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Ammonia-oxidizing archaea (AOA) play a key role in the aquatic nitrogen cycle. Their genetic diversity is viewed as the outcome of evolutionary processes that shaped ancestral transition from terrestrial to marine habitats. However, current genome-wide insights into AOA evolution rarely consider brackish and freshwater representatives or provide their divergence timeline in lacustrine systems. An unbiased global assessment of lacustrine AOA diversity is critical for understanding their origins, dispersal mechanisms, and ecosystem roles. Here, we leveraged continental-scale metagenomics to document that AOA species diversity in freshwater systems is remarkably low compared to marine environments. We show that the uncultured freshwater AOA, "Candidatus Nitrosopumilus limneticus," is ubiquitous and genotypically static in various large European lakes where it evolved 13 million years ago. We find that extensive proteome remodeling was a key innovation for freshwater colonization of AOA. These findings reveal the genetic diversity and adaptive mechanisms of a keystone species that has survived clonally in lakes for millennia.

Background: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. Results: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. Con

[Background] Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. [Results] Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. [Conclusions] Our novel ap

Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists., QC 20210415

Members of the bacterial phylum Gemmatimonadota are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with Gemmatimonadota were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency, Gemmatimonadota represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms (Fragilaria sp.) or cyanobacteria (Microcystis sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to Gemmatimonadota. They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to Gemmatimonas phototrophica and represented the majority of Gemmatimonadota retrieved from the lakes’ epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic Gemmatimonadota distinct from the previously described species isolated from soils or sediments.