The oxygenated hypolimnion accounts for a volumetrically significant part of the global freshwater systems. Previous studies have proposed the presence of hypolimnion-specific bacterioplankton lineages that are distinct from those inhabiting the epilimnion. To date, however, no consensus exists regarding their ubiquity and abundance, which is necessary to evaluate their ecological importance. The present study investigated the bacterioplankton community in the oxygenated hypolimnia of 10 deep freshwater lakes. Despite the broad geochemical characteristics of the lakes, 16S rRNA gene sequencing demonstrated that the communities in the oxygenated hypolimnia were distinct from those in the epilimnia and identified several predominant lineages inhabiting multiple lakes. Catalyzed reporter deposition fluorescence in situ hybridization revealed that abundant hypolimnion-specific lineages, CL500-11 (Chloroflexi), CL500-3, CL500-37, CL500-15 (Planctomycetes) and Marine Group I (Thaumarchaeota), together accounted for 1.5–32.9% of all bacterioplankton in the hypolimnion of the lakes. Furthermore, an analysis of single-nucleotide variation in the partial 16S rRNA gene sequence (oligotyping) suggested the presence of different sub-populations between lakes and water layers among the lineages occurring in the entire water layer (for example, acI-B1 and acI-A7). Collectively, these results provide the first comprehensive overview of the bacterioplankton community in the oxygenated hypolimnion of deep freshwater lakes.

Freshwater bacterioplankton in the oxygenated hypolimnion are reportedly dominated by specific members that are distinct from those in the epilimnion. However, no consensus exists regarding the ubiquity and abundance of these bacterioplankton, which is necessary to evaluate their ecological importance. The present study investigated the bacterioplankton community in the oxygenated hypolimnia of 10 deep freshwater lakes. Despite the broad geochemical characteristics of the lakes, 16S rRNA gene sequencing demonstrated that many predominant lineages in the hypolimnion were shared by several lakes and consisted of members occurring in the entire water layer and members specific to the hypolimnion. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) revealed that representative hypolimnion-specific lineages, CL500-11 (Chloroflexi), CL500-3, CL500-37, CL500-15 (Planctomycetes), and the MGI group (Thaumarchaeota), together accounted for 1.5-32.9% of all bacterioplankton in the hypolimnion of the lakes. Furthermore, an analysis of micro-diversification based on single-nucleotide variation in the partial 16S rRNA gene sequence (oligotyping) suggested the presence of hypolimnion-specific ecotypes among the lineages occurring in the entire water layer (e.g., acI and Limnohabitans). Collectively, these results demonstrate the uniqueness, ubiquity, and quantitative significance of bacterioplankton in the oxygenated hypolimnion, motivating future studies to focus on their eco-physiological characteristics.

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 approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

Lake Tazawa, the deepest lake (423.4 m depth at maximum) in Japan underwent drastically changed water quality in 1940, because volcanic water from two active volcanos was then drawn into the lake for power generation and irrigation. Thereby, the pH of lake water decreased from 6.7 to 4.2, which exterminated a land-locked type of sockeye salmon, Oncorhynchus nerkakawamurae (locally called Kunimasu trout). Additionally, the mean residence time of lake water changed from 195 years to 8.9 years by rapidly increasing the outflow for power generation and irrigation. In this study, long-term chemical fluxes controlling lake water chemistry were obtained, and a groundwater water cycle system between the lake and the volcano was explored by estimating hydrological and chemical budgets of the lake. In the chemical budget estimate, two ionic species, SO42− and Cl−, in volcanic fluids were chosen and each mass conservation equation was yielded. The hydrological budget estimate gave us the net groundwater inflow at −1.36 m3/s on average over three periods in 2020–2021, and then the simultaneous equation coupled with the chemical budget equation allowed us to separate into groundwater inflow and outflow at 6.01 m3/s and 7.37 m3/s, averaged over the three periods, respectively. The evaluated groundwater inflow and outflow were compared with those of the other crater or caldera lakes. The linear relationship between the lake volume and the magnitude of groundwater inflow or outflow suggests that the groundwater cycle scale in such a lake increases with the magnitude of the volcanic eruption to have formed the lake.

Kuttara Volcano, Hokkaido, Japan, consists of temperate Lake Kuttara and the western Noboribetsu geothermal area. In order to explore geothermal relations between Lake Kuttara and the geothermal area, the heat budget of a hydrothermal pond, Okunoyu, was evaluated, and the heat storage change in the lower layer of Lake Kuttara was calculated by monitoring the water temperature at the deepest point. The lake water temperature consistently increased during the thermal stratification in June–November of 2013–2016. The heat flux QB at lake bottom was then calculated at a range of 4.1–10.9 W/m2, which is probably due to the leakage from a hydrothermal reservoir below the lake bottom. Meanwhile, the heat flux HGin by geothermal groundwater input in Okunoyu was evaluated at 3.5–8.5 kW/m2, which is rapidly supplied through faults from underlying hydrothermal reservoirs. With a time lag of 5 months to monthly mean QB values in Lake Kuttara, the correlation with monthly mean HGin in Okunoyu was significant (R2 = 0.586; p < 0.01). Applying Darcy’s law to the leakage from the hydrothermal reservoir at 260–310 m below the lake bottom, the time needed for groundwater’s passage through the media 260–310 m thick was evaluated at 148–149 days (ca. 5 months). These findings suggest that the hydrothermal reservoir below lake bottom and the underlying hydrothermal reservoirs in the western geothermal area are both connected to a unique geothermal source in the deeper zone as a geothermal flow system of Kuttara Volcano.