The microbiome of the Black Sea water column analyzed by shotgun and genome centric metagenomics.

BACKGROUND: The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former, albeit with lower salinity and temperature. Despite its well-known hydrology and physicochemical features, this enormous water mass remains poorly studied at the microbial genomics level. RESULTS: We have sampled its different water masses and analyzed the microbiome by shotgun and genome-resolved metagenomics, generating a large number of metagenome-assembled genomes (MAGs) from them. We found various similarities with previously described Black Sea metagenomic datasets, that show remarkable stability in its microbiome. Our datasets are also comparable to other marine anoxic water columns like the Cariaco Basin. The oxic zone resembles to standard marine (e.g. Mediterranean) photic zones, with Cyanobacteria (Synechococcus but a conspicuously absent Prochlorococcus), and photoheterotrophs domination (largely again with marine relatives). The chemocline presents very different characteristics from the oxic surface with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. The euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy-generating metabolism, a few (but detectable) methanogenesis marker genes, and a large number of "dark matter" streamlined genomes of largely unpredictable ecology. CONCLUSIONS: The Black Sea oxic zone presents many similarities to the global ocean while the redoxcline and euxinic water masses have similarities to other similar aquatic environments of marine (Cariaco Basin or other Black Sea regions) or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environment. We are adding critical information about this unique and important ecosystem and its microbiome.

Genomic Comparison and Spatial Distribution of Different Synechococcus Phylotypes in the Black Sea.

Picocyanobacteria of the genus Synechococcus are major contributors to global primary production and nutrient cycles due to their oxygenic photoautotrophy, their abundance, and the extensive distribution made possible by their wide-ranging biochemical capabilities. The recent recovery and isolation of strains from the deep euxinic waters of the Black Sea encouraged us to expand our analysis of their adaptability also beyond the photic zone of aquatic environments. To this end, we quantified the total abundance and distribution of Synechococcus along the whole vertical profile of the Black Sea by flow cytometry, and analyzed the data obtained in light of key environmental factors. Furthermore, we designed phylotype-specific primers using the genomes of two new epipelagic coastal strains - first described here - and of two previously described mesopelagic strains, analyzed their presence/abundance by qPCR, and tested this parameter also in metagenomes from two stations at different depths. Together, whole genome sequencing, metagenomics and qPCR techniques provide us with a higher resolution of Synechococcus dynamics in the Black Sea. Both phylotypes analyzed are abundant and successful in epipelagic coastal waters; but while the newly described epipelagic strains are specifically adapted to this environment, the strains previously isolated in mesopelagic waters are able, in low numbers, to withstand the aphotic and oxygen depleted conditions of deep layers. This heterogeneity allows different Synechococcus phylotypes to occupy different niches and underscores the importance of a more detailed characterization of the abundance, distribution, and dynamics of individual populations of these picocyanobacteria.

Comparative analysis of morphological and molecular approaches integrated into the study of the dinoflagellate biodiversity within the recently deposited Black Sea sediments - benefits and drawbacks.

One of the assets, assigned to the phytoplankton resting stages, is that of serving as the "memory" of the aquatic ecosystems and preserved biodiversity in the course of time. However, an accurate cyst identification proves to be a more difficult and extremely challenging process, even today. In order to gain a better taxonomic coverage of cyst assemblages in the Black Sea, an integrated approach of the classical morphological identification with metabarcoding methods (MySeq sequencing of V7-V9 regions of the 18S rDNA) was applied on thirteen surface sediment samples collected from different sites. A total number of 112 dinoflagellate taxa was detected at the species level and ascribed to 51 genera. In general, it is the molecular analysis that yields a higher number of taxa as compared to those obtained through the morphological taxonomy (66 taxa based on the DNA sequences versus 56 morphologically-identified taxa). Besides, it should be pointed out that the integrated dataset includes 14 potentially toxic dinoflagellate species. Discerned, subsequently, was a good dataset consistency for ten species, followed by some discrepancies as to a number of taxa, identified with one of the methods only, due to specific methodological biases. On the whole, it could be concluded that the combination of morphological and molecular methods is likely to increase the potential for a more reliable taxonomic assessment of phytoplankton diversity in marine sediments which, in turn, proves conclusively the utmost importance of the integrated approach.

The mesopelagic anoxic Black Sea as an unexpected habitat for Synechococcus challenges our understanding of global "deep red fluorescence".

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml-1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.