Corals, metagenomic analysis showed that the most obvious response to bleaching was the increase in abundance of Bacterial taxa, affiliated with Proteobacteria (Alphaproteobacteria, Deltaproteobacteria,
Epsilonproteobacteria, and Gammaproteobacteria), Bacteroidetes (Cytophagia) and Green Suplhur Bacteria (Chlorobia), in addition to a significant decrease in Dinophyceae (Dinoflagellata) abundance. 

A colony of Goniastrea minuta in the Philippines. Charlie Veron/Corals of the World.

Furthermore, distinct differences were observed at the class level between unbleached and bleached Corals. The dominant class of symbionts in Pocillopora verrucosa and Pocillopora meandrina were Dinophyceae, Eurotiomycetes (Ascomycotae Fungi) and Pucciniomycetes (Basidiomycote Fungi), and Bacilli. In the case of both unbleached and bleached Pocillopora verrucosa and Pocillopora meandrina Corals, there was a very low abundance of Prokaryotes compared with other symbiont components. Apart from the decreased abundance of Dinophyceae, another feature of bleachied Pocillopora verrucosa and Pocillopora meandrina Corals was the higher abundance of Eukaryotes, especially Pucciniomycetes, when compared with unbleached Corals. The major genera identified at the RNA and DNA levels varied with samples. For Acropora tenuis and Goniastrea minuta Corals, the most obvious response to bleaching in Coral symbionts was the shift in abundance of Symbiodiniaceae and Bacterial taxa. Bleached Corals exhibited a high diversity and abundance of Bacterial taxa at the DNA and RNA level. These taxa included Chlorobiales (Green Sulphur Bacteria), Rhodobacterales (Alphaproteobacteria), Alteromonadales (Gammaproteobacteria), Oceanospirillales (Gammaproteobacteria), and Vibrionales (Gammaproteobacteria). More Bacterial orders were recorded from Acropora tenuis and Goniastrea minuta than Pocillopora verrucosa and Pocillopora meandrina at the DNA and RNA levels. A higher amount of Campylobacterales (Epsilonproteobacteria), Alteromonadales and Streptomycetale (Actinobacteria) were revealed at the RNA level, in contrast to the results for Rhodobacterales and Vibrionales, which were highly abundant at both the RNA and DNA levels.

A colony of Pocillopora verrucosa at the Birch Aquarium in San Diego. Wikimedia Commons.

For Pocillopora verrucosa and Pocillopora meandrina Corals, the symbiotic component was mainly composed of Microeukaryotes, and there was a distinct difference in the abundance and composition for unbleached and bleached Corals. Most notably, Dinophyceae-like genera had a significantly lower abundance in bleached Corals, including Symbiodiniaceae, Crypthecodinium, Amphidinium, Karlodinium, Heterocapsa, Pfiesteria, and Prorocentrum. The Puccinia Fungus genus (Basidiomycota) was dominant at the RNA level, and was more abundant in bleached than unbleached corals. In unbleached Corals, Dinoflagellates of the orders Gymnodiniales, Cantharellales, Peridiniales, Prorocentrales and Gonyaulacales, all of which are affiliated with Dinophyceae, were observed to be more abundant at the RNA and DNA levels in comparison with bleached Corals.

A colony of the Coral Pocillopora meandrina in American Samoa. Douglas Fenner/NOAA Fisheries.

For Acropora tenuis and Goniastrea minuta Corals, gene composition and transcriptional abundance increased significantly in bleached Corals in comparison with unbleached Corals, including ribosomal, carbon fixation, cofactor and vitamin biosynthesis, and ATP synthesis. Both the metagenomic and metatranscriptome results of Sun et al.'s study indicated that the main contributing prokaryotes for these functions were Proteobacteria, Bacteroidetes, Chlorobi and Actinobacteria, and the abundance of Bacterial orders was significantly correlated with function, especially at the RNA level. Rhodobacterales, Campylobacterales, Vibrionales and Alteromonadales contributed more to gene function at the RNA level, in contrast to the finding that dominant of Flavobacteriales, Oceanospirillales and Cellvibrionales had a slightly higher contribution to gene function at the DNA level. For Pocillopora verrucosa and Pocillopora meandrina Corals, the Bacterial contribution to gene function was very low in both bleached and unbleached Corals. 

The abundance of Prokaryotes was significantly correlated with functional abundance at the RNA and DNA levels. For Acropora tenuis and Goniastrea minuta Corals, carbon fixation pathways, including the reductive citrate cycle (Arnon-Buchanan cycle), the reductive pentose phosphate cycle and the 3-hydroxypropionate bi-cycle, were contributed by Proteobacteria and Bacteroidetes. However, the gene abundance of carbon fixation was very low in unbleached and bleached Pocillopora verrucosa and Pocillopora meandrina Corals. Dissimilatory nitrate reduction was the main pathway of nitrogen metabolism identified. Proteobacteria, Bacteroidetes, Chlorobi and Tectomicrobia mainly contributed to sulphur metabolism in Acropora tenuis and Goniastrea minuta Corals. Two of three high abundance sulphur metabolism pathways, assimilatory sulphate reduction and dissimilatory sulphate reduction, were detected in Acropora tenuis and Goniastrea minuta Corals only. Dominant bacteria, such as Campylobacterales (Arcobacter and Sulfurimonas) mainly contributed to assimilatory sulphate reduction.

Sulfurimonas Bacteria were found to contribute to assimilatory sulphate reduction in bleached Acropora tenuis and Goniastrea minuta Corals. Sikorski et al. (2010).

The results of metagenomic and metatranscriptomic analysis showed that 24 Symbiodiniaceae species were detectable in the Corals. According to the results, all Symbiodiniaceae species decreased in abundance in bleaching corals in comparison with unbleached corals, especially for dominant species Cladocopium and Symbiodinium. 

Of all Symbiodinium types, Cladocopium and Symbiodinium were the two genera with the highest abundance of gene composition and transcription. Among the functions performed by Symbiodiniaceae, photosynthesis and ATP synthesis were the most important functions. The abundance of Symbiodiniaceae was significantly correlated with functional abundance at the RNA and DNA levels. In comparison with unbleached Corals, Symbiodiniaceae were less abundant in almost all functional genes in bleached Corals. Cladocopium and Symbiodinium were the main contributors to photosynthesis. Cladocopium and Symbiodinium minutum were the main contributors to ATP synthesis. At the RNA level, Cladocopium was the main contributor to ATP synthesis in Acropora tenuis Corals, while Cladocopium and Symbiodinium minutum were the main contributors for Pocillopora verrucosa and Pocillopora meandrina Corals. Cladocopium was the only performer for RNA processing and Spliceosome at the RNA level. Spliceosome genes in unbleached Corals had a higher expression of abundance than bleached Corals, the majority of which belonged to genes involving the heat shock gene (HSPA1_8). There was one carbon fixation pathway (Calvin-Benson cycle) detected in the Symbiodiniaceae. It had a high abundance in all unbleached Corals, and exhibited a reduced gene abundance in bleached Corals.

Symbiodinium minutum Dinoflagellates, major contributers to ATP production in the Corals Pocillopora verrucosa and Pocillopora meandrina. LaJeunesse et al. (2012).

The results indicated that most eukaryotes displayed reduced function in bleached Corals compared with unbleached Corals. This decline in function resulted in decreased proteasome function, carbon fixation, ATP synthesis and central carbohydrate metabolism. However, photosynthetic activity was found to increase in all bleached Corals, in particular Acropora tenuis. The main contributors to function are the dominant Dinophyceae (including Gymnodiniales, Peridiniales, Prorocentrales, and Gonyaulacales) and Eurotiales (Ascomycote Fungi). A higher number of Gymnodiniales, Peridiniales, Prorocentrales and Gonyaulacales contributed to function at the RNA level, in contrast to the DNA level, where Eurotiales mostly contributed to the function. It was unexpectedly found that Dinophyceae were the main contributors to all functions except photosynthesis. Interestingly, there was an obvious increase in the abundance of genes involved in photosynthesis in bleached Coral, mainly attributed to Bacillariophyceae (Diatoms), Florideophyceae (Red Algae), and Trebouxiophyceae (Green Algae). The Calvin cycle is the main pathway for carbon fixation of these Eukaryotes. There was a lower abundance of genes involved in carbon fixation in bleached Corals compared with unbleached Corals. Dinophyceae were the main contributor to Spliceosomes, and the abundance of the heat shock 70 gene was significantly reduced in bleached Corals compared with unbleached Corals.

Almost all of the functions of bleached Acropora tenuis Coral were reduced compared with unbleached Corals, in particular cell signaling, ribosomal activity, spliceosome activity, glycan metabolism, RNA processing and ATP synthesis. In contrast to Acropora tenuis Coral, almost all functions of bleached Pocillopora verrucosa and Pocillopora meandrina were increased in comparison with unbleached Corals.

An obvious response to bleaching displayed by Coral symbionts was the shift in abundance of Bacteria. Bleached Corals exhibited a higher diversity and abundance of Bacterial taxa at the DNA and RNA level than unbleached Corals, indicating that Bacteria were easily affected by Coral bleaching. Many studies have shown that potentially opportunistic microbes can sharply increase in abundance and become dominant in bleached Corals, causing the coral to move toward an unstable state, even inducing Coral disease. Coral bleaching can alter the chemical composition of Coral mucus, increase organic matter and mucus production, which induces a shift in the Coral-associated microbial community, The results of the current study indicated that these opportunistic microbes were highly abundant in bleached Coral, and may have resulted in an elevation of bacterial-organic matter coupling.

Almost all functional genes of Prokaryotes were improved in bleached Acropora tenuis and Goniastrea minuta Corals. Previous studies demonstrated that the metabolism of the microbial community could shift from autotrophy to heterotrophy under stress, resulting in an increase in the abundance of microbial genes involved in sulphur and nitrogen metabolism, and secondary metabolism. As the microbial community shifts from autotrophy to heterotrophy, Bacterial consumption of organic matter becomes greatly enhanced, and the contribution of fixed nitrogen and photosynthesis for nitrogen and carbon budgets became less obvious. High abundance sulphur metabolism pathways (assimilatory sulphate reduction and dissimilatory sulphate reduction) were detected in bleaching bleached Acropora tenuis and Goniastrea minuta Corals, increasing the possibility of producing sulphide. High abundance of these heterotrophic Bacteria could deplete nutrients in Coral, and deteriorate the microenvironment, ultimately making Coral bleaching irreversible.

According to the results of Sun et al.'s study, the abundance of all Symbiodiniaceae obviously decreased in all bleached Corals in comparison with unbleached Corals, indicating that the nutrition supplied to Coral by Symbiodiniaceae decreased. Exocytosis or in situ symbiotic degradation during bleaching seemed to be less invasive and cost effective than host cell degradation. Sun et al.'s tudy identified that the four Coral species could simultaneously host a very high diversity of genotypic Symbiodiniaceae phylotypes, more than described by other studies. Cladocopium is often regarded as a sensitive species to temperature or bleaching, and is dominant in Scleractinian Corals in the South China Sea. Cladocopium were the main contributors to photosynthesis and ATP synthesis in Coral.

This result provided direct evidence that bleaching may have important effects on photosynthesis via the inhibition of the Calvin cycle, limiting carbon fixation in Symbiodiniaceae, as described in previous studies. It had been found that carbon fixation via the Calvin cycle is sensitive to heat stress. Previous studies reported that the maximum quantum yield of photosystem II was significantly lower and highly variable in bleached Corals in comparison with healthy Corals. This occurred with a corresponding loss of electron flow supporting carbon fixation. ATP synthesis was essential for repair in photosystem II, therefore the repair rate that this study suggested was decreased through the inhibition of ATP synthesis in bleached Coral. In addition, a low abundance of the heat shock gene indicated that bleaching inhibited the activity of heat-inducible genes and heat acclimation of Symbiodiniaceae resulting in the reduced ability of Symbiodiniaceae to resist thermal stresses.

Another finding of Sun et al.'s study was that, when Corals are bleaching, they not only expel Symbiodiniaceae, but all of the identified Dinophyceae genera, which indicates that these Algae have the same response mechanism to Coral bleaching. Other Eukaryotic Algae also showed distinct changes in bleached Corals compared to unbleached Corals. It has been reported that when Symbiodiniaceae are absent, these Algae could provide an alternative source of photoassimilates, and provide nutrients that increase Coral survival during stress. Sun et al.'s study also suggested that Fungi displayed high abundance of RNA in bleached Pocillopora verrucosa and Pocillopora meandrina Corals, and bleaching stimulated Fungal growth. Some studies have suggested that Fungi were also thought to be opportunistic pathogens, and their abundance depended on Coral health.

The functional genes of other Eukaryote Algae increased in abundance during bleaching, indicating that the photosystems of other Eukaryotes (such as Bacillariophyta, Chlorophyta) used a different mechanism and had higher levels of thermal tolerance compared to Dinophyceae Algae. It was also thought that other endosymbiotic Algae benefit the host Coral during periods of stress. This may be because during bleaching, the shading effect of Symbiodiniaceae was lost, allowing increased light to penetrate the Coral skeleton, possibly resulting in increased photosynthetic activity of these Algae. Given the ability of Microalgae to adapt rapidly to heat stress, these populations might become important as 'secondary' symbionts, and continue to provide nutrients for coral through photosynthesis.

The results of Sun et al.'s study indicated that the Coral symbionts under investigation were at different stages of bleaching. Almost all of the functions of bleached Acropora tenuis and Goniastrea minuta Corals were reduced compared with unbleached Corals, suggesting that the nucleic acid of bleaching Corals was being degraded and was in an apoptotic state. A reduction in mRNA abundance of cytochrome c and ATP synthase, which were central components of the respiratory electron transport chain inhibited the ability of the host to survive or recover from thermal stress. This suggested that bleached Acropora tenuis and Goniastrea minuta Corals lost most of their physiological metabolic activity and function.

However, almost all functions of bleached Pocillopora verrucosa and Pocillopora meandrina Corals were increased in comparison with unbleached Corals. This indicated that Coral was in a temporary stage of transformation from an unbleached to a seriously bleached state. At this stage, Corals exhibit symptoms of bleaching as they have expelled most symbiotic Algae; however, the symbiotic structure in bleached Corals has certain similarities in composition to unbleached corals. The remaining Symbiodiniaceae and other Algae play an important role in bleached Coral, providing nutrition to the host and maintaining Coral activities under stress.

For both unbleached and bleached Coral, it was found that different Coral species have common symbiotic taxa that perform biological functions in vivo. Overall, different Coral species were found to have common characteristics when bleached: a decreased abundance of Symbiodiniaceae and associated function and the exclusion of Dinophyceae-like Eukaryotes. Furthermore, Sun et al.'s study might reflect the different stages of the Coral bleaching process. In the early stages of Coral bleaching, Algae such as Symbiodiniaceae and other Dinophyceae were expelled from Corals. If the Coral microbiome could maintain a level of stability similar to that of an unbleached Coral, the Coral itself could retain its functional activity. Otherwise, the Coral itself gradually decreased in activity due to the lack of nutrients usually provided by Algae. Opportunistic Bacteria then multiply in large numbers, and result in the deterioration of the Coral microenvironment.

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Seven new species of Marine Fungi from the Mediterranean.

Marine Fungi are an important and active component of the microbial communities that inhabit the oceans. Fungi in the marine environment live as mutualists, parasites, pathogens and saprobes, and are pivotal to marine food webs because of the recycling of tough organic materials that other organisms cannot break down; besides which, these widely dispersed organisms are a source of novel bioactive compounds. Marine Fungi have been recovered worldwide from a broad range of biotic and abiotic substrata, such as driftwood algae, sponges, corals, sediments, etc. A 'Marine Fungus' is defined as any fungus retrieved repeatedly from marine environment and that reproduces in the marine environment. There are currently about 1680 described Marina Fungal species belonging to 693 genera, 223 families, 87 orders, 21 classes and six phyla. However, considering that the total number of Marine Fungi has been estimated to exceed 10 000 taxa, fungal diversity remains largely undescribed. With more than 900 species, the Ascomycota are the dominant Fungal phylum in the sea.

In a paper published in the journal Diversity on 6 April 2020, Anna Poli, Elena Bovio, Lucrezia Ranieri, Giovanna Cristina Varese, and and Valeria Prigione of the Department of Life Sciences and Systems Biology at the University of Torino describe seven ew species of Marine Ascomycote Fungi from the Mediterranean.

The first new species is placed in a new genus, Parathyridariella, which means 'beside Thyridariella', in reference to a previously described species, to which the new genus is closely related, and given the specific name dematiacea, meaning darkly pigmented, in reference to the colour of the colony on culture media  This species was isolated from a Green Seaweed, Flabellia petiolata, found growing at a depth of 14-15 m off the coast of Ghiaie Beach on the island of Elba, and a Seagrass, Posidonia oceanica, growing at a depth of 5-21 m off the coast of Punta Manara in the Province of Genoa, Italy. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 28–34 mm in diameter after 28 days at 24 °C, grown on Oatmeal Agar-sea water reached 40-34 mm in diameter after 28 days at 24°C, and on Potato Dextrose Agar-sea water reached 36-49 mm in diameter after 28 days at 24 °C, and 15.5–22.5 mm in diameter after 28 days at 15°C. The species grew actively on Pine wood and cork. The mycelium varies in colour from dark grey/black to dark green, and is dense with radial grooves and concentric rings, and submerged edges; the reverse is dark green. A brown exudate present above the concentric rings. The hyphae are 2.8–4.8 m wide, septate, hyaline to lightly pigmented. Parathyridariella dematiacea produces numerous Chlamydospores (thick-walled hyphal cells which function like spores), but neither sexual morphs or asexual conidiogenesis (spore production) were seen.

Parathyridariella dematiacea, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); solitary (C) and in chain (D) chlamydospores. Scale bars are 10 μ m (C), (D). Poli et al. (2020).

The second new species is also placed in the genus Parathyridariella, and given the specific name tyrrhenica, in reference to the Tyrrhenian Sea, where it was discovered. This species was isolated from a Brown Seaweed, Padina pavonica, (Peacock's Tail), and a Green Seaweed, Flabellia petiolata, both found growing at a depth of 14-15 m off the coast of Ghiaie Beach on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 10 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 48-50 mm in diameter after 28 days at 24°C, and 26-29 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 31–46 mm in diameter after 28 days at 24 °C, and 16–19 mm in diameter after 28 days at 15°C. The species grew actively on Pine wood and cork. The mycelium is funiculose (made up of rope-like strands), yellowish, land ightly ochre at the edges; the reverse is light yellow, lighter at the edges. The hyphae are 5 μm diameter, septate, hyaline to brownish, sometimes wavy or swollen, forming hyphal strands. No reproductive structures were observed.

Parathyridaria tyrrhenica, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); mycelium (C), black and white arrows indicate hyphal strands and wavy hyphae, respectively. Scale bar is 10 μ m. Poli et al. (2020).

The third species described is also placed in the genus Parathyridaria, and given the specific name flabelliae, in reference to the Green Seaweed, Flabellia petiolata, on which it was found growing, at a depth of 14-15 m off the coast of Ghiaie Beach on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 37–44 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 60 mm in diameter after 28 days at 24°C, and 33–35 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 53–64 mm in diameter after 28 days at 24 °C, and 23–24 mm in diameter after 28 days at 15°C. The species grew actively on Pine wood and cork. The mycelium is funiculose (made up of rope-like strands), and whitish with submerged edges; the reverse is brown in the middle, lighter at edges. The hyphae are 2.6-5 μ m wide, septate and hyaline. Parathyridariella flabelliae produces numerous Chlamydospores, which are globose or subglobose, from light to dark brown in colour, and either unicellular (4 x 5 μ m diameter) or multicellular (up to four-celled and 8 x 12 μm diameter), but neither sexual morphs or asexual conidiogenesis (spore production) were seen.

Parathyridaria flabelliae, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); unicellular and multicellular chlamydospores (C). Scale bar is 10 μm. Poli et al. (2020).

The fourth  new species described is placed in the genus Neoroussoella, and given the specific name lignicola, which implies it grows on dead wood.  This species was isolated from a Brown Seaweed, Padina pavonica, (Peacock's Tail), and a Seagrass, Posidonia oceanica, both found growing at a depth of 14-15 m off the coast of Ghiaie Beach on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 28–29 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 27-40 mm in diameter after 28 days at 24°C, and 14.5-26 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 38–45 mm in diameter after 28 days at 24 °C, and 19–29 mm in diameter after 28 days at 15°C. This species grew efficiently on Pine wood. The mycelium is grey to dark green and floculose, with irregular edges, the reverse is dark grey. A clear exudate is often present. Hyphae are 2–4.4 m wide, septate, hyaline, and assume a toruloid aspect when growing into wood vessels; they form chains of two-celled chlamydospores which, at maturity, protrude from the vessels. The chlamydospores are 7.4 x 5.2 μ m, from light to dark brown, and globose or subglobose. Neither sexual morphs or asexual conidiogenesis (spore production) was seen.

Neoroussoella lignicola, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); two-celled chlamydospores inside wood vessels (C). Scale bar is 10 μm. Poli et al. (2020).

The fifth new species described is placed in the genus Roussoella, and given the specific namemargidorensis, meaning 'from Margidore'; the species was isolated from  a Brown Seaweed, Padina pavonica, (Peacock's Tail), found growing at a depth of 14-15 m off the coast of Margidore on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 33-34 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 45 mm in diameter after 28 days at 24°C, and 27 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 45 mm in diameter after 28 days at 24 °C, and 23 mm in diameter after 28 days at 15°C. This species grew actively on Pine wood. The mycelium is whitish, lighter to the edge, and umbonate (having a rounded knob or protuberance) in the middle, the reverse is ochre. Hyphae are approximately 2 μm wide, septate and brownish. Neither sexual morphs or asexual conidiogenesis (spore production) was seen.

Roussoella margidorensis, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); chlamydospores (C). Scale bar is 10 μ m. Poli et al. (2020).

The sixth new species described is also placed in the genus Roussoella, and given the specific name mediterranea, in reference to the Mediterranean Sea. The species was isolated from  a Brown Seaweed, Padina pavonica, (Peacock's Tail), found growing at a depth of 14-15 m off the coast of Margidore on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 55 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 67–72 mm in diameter after 28 days at 24°C, and 33–38 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 69–76 mm in diameter after 28 days at 24 °C, and 32.5–39 mm in diameter after 28 days at 15°C. This species grew actively on Pine wood, and poorly on cork. The mycelium is light grey, and floccose, with an umbonate area in the middle, the reverse is brown with lighter edges. A dark exudate present. Hyphae are 2.4 μm wide, septate and dematiaceous. Branched chains of light to dark brown chlamydospores often present, these are 4.5 x 5.7 μm, and from unicellular to 4-celled. Neither sexual morphs or asexual conidiogenesis (spore production) was seen.

Roussoella mediterranea, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); unicellular and multicellular chlamydosporesn indicated by a black arrow (C). Scale bar is 10 μ m. Poli et al. (2020).

The final species is also placed in the genus Roussoella, and given the specific name padinae, in reference to the Brown Seaweed, Padina pavonica, (Peacock's Tail), upon which it was found growing, at a depth of 14-15 m off the coast of Margidore on the island of Elba. Colonies of this Fungus grown on Malt Extract Agar-sea water media reached 53 mm in diameter after 28 days, at 21° C, grown on Oatmeal Agar-sea water reached 57.5–65 mm in diameter after 28 days at 24°C, and 30–35 mm in diameter after 28 days at 15°C, and on Potato Dextrose Agar-sea water reached 60–69 mm in diameter after 28 days at 24 °C, and 30–34 mm in diameter after 28 days at 15°C. This species grew poorly on Pine wood, and efficiantly on cork. The mycelium is from grey to dark green, floccose in the middle, with radial grooves, and fimbriate edges; the reverse is brown. Hyphae are 3 μm wide, septate, brownish and assume a toluroid aspect when growing into wood vessels, and form chains of two-celled chlamydospores which, at maturity, protrude from the vessels. These chlamydospores are 5–7 x 4 μm, from light to dark brown in colour, subglobose, ellipsoidal or cylindrical. Neither sexual morphs or asexual conidiogenesis (spore production) was seen.

 Roussoella padinae, 28-days-old colony at 21°C on Malt Extract Agar-sea water media (A) and reverse (B); toruloid hyphae (C) and two-celled chlamydospores (D) inside wood vessels. Scale bars are 10 μm. Poli et al. (2020).

The description of these new taxa was particularly challenging because neither asexual nor sexual reproductive structures developed in axenic conditions. Therefore, Poli et al. were unable to describe the range of anatomical variations and diagnostic features among these newly recognized phylogenetic lineages. Indeed, strictly vegetative growth without sporulation is a common feature of many marine Fungal strains. Possibly, these organisms rely on hyphal fragmentation for their dispersal, or alternatively, the di erentiation of reproductive structures may be obligatorily dependent on the peculiar environmental conditions under which they live (e.g., wet-dry cycles, high salinity, low temperature, high pressure, etc.). During the study of these fungi, Poli et al. tried to mimic the saline environment by using di erent culture media supplemented with natural sea water or sea salts. Although these culture methods were applied to induce sporulation, they observed that only media supplemented with sea water supported a measurable growth of vegetative mycelium. A method tried previously with other Marine Fungi, to induce sporulation by placing wood and cork specimens on the colony surface with their subsequent transfer into sea water, was only partially successful: out of seven species, three (Parathyridariella dematiacea, Parathyridariella flabelliae, Roussoella mediterranea) developed chlamydospores in the mycelium above the wood surface, two (Neoroussoella lignicola, Roussoella padinae) gave rise to resting spores inside wood vessels. Most of the strains preferred to colonise wood rather than cork. These structures were interpreted as 'chlamydospores' instead of 'conidia' for the following reasons: (i) They were characterized by a very thick cell wall, a typical feature of resting spores; (ii) conidiogenous cells were never observed. Additional e orts to force the development of reproductive structures by using Syntetic Nutrient Agar-sea water and Pine needles, were also unsuccessful.

Both Roussoella padinae and Neoroussoella lignicola displayed a similar lignicolous behavior, growing and producing chlamydospores inside wooden vessels, although of di erent size and shape. The ability to form hyphae and to grow inside the wood vessels has been reported for a number of dark septate endophyte Fungi in terrestrial environments, and, recently, for Posidoniomyces atricolor, marine endophyte that lives in association with the roots of the Seagrass, Posidonia oceanica. By definition, endophytes live inside living plant tissues. To induce sporulation, sterilized specimens of dead wood were employed, therefore Roussoella padinae and Neoroussoella lignicola were inferred to be 'lignicolous Fungi' rather than 'endophytes'. The observation of this growth characteristic in two di erent genera, may find its reason in an evolutionary adaptation to marine life in association with lignocellulosic matrices. Therefore, Poli et al. hypothesise their ecological role as saprobes involved in degrading organic matter.

Most of the Roussoellaceae (the family that includes the genera Roussoella and Neoroussoella) and Thyridariaceae (the family that includes the genus Parathyridariella) described to date are associated with terrestrial plants, especially Bamboo and Palm species. In fact, only two species, Roussoella mangrovei and Roussoella nitidula have previously been retrieved from the marine environment. However, Poli et al. infer that these families may be well represented in the sea, thus improving our knowledge on the largely unexplored Fungal marine biodiversity.

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