Hypersaline meromictic lakes are extreme environments in which water stratification is associated with powerful physicochemical gradients and high salt concentrations. Furthermore, their physical stability coupled with vertical water column partitioning makes them important research model systems in microbial niche differentiation and biogeochemical cycling. Here, we compare the prokaryotic assemblages from Ursu and Fara Fund hypersaline meromictic lakes (Transylvanian Basin, Romania) in relation to their limnological factors and infer their role in elemental cycling by matching taxa to known taxon-specific biogeochemical functions. To assess the composition and structure of prokaryotic communities and the environmental factors that structure them, deepcoverage small subunit (SSU) ribosomal RNA (rDNA) amplicon sequencing, community domainspecific quantitative PCR and physicochemical analyses were performed on samples collected along depth profiles. The analyses showed that the lakes harbored multiple and diverse prokaryotic communities whose distribution mirrored the water stratification patterns. Ursu Lake was found to be dominated by Bacteria and to have a greater prokaryotic diversity than Fara Fund Lake that harbored an increased cell density and was populated mostly by Archaea within oxic strata. In spite of their contrasting diversity, the microbial populations indigenous to each lake pointed to similar physiological functions within carbon degradation and sulfate reduction. Furthermore, the taxonomy results coupled with methane detection and its stable C isotope composition indicated the presence of a yet-undescribed methanogenic group in the lakes' hypersaline monimolimnion. In addition, ultrasmall uncultivated archaeal lineages were detected in the chemocline of Fara Fund Lake, where the recently proposed Nanohaloarchaeota phylum was found to thrive.

Perennially stratified salt lakes situated in the Transylvanian Basin (Central Romania) were surveyed for the diversity of culturable halophilic archaea (Fam. Halobacteriaceae). The physical and chemical characteristics of the waters indicated that all the investigated lakes were meromictic and neutral hypersaline. Samples collected from upper, intermediate, and deeper water layers and sediments were used for the isolation of halophilic strains followed by 16S rRNA gene-based identification and phenotypic characterization. The phylogenetic analysis of the 16S rRNA gene sequences revealed that all 191 isolates reported in this study and 43 strains previously isolated were affiliated with the family Halobacteriaceae and classified to 18 genera. Haloferax was the most frequently isolated genus (~47 %), followed by Halobacterium spp. (~12 %), and Halorubrum spp. (~11 %). Highest culturable diversity was detected in Brâncoveanu Lake, the oldest and saltiest of all studied lakes, while the opposite was observed in the most stable and least human-impacted Fără Fund Lake. One strain from Ursu Lake might possibly constitute a novel Halorubrum species as shown by phylogenetic analysis. Several haloarchaeal taxa recently described in Asian (i.e., Iran, China) saline systems were also identified as inhabiting the Transylvanian salt lakes thus expanding our knowledege on the geographic distribution of Halobacteriaceae.

Summary Ursu Lake is located in the Middle Miocene salt deposit of Central Romania. It is stratified, and the water column has three distinct water masses: an upper freshwater‐to‐moderately saline stratum (0–3 m), an intermediate stratum exhibiting a steep halocline (3–3.5 m), and a lower hypersaline stratum (4 m and below) that is euxinic (i.e. anoxic and sulphidic). Recent studies have characterized the lake's microbial taxonomy and given rise to intriguing ecological questions. Here, we explore whether the communities are dynamic or stable in relation to taxonomic composition, geochemistry, biophysics, and ecophysiological functions during the annual cycle. We found: (i) seasonally fluctuating, light‐dependent communities in the upper layer (≥0.987–0.990 water‐activity), a stable but phylogenetically diverse population of heterotrophs in the hypersaline stratum (water activities down to 0.762) and a persistent plate of green sulphur bacteria that connects these two (0.958–0.956 water activity) at 3–3.5 to 4 m; (ii) communities that might be involved in carbon‐ and sulphur‐cycling between and within the lake's three main water masses; (iii) uncultured lineages including Acetothermia (OP1), Cloacimonetes (WWE1), Marinimicrobia (SAR406), Omnitrophicaeota (OP3), Parcubacteria (OD1) and other Candidate Phyla Radiation bacteria, and SR1 in the hypersaline stratum (likely involved in the anaerobic steps of carbon‐ and sulphur‐cycling); and (iv) that species richness and habitat stability are associated with high redox‐potentials. Ursu Lake has a unique and complex ecology, at the same time exhibiting dynamic fluctuations and stability, and can be used as a modern analogue for ancient euxinic water bodies and comparator system for other stratified hypersaline systems.

Geography, babeş-bolyai university, 5-7 clinicilor str., 400006 cluj-napoca, romania; 2 national institute of research and development for biological sciences (nirdbs), institute of biological research, 48 republicii str., 400015 cluj-napoca, romania; 3 department of Aquatic Microbial ecology, institute of Hydrobiology, biology center of the Academy of sciences of the czech republic, na sádkách 7, 370 05 České budějovice, czech republic; 4 department of Molecular biology and biotechnology, Faculty of biology and Geology, babeş-bolyai university, 5-7 clinicilor str., 400006 cluj-napoca, romania; 5 5department of taxonomy and ecology, Faculty of biology and Geology, babeş-bolyai university, 5-7 clinicilor str., 400006 cluj-napoca, romania; 6 electron Microscopy center, babeș-bolyai university, 5-7 clinicilor street, clujnapoca, romania; 7 center for systems biology, biodiversity, and bioresources, babes-bolyai university, 5-7 clinicilor str., 400006 cluj-napoca, romania *corresponding author: gheorghe.serban@ubbcluj.ro ABSTRACTin the present work, we review the current knowledge on genesis, limnology and biodiversity of salt lakes distributed around the inner contour of eastern carpathian arc (transylvanian basin, central romania). transylvanian salt lakes formed on ancient halite (nacl) deposits following natural processes or quarrying activities. Most of these lakes are located in eastern (sovata area), southern (ocna sibiului), and western (turda-cojocna) parts of the transylvanian basin, have small surfaces (0.1-4 ha), variable depths (2-100 m), are hypersaline (>10%, w/v, total salts, mainly nacl) and permanently stratified. As consequence of steady salinity/density gradient, heat entrapment below surface layer (i.e., heliothermy) develops in several transylvanian lakes. the physical and chemical water stratification is mirrored in the partition of plankton diversity. lakes with less saline (2-10% salinity) water layers appear to harbor halotolerant representatives of phyto-(e.g., marine native Picochlorum spp. and Synechococcus spp.), zoo-(e.g., Moina salina), and bacterioplankton (e.g., Actinobacteria, Verrucomicobia), whereas halophilic plankton communities (e.g., green algae Dunaliella sp., brine shrimp Artemia sp., and members of Halobacteria class) dominate in the oxic surface of hypersaline (>10% salinity) lakes. Molecular approaches (e.g., Pcr-dGGe, 16s rrnA gene-based clone libraries, and dnA metabarcoding) showed that the o 2 -depleted bottom brines of deep meromictic transylvanian lakes are inhabited by known extremely halophilic anaerobes (e.g. sulfate-reducing delta-Proteobacteria, fermenting clostridia, methanogenic and polymer-degrading archaea) in addition to representatives of uncultured/unclassified prokaryotic lineages. overall, the plankton communities thriving in saline transylvanian lakes seem to drive full biogeochemical cycling of main elements. However, the trophic interactions (i.e., food web structure and energy flow) as well as impact of human activities and predicted climate chang...

Saline, meromictic lakes with significant depth are usually formed as a result of salt mining activity. Ocnei Lake is one of the largest Transylvanian (Central Romania) neutral, hypersaline lake of man-made origin. We aimed to survey the seasonal dynamics of archaeal diversity in the water column of Ocnei Lake by employing microbiological methods as well as molecular techniques based on the sequence analysis of the 16S rRNA gene. We found that archaeal diversity in the water column increased with depth and salinity, with 8 OTUs being detected in the epilimnion compared to 21 found in the chemocline, and 32 OTUs in the monimolimnion. Down to 3.5 m depth, the archaeal community was markedly dominated by the presence of an unclassified archaeon sharing 93% sequence identity to Halogeometricum spp. At the chemocline, the shift in archaeal community composition was associated with an increase in salinity, the main factor affecting the vertical distribution of archaeal assemblages. It appears that the microoxic and hypersaline monimolimnion is populated by several major haloarchaeal taxa, with minor fluctuations in their relative abundances throughout all seasons. The culturable diversity was reasonably correlated to the dominant OTUs obtained by molecular methods. Our results indicate that Ocnei Lake represents a relatively stable extreme habitat, accommodating a diverse and putatively novel archaeal community, as 30% of OTUs could not be classified at the genus level.