Uncultured bacteria affiliated with the CL500‐11 cluster (phylum Chloroflexi) were first reported from the oxygenated hypolimnion of Crater Lake (USA) as a predominant bacterioplankton, although this dominance has not been reported in other environments. In this study, we showed that CL500‐11 is also dominant in the oxygenated hypolimnion of Lake Biwa (Japan) and followed its spatiotemporal succession using fluorescent in situ hybridization. CL500‐11 cells were almost absent [< 1% of 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained cells] at the beginning of the stratification period, dominated (> 10% of DAPI‐stained cells; maximum = 16.5%) in the hypolimnion during the stratification period, and decreased to below the detection limit with the collapse of the thermocline. This pattern was observed over two annual cycles. A longitudinal assessment also showed that CL500‐11 was the dominant bacterium in the hypolimnion over the whole lake, but was generally undetectable in the stratified epilimnion. These data suggest that CL500‐11 is acclimated to the oxygenated hypolimnion and is a potentially important component of the pelagic biogeochemical cycling of the lake. A comparative analysis of 16S rRNA gene sequences revealed that almost all CL500‐11 sequences previously deposited in the database were detected from hypolimnion or holomictic water in deep oxic freshwater lakes, suggesting that the bacteria may form one of the common lineages residing in an aerobic hypolimnetic niche.

Microcystis aeruginosa is a bloom-forming cyanobacterium found in eutrophic water bodies worldwide. M. aeruginosa blooms usually occur in freshwater; however, they have also been reported to occur in brackish water. Because M. aeruginosa often produces the cyanotoxin microcystin, they are a major concern to public health and environment. Despite this, the ecology, genomic basis, and evolutionary process underlying the M. aeruginosa bloom invasion from fresh to brackish water have been poorly investigated. Hence, in the present study, we have sequenced and characterized genomes of two newly discovered salt-tolerant M. aeruginosa strains obtained from Japanese brackish water lakes (Lakes Shinji and Tofutsu). Both genomes contain a set of genes for the synthesis of osmolyte sucrose (sppA, spsA, and susA), hitherto identified in only one strain (PCC 7806) of M. aeruginosa. Chemical and gene expression analyses confirmed sucrose accumulation induced by salt. A comprehensive genetic survey of >200 strains indicated that sucrose genes are extremely rare in M. aeruginosa. Most surprisingly, comparative genome analyses of the three strains indicated extremely low genetic diversity in the sucrose genes compared with other core genome genes, suggesting very recent acquisitions via horizontal transfer. Invasion of M. aeruginosa blooms into brackish water may be a recent event triggered by anthropogenic eutrophication of brackish water.

Kinetoplastid flagellates, microscopically often detected from various aquatic environments and considered ubiquitous are seldom reported in molecular diversity studies with universal eukaryote DNA primers. To investigate this inconsistency, we examined nanoflagellate diversity in Lake Biwa, Japan by 18S rRNA gene clone libraries using universal eukaryote and kinetoplastid-specific primers. We also examined the abundance of kinetoplastids by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization. No, kinetoplastid sequences were detected in the universal eukaryote primers library from epilimnion and hypolimnion in different seasons. However, kinetoplastid flagellates were detected with kinetoplastid-specific probe from all of the seasons and contributed up to 11.9 and 36.0% of total eukaryotes in the epilimnion and hypolimnion, respectively. Thus, kinetoplastids probably are a significant, sometimes dominant, group in the hypolimnion, contributing up to 43.7% of the total flagellates. Using group-specific primers, kinetoplastid sequences were also obtained from both epilimnion and hypolimnion library. Therefore, we attributed the inconsistency to the divergent nature of 18S rRNA gene of kinetoplastids, which lead to their undetection in the universal eukaryote primer libraries. This study revealed that kinetoplastids have significant ecological importance in the hypolimnion of Lake Biwa, suggesting that these flagellates have been overlooked in other studies using universal eukaryote primers.

Kinetoplastid flagellates are generally abundant in the deep sea and recently they were even found to be dominant in the hypolimnion of a deep freshwater lake. Therefore, to understand the distribution of kinetoplastids in deep freshwater lakes, we have collected vertical samples from five lakes in Japan. The abundance of kinetoplastids was enumerated by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization, and the diversity was determined by 18S amplicon sequencing using universal eukaryote and kinetoplastid-specific primers. Kinetoplastids were abundant in the deep waters of all the lakes, contributing up to 53.6% of total nanoeukaryotes. Despite this significant contribution, kinetoplastids remain undetected by amplicon sequencing using universal primers that are widely used in eukaryotic diversity studies. However, they were detected with specific primers, and the communities were characterized by both ubiquitous and lake-specific unique OTUs. Oligotyping of a ubiquitous and dominant OTU revealed the presence of lake-specific sequence types (oligotypes). Remarkably, we also detected diplonemids (a sister group of kinetoplastids and considered to be specific in the marine habitat) using kinetoplastid-specific primers, showing their presence in freshwaters. Underestimation of kinetoplastids and diplonemids using universal primers indicates that euglenozoan flagellates are overlooked in diversity studies worldwide. The present study highlighted the importance of kinetoplastids in the hypolimnion of deep lakes, thereby indicating their role in material cycling in deep waters.

The dynamics of fluorescent dissolved organic matter (FDOM) in the large monomictic freshwater Lake Biwa (surface area 675 km 2 , maximum depth 104 m) was studied from December 2010 to December 2011. The proteinlike FDOM (FDOM T ) and dissolved organic carbon (DOC) showed epilimnetic accumulation (FDOM T from 4.42 6 0.22 quinine sulfate units [QSU] to 6.30 6 0.04 QSU; DOC from 80.8 6 2.7 mmol L 21 to 102.7 6 3.5 mmol L 21 ) between nutrient-replete winter mixing to nutrient-depleted stratified periods. This accumulation is attributed to the reduced heterotrophic activity following severe P-limitation. The positive correlation between accumulated DOC and FDOM T in the epilimnion and their uniform reduction in the hypolimnion (, 9%) suggest FDOM T as a proxy for semi-labile DOM. The humic-like FDOM (FDOM M ) generally increased with depth, a pattern similar to nutrients and total carbon dioxide (TCO 2 ), but adverse to dissolved oxygen. The significant positive correlations of FDOM M with apparent oxygen utilization (r 5 0.86, p , 0.001), TCO 2 (r 5 0.91, p , 0.001), nitrate (r 5 0.83, p , 0.001), and phosphate (r 5 0.76, p , 0.001) in the deeper layers suggest that FDOM M is formed during hypolimnetic mineralization. We estimated that , 8% of the organic carbon degraded in the hypolimnion is transferred into humic substances. The minor contribution of DOC (6.4%) to hypolimnetic mineralization suggests that production of humic substances is mainly fueled by the mineralization of sinking biogenic particles. The production and consumption of FDOM in freshwater lakes may influence the quality and bioavailability of carbon exported from these systems.