| Summary: | Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of <i>Bacteria</i> and <i>Archaea</i>, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of <i>Archaea</i> in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for <i>Bacteria</i> and at 24.1% salinity for <i>Archaea</i>. While <i>Bacteria</i> showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, <i>Salinibacter</i> was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal <i>Haloquadratum</i> (~43% at 36.0% salinity) and <i>Natronomonas</i> (~18% at 13.1% salinity) and the bacterial “<i>Candidatus</i> Aquiluna” (~19% at 14.5% salinity). Interestingly, “<i>Candidatus</i> Aquiluna” had not been identified before in thalassohaline waters.
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