Distinct and rich assemblages of giant viruses in Arctic and Antarctic lakes.

Giant viruses (GVs) are key players in ecosystem functioning, biogeochemistry, and eukaryotic genome evolution. GV diversity and abundance in aquatic systems can exceed that of prokaryotes, but their diversity and ecology in lakes, especially polar ones, remain poorly understood. We conducted a comprehensive survey and meta-analysis of GV diversity across 20 lakes, spanning polar to temperate regions, combining our extensive lake metagenome database from the Canadian Arctic and subarctic with publicly available datasets. Leveraging a novel GV genome identification tool, we identified 3304 GV metagenome-assembled genomes, revealing lakes as untapped GV reservoirs. Phylogenomic analysis highlighted their dispersion across all Nucleocytoviricota orders. Strong GV population endemism emerged between lakes from similar regions and biomes (Antarctic and Arctic), but a polar/temperate barrier in lacustrine GV populations and differences in their gene content could be observed. Our study establishes a robust genomic reference for future investigations into lacustrine GV ecology in fast changing polar environments.

Lower viral evolutionary pressure under stable versus fluctuating conditions in subzero Arctic brines.

BACKGROUND: Climate change threatens Earth's ice-based ecosystems which currently offer archives and eco-evolutionary experiments in the extreme. Arctic cryopeg brine (marine-derived, within permafrost) and sea ice brine, similar in subzero temperature and high salinity but different in temporal stability, are inhabited by microbes adapted to these extreme conditions. However, little is known about their viruses (community composition, diversity, interaction with hosts, or evolution) or how they might respond to geologically stable cryopeg versus fluctuating sea ice conditions. RESULTS: We used long- and short-read viromics and metatranscriptomics to study viruses in Arctic cryopeg brine, sea ice brine, and underlying seawater, recovering 11,088 vOTUs (~species-level taxonomic unit), a 4.4-fold increase of known viruses in these brines. More specifically, the long-read-powered viromes doubled the number of longer (≥25 kb) vOTUs generated and recovered more hypervariable regions by >5-fold compared to short-read viromes. Distribution assessment, by comparing to known viruses in public databases, supported that cryopeg brine viruses were of marine origin yet distinct from either sea ice brine or seawater viruses, while 94% of sea ice brine viruses were also present in seawater. A virus-encoded, ecologically important exopolysaccharide biosynthesis gene was identified, and many viruses (~half of metatranscriptome-inferred "active" vOTUs) were predicted as actively infecting the dominant microbial genera Marinobacter and Polaribacter in cryopeg and sea ice brines, respectively. Evolutionarily, microdiversity (intra-species genetic variations) analyses suggested that viruses within the stable cryopeg brine were under significantly lower evolutionary pressures than those in the fluctuating sea ice environment, while many sea ice brine virus-tail genes were under positive selection, indicating virus-host co-evolutionary arms races. CONCLUSIONS: Our results confirmed the benefits of long-read-powered viromics in understanding the environmental virosphere through significantly improved genomic recovery, expanding viral discovery and the potential for biological inference. Evidence of viruses actively infecting the dominant microbes in subzero brines and modulating host metabolism underscored the potential impact of viruses on these remote and underexplored extreme ecosystems. Microdiversity results shed light on different strategies viruses use to evolve and adapt when extreme conditions are stable versus fluctuating. Together, these findings verify the value of long-read-powered viromics and provide foundational data on viral evolution and virus-microbe interactions in Earth's destabilized and rapidly disappearing cryosphere. Video Abstract.

Climate-Endangered Arctic Epishelf Lake Harbors Viral Assemblages with Distinct Genetic Repertoires.

Milne Fiord, located on the coastal margin of the Last Ice Area (LIA) in the High Arctic (82°N, Canada), harbors an epishelf lake, a rare type of ice-dependent ecosystem in which a layer of freshwater overlies marine water connected to the open ocean. This microbe-dominated ecosystem faces catastrophic change due to the deterioration of its ice environment related to warming temperatures. We produced the first assessment of viral abundance, diversity, and distribution in this vulnerable ecosystem and explored the niches available for viral taxa and the functional genes underlying their distribution. We found that the viral community in the freshwater layer was distinct from, and more diverse than, the community in the underlying seawater and contained a different set of putative auxiliary metabolic genes, including the sulfur starvation-linked gene tauD and the gene coding for patatin-like phospholipase. The halocline community resembled the freshwater more than the marine community, but harbored viral taxa unique to this layer. We observed distinct viral assemblages immediately below the halocline, at a depth that was associated with a peak of prasinophyte algae and the viral family Phycodnaviridae. We also assembled 15 complete circular genomes, including a putative Pelagibacter phage with a marine distribution. It appears that despite its isolated and precarious situation, the varied niches in this epishelf lake support a diverse viral community, highlighting the importance of characterizing underexplored microbiota in the Last Ice Area before these ecosystems undergo irreversible change. IMPORTANCE Viruses are key to understanding polar aquatic ecosystems, which are dominated by microorganisms. However, studies of viral communities are challenging to interpret because the vast majority of viruses are known only from sequence fragments, and their taxonomy, hosts, and genetic repertoires are unknown. Our study establishes a basis for comparison that will advance understanding of viral ecology in diverse global environments, particularly in the High Arctic. Rising temperatures in this region mean that researchers have limited time remaining to understand the biodiversity and biogeochemical cycles of ice-dependent environments and the consequences of these rapid, irreversible changes. The case of the Milne Fiord epishelf lake has special urgency because of the rarity of this type of "floating lake" ecosystem and its location in the Last Ice Area, a region of thick sea ice with global importance for conservation efforts.

Evolutionary Divergence of Marinobacter Strains in Cryopeg Brines as Revealed by Pangenomics.

Marinobacter spp. are cosmopolitan in saline environments, displaying a diverse set of metabolisms that allow them to competitively occupy these environments, some of which can be extreme in both salinity and temperature. Here, we introduce a distinct cluster of Marinobacter genomes, composed of novel isolates and in silico assembled genomes obtained from subzero, hypersaline cryopeg brines, relic seawater-derived liquid habitats within permafrost sampled near Utqiagvik, Alaska. Using these new genomes and 45 representative publicly available genomes of Marinobacter spp. from other settings, we assembled a pangenome to examine how the new extremophile members fit evolutionarily and ecologically, based on genetic potential and environmental source. This first genus-wide genomic analysis revealed that Marinobacter spp. in general encode metabolic pathways that are thermodynamically favored at low temperature, cover a broad range of organic compounds, and optimize protein usage, e.g., the Entner-Doudoroff pathway, the glyoxylate shunt, and amino acid metabolism. The new isolates contributed to a distinct clade of subzero brine-dwelling Marinobacter spp. that diverged genotypically and phylogenetically from all other Marinobacter members. The subzero brine clade displays genomic characteristics that may explain competitive adaptations to the extreme environments they inhabit, including more abundant membrane transport systems (e.g., for organic substrates, compatible solutes, and ions) and stress-induced transcriptional regulatory mechanisms (e.g., for cold and salt stress) than in the other Marinobacter clades. We also identified more abundant signatures of potential horizontal transfer of genes involved in transcription, the mobilome, and a variety of metabolite exchange systems, which led to considering the importance of this evolutionary mechanism in an extreme environment where adaptation via vertical evolution is physiologically rate limited. Assessing these new extremophile genomes in a pangenomic context has provided a unique view into the ecological and evolutionary history of the genus Marinobacter, particularly with regard to its remarkable diversity and its opportunism in extremely cold and saline environments.

Divergent Genomic Adaptations in the Microbiomes of Arctic Subzero Sea-Ice and Cryopeg Brines.

Subzero hypersaline brines are liquid microbial habitats within otherwise frozen environments, where concentrated dissolved salts prevent freezing. Such extreme conditions presumably require unique microbial adaptations, and possibly altered ecologies, but specific strategies remain largely unknown. Here we examined prokaryotic taxonomic and functional diversity in two seawater-derived subzero hypersaline brines: first-year sea ice, subject to seasonally fluctuating conditions; and ancient cryopeg, under relatively stable conditions geophysically isolated in permafrost. Overall, both taxonomic composition and functional potential were starkly different. Taxonomically, sea-ice brine communities (∼105 cells mL-1) had greater richness, more diversity and were dominated by bacterial genera, including Polaribacter, Paraglaciecola, Colwellia, and Glaciecola, whereas the more densely inhabited cryopeg brines (∼108 cells mL-1) lacked these genera and instead were dominated by Marinobacter. Functionally, however, sea ice encoded fewer accessory traits and lower average genomic copy numbers for shared traits, though DNA replication and repair were elevated; in contrast, microbes in cryopeg brines had greater genetic versatility with elevated abundances of accessory traits involved in sensing, responding to environmental cues, transport, mobile elements (transposases and plasmids), toxin-antitoxin systems, and type VI secretion systems. Together these genomic features suggest adaptations and capabilities of sea-ice communities manifesting at the community level through seasonal ecological succession, whereas the denser cryopeg communities appear adapted to intense bacterial competition, leaving fewer genera to dominate with brine-specific adaptations and social interactions that sacrifice some members for the benefit of others. Such cryopeg genomic traits provide insight into how long-term environmental stability may enable life to survive extreme conditions.

Comparison of Two 16S rRNA Primers (V3-V4 and V4-V5) for Studies of Arctic Microbial Communities.

Microbial communities of the Arctic Ocean are poorly characterized in comparison to other aquatic environments as to their horizontal, vertical, and temporal turnover. Yet, recent studies showed that the Arctic marine ecosystem harbors unique microbial community members that are adapted to harsh environmental conditions, such as near-freezing temperatures and extreme seasonality. The gene for the small ribosomal subunit (16S rRNA) is commonly used to study the taxonomic composition of microbial communities in their natural environment. Several primer sets for this marker gene have been extensively tested across various sample sets, but these typically originated from low-latitude environments. An explicit evaluation of primer-set performances in representing the microbial communities of the Arctic Ocean is currently lacking. To select a suitable primer set for studying microbiomes of various Arctic marine habitats (sea ice, surface water, marine snow, deep ocean basin, and deep-sea sediment), we have conducted a performance comparison between two widely used primer sets, targeting different hypervariable regions of the 16S rRNA gene (V3-V4 and V4-V5). We observed that both primer sets were highly similar in representing the total microbial community composition down to genus rank, which was also confirmed independently by subgroup-specific catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) counts. Each primer set revealed higher internal diversity within certain bacterial taxonomic groups (e.g., the class Bacteroidia by V3-V4, and the phylum Planctomycetes by V4-V5). However, the V4-V5 primer set provides concurrent coverage of the archaeal domain, a relevant component comprising 10-20% of the community in Arctic deep waters and the sediment. Although both primer sets perform similarly, we suggest the use of the V4-V5 primer set for the integration of both bacterial and archaeal community dynamics in the Arctic marine environment.

Viral Ecogenomics of Arctic Cryopeg Brine and Sea Ice.

Arctic regions, which are changing rapidly as they warm 2 to 3 times faster than the global average, still retain microbial habitats that serve as natural laboratories for understanding mechanisms of microbial adaptation to extreme conditions. Seawater-derived brines within both sea ice (sea-ice brine) and ancient layers of permafrost (cryopeg brine) support diverse microbes adapted to subzero temperatures and high salinities, yet little is known about viruses in these extreme environments, which, if analogous to other systems, could play important evolutionary and ecosystem roles. Here, we characterized viral communities and their functions in samples of cryopeg brine, sea-ice brine, and melted sea ice. Viral abundance was high in cryopeg brine (1.2 × 108 ml-1) and much lower in sea-ice brine (1.3 × 105 to 2.1 × 105 ml-1), which roughly paralleled the differences in cell concentrations in these samples. Five low-input, quantitative viral metagenomes were sequenced to yield 476 viral populations (i.e., species level; ≥10 kb), only 12% of which could be assigned taxonomy by traditional database approaches, indicating a high degree of novelty. Additional analyses revealed that these viruses: (i) formed communities that differed between sample type and vertically with sea-ice depth; (ii) infected hosts that dominated these extreme ecosystems, including Marinobacter, Glaciecola, and Colwellia; and (iii) encoded fatty acid desaturase (FAD) genes that likely helped their hosts overcome cold and salt stress during infection, as well as mediated horizontal gene transfer of FAD genes between microbes. Together, these findings contribute to understanding viral abundances and communities and how viruses impact their microbial hosts in subzero brines and sea ice.IMPORTANCE This study explores viral community structure and function in remote and extreme Arctic environments, including subzero brines within marine layers of permafrost and sea ice, using a modern viral ecogenomics toolkit for the first time. In addition to providing foundational data sets for these climate-threatened habitats, we found evidence that the viruses had habitat specificity, infected dominant microbial hosts, encoded host-derived metabolic genes, and mediated horizontal gene transfer among hosts. These results advance our understanding of the virosphere and how viruses influence extreme ecosystems. More broadly, the evidence that virally mediated gene transfers may be limited by host range in these extreme habitats contributes to a mechanistic understanding of genetic exchange among microbes under stressful conditions in other systems.

Diversity and metabolism of Woeseiales bacteria, global members of marine sediment communities.

Surveys of 16S rRNA gene sequences derived from marine sediments have indicated that a widely distributed group of Gammaproteobacteria, named "JTB255-Marine Benthic Group" (now the candidate order Woeseiales), accounts for 1-22% of the retrieved sequences. Despite their ubiquity in seafloor communities, little is known about their distribution and specific ecological niches in the deep sea, which constitutes the largest biome globally. Here, we characterized the phylogeny, environmental distribution patterns, abundance, and metabolic potential of Woeseiales bacteria with a focus on representatives from the deep sea. From a phylogenetic analysis of publicly available 16S rRNA gene sequences (≥1400 bp, n = 994), we identified lineages of Woeseiales with greater prevalence in the deep sea than in coastal environments, a pattern corroborated by the distribution of 16S oligotypes recovered from 28 globally distributed sediment samples. Cell counts revealed that Woeseiales bacteria accounted for 5 ± 2% of all microbial cells in deep-sea surface sediments at 23 globally distributed sites. Comparative analyses of a genome, metagenome bins, and single-cell genomes suggested that members of the corresponding clades are likely to grow on proteinaceous matter, potentially derived from detrital cell membranes, cell walls, and other organic remnants in marine sediments.

Distinctive microbial communities in subzero hypersaline brines from Arctic coastal sea ice and rarely sampled cryopegs.

Hypersaline aqueous environments at subzero temperatures are known to be inhabited by microorganisms, yet information on community structure in subzero brines is very limited. Near Utqiagvik, Alaska, we sampled subzero brines (-6°C, 115-140 ppt) from cryopegs, i.e. unfrozen sediments within permafrost that contain relic (late Pleistocene) seawater brine, as well as nearby sea-ice brines to examine microbial community composition and diversity using 16S rRNA gene amplicon sequencing. We also quantified the communities microscopically and assessed environmental parameters as possible determinants of community structure. The cryopeg brines harbored surprisingly dense bacterial communities (up to 108 cells mL-1) and millimolar levels of dissolved and particulate organic matter, extracellular polysaccharides and ammonia. Community composition and diversity differed between the two brine environments by alpha- and beta-diversity indices, with cryopeg brine communities appearing less diverse and dominated by one strain of the genus Marinobacter, also detected in other cold, hypersaline environments, including sea ice. The higher density and trend toward lower diversity in the cryopeg communities suggest that long-term stability and other features of a subzero brine are more important selective forces than in situ temperature or salinity, even when the latter are extreme.

Effects of Ice-Algal Aggregate Export on the Connectivity of Bacterial Communities in the Central Arctic Ocean.

In summer 2012, Arctic sea ice declined to a record minimum and, as a consequence of the melting, large amounts of aggregated ice-algae sank to the seafloor at more than 4,000 m depth. In this study, we assessed the composition, turnover and connectivity of bacterial and microbial eukaryotic communities across Arctic habitats from sea ice, algal aggregates and surface waters to the seafloor. Eukaryotic communities were dominated by diatoms, dinoflagellates and other alveolates in all samples, and showed highest richness and diversity in sea-ice habitats (∼400-500 OTUs). Flavobacteriia and Gammaproteobacteria were the predominant bacterial classes across all investigated Arctic habitats. Bacterial community richness and diversity peaked in deep-sea samples (∼1,700 OTUs). Algal aggregate-associated bacterial communities were mainly recruited from the sea-ice community, and were transported to the seafloor with the sinking ice algae. The algal deposits at the seafloor had a unique community structure, with some shared sequences with both the original sea-ice community (22% OTU overlap), as well as with the deep-sea sediment community (17% OTU overlap). We conclude that ice-algal aggregate export does not only affect carbon export from the surface to the seafloor, but may change microbial community composition in central Arctic habitats with potential effects for benthic ecosystem functioning in the future.

Diazotroph Diversity in the Sea Ice, Melt Ponds, and Surface Waters of the Eurasian Basin of the Central Arctic Ocean.

The Eurasian basin of the Central Arctic Ocean is nitrogen limited, but little is known about the presence and role of nitrogen-fixing bacteria. Recent studies have indicated the occurrence of diazotrophs in Arctic coastal waters potentially of riverine origin. Here, we investigated the presence of diazotrophs in ice and surface waters of the Central Arctic Ocean in the summer of 2012. We identified diverse communities of putative diazotrophs through targeted analysis of the nifH gene, which encodes the iron protein of the nitrogenase enzyme. We amplified 529 nifH sequences from 26 samples of Arctic melt ponds, sea ice and surface waters. These sequences resolved into 43 clusters at 92% amino acid sequence identity, most of which were non-cyanobacterial phylotypes from sea ice and water samples. One cyanobacterial phylotype related to Nodularia sp. was retrieved from sea ice, suggesting that this important functional group is rare in the Central Arctic Ocean. The diazotrophic community in sea-ice environments appear distinct from other cold-adapted diazotrophic communities, such as those present in the coastal Canadian Arctic, the Arctic tundra and glacial Antarctic lakes. Molecular fingerprinting of nifH and the intergenic spacer region of the rRNA operon revealed differences between the communities from river-influenced Laptev Sea waters and those from ice-related environments pointing toward a marine origin for sea-ice diazotrophs. Our results provide the first record of diazotrophs in the Central Arctic and suggest that microbial nitrogen fixation may occur north of 77°N. To assess the significance of nitrogen fixation for the nitrogen budget of the Arctic Ocean and to identify the active nitrogen fixers, further biogeochemical and molecular biological studies are needed.

Microbial ecology of the cryosphere: sea ice and glacial habitats.

The Earth's cryosphere comprises those regions that are cold enough for water to turn into ice. Recent findings show that the icy realms of polar oceans, glaciers and ice sheets are inhabited by microorganisms of all three domains of life, and that temperatures below 0 °C are an integral force in the diversification of microbial life. Cold-adapted microorganisms maintain key ecological functions in icy habitats: where sunlight penetrates the ice, photoautotrophy is the basis for complex food webs, whereas in dark subglacial habitats, chemoautotrophy reigns. This Review summarizes current knowledge of the microbial ecology of frozen waters, including the diversity of niches, the composition of microbial communities at these sites and their biogeochemical activities.

Expanding the World of Marine Bacterial and Archaeal Clades.

Determining which microbial taxa are out there, where they live, and what they are doing is a driving approach in marine microbial ecology. The importance of these questions is underlined by concerted, large-scale, and global ocean sampling initiatives, for example the International Census of Marine Microbes, Ocean Sampling Day, or Tara Oceans. Given decades of effort, we know that the large majority of marine Bacteria and Archaea belong to about a dozen phyla. In addition to the classically culturable Bacteria and Archaea, at least 50 "clades," at different taxonomic depths, exist. These account for the majority of marine microbial diversity, but there is still an underexplored and less abundant portion remaining. We refer to these hitherto unrecognized clades as unknown, as their boundaries, names, and classifications are not available. In this work, we were able to characterize up to 92 of these unknown clades found within the bacterial and archaeal phylogenetic diversity currently reported for marine water column environments. We mined the SILVA 16S rRNA gene datasets for sequences originating from the marine water column. Instead of the usual subjective taxa delineation and nomenclature methods, we applied the candidate taxonomic unit (CTU) circumscription system, along with a standardized nomenclature to the sequences in newly constructed phylogenetic trees. With this new phylogenetic and taxonomic framework, we performed an analysis of ICoMM rRNA gene amplicon datasets to gain insights into the global distribution of the new marine clades, their ecology, biogeography, and interaction with oceanographic variables. Most of the new clades we identified were interspersed by known taxa with cultivated members, whose genome sequences are available. This result encouraged us to perform metabolic predictions for the novel marine clades using the PICRUSt approach. Our work also provides an update on the taxonomy of several phyla and widely known marine clades as our CTU approach breaks down these randomly lumped clades into smaller objectively calculated subgroups. Finally, all taxa were classified and named following standards compatible with the Bacteriological Code rules, enhancing their digitization, and comparability with future microbial ecological and taxonomy studies.