eng Microbial communities in aerobic brines of hypersaline ecosystems are typically resistant and resilient to single environmental fluctuations in salinity or light. However, the effects of recurrent disturbances of the same nature on microbial stability, composition, and diversity remain poorly understood. In this Ph.D. thesis I explored microbial and viral dynamics at inter- and intraspecies levels across over two years of mesocosm-scale experimentation, combining metagenomic and culture-based approaches. To evaluate the impact of repeated perturbations, in two mesocosm experiments we challenged salt-saturated brines (>36% salts) to cycles of dilution down to 13% (D13) or 20% (D20) salinities once saturation was reached by evaporation. These cycles, 10 for D13 and 17 for D20, comprised 813 days. Community responses varied with disturbance intensity: under moderate stress (D20), dominant taxa such as Haloquadratum walsbyi and Salinibacter ruber prevailed, resembling patterns observed globally in hypersaline environments. In contrast, stronger stress (D13) led to their replacement by congeneric species better adapted to osmotic fluctuations. This pattern was consistent across genera, highlighting that genus-level diversity, facilitated by functional gene redundancy, plays an ecological role beyond taxonomy. Given that accurate strain-level diversity estimates are key to the understanding such dynamics, we tested how sequencing coverage affects two widely used microdiversity metrics using Metagenome-Assembled Genomes (MAGs): the nucleotide diversity (π, based on SNV frequency) and the aggregated Average Nucleotide Identity of reads (ANIr). Using 53 metagenomes from Illumina, PacBio, and Oxford Nanopore platforms across marine, gut, and hypersaline microbiomes, we found π to be highly sensitive to coverage variability, whereas ANIr remained robust above 10X and was computationally more efficient, making it a more reliable metric for cross-study comparisons. A specific focusing on Haloquadratum walsbyi, the most abundant species in these environments, genomic comparison of 140 MAGs and two isolates revealed a high genomic homogeneity (98.25-99.88% ANI), composed by four dominant genomovars (>99.5% ANI). Under moderate disturbances (D20), the globally dominant genomovar Hqrw1 increased in abundance, reducing intraspecific diversity. In contrast, strong dilution (D13) led to the decline of Hqrw1 and its replacement by Hqrw2, which encodes more genes linked to osmotic resistance. We propose that Hqr. walsbyi's global dominance in thalassohaline sites results from Hqrw1's success under stable salinities and Hqrw2’s adaptation to osmotic stress. A culture-based approach was further used to resolve intraspecies diversity within Salinibacter species. Our results showed that thousands of genomovars coexisted in a single brine, and shifts in their abundance revealed that species and genomovar turnover ensured lineage persistence over time. Environmental pressures drove these shifts, which were closely mirrored by changes in associated viral diversity, indicating viral regulation throughout the time series. One of the most resilient genomovars under intense dilutions in D13, Salr84, showed that its control by specific viruses was following the classical Kill-the-Winner dynamics. This viral-mediated turnover seems to foster ongoing microbial and viral diversification and adaptation, underscoring the central role of viruses in maintaining diversity in hypersaline ecosystems. Altogether, this study reveals how microbial communities of hypersaline environments respond to repeated disturbances. By integrating metagenomics and cultivation, we show that community stability is maintained through species and genomovar replacements. These mechanisms, together with virus-driven regulation, explain the remarkable ecological resilience of microbial life in extreme environments.
Esteban Bustos Caparrós (Fri,) studied this question.