A freshwater radiation of diplonemids.

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Japanese deep freshwater lakes. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and catalysed reporter deposition-fluorescent in situ hybridization (CARD-FISH). We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to a marine clade, which are mainly isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single-cell genomes of marine diplonemids show they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.

Distribution of the Harmful Bloom-Forming Cyanobacterium, Microcystis aeruginosa, in 88 Freshwater Environments across Japan.

Microcystis aeruginosa was quantitatively surveyed in 88 freshwater environments across Japan within 3| |weeks in 2011. In order to clarify the distribution pattern of M. aeruginosa at the intra-species level, three major genotypes, which were defined by 16S-23S rRNA inter-transcribed-spacer (ITS) regions, were selectively detected using quantitative real-time PCR assays. Of the 68 sites at which the Microcystis intergenic-spacer region of the phycocyanin (IGS-PC) gene was detected, the M. aeruginosa morphotype-related genotype (MG1) dominated in 41 sites, followed by the non-toxic M. wesenbergii-related genotype (MG3). A correlation analysis showed that total nitrogen and phosphate positively correlated with the abundance of IGS-PC, which positively correlated with microcystin synthetase gene abundance. A redundancy analysis of genotype compositions showed that pH positively correlated with the dominance of MG3 and negatively correlated with MG1, i.e., both toxic and non-toxic genotypes. Our survey of Microcystis populations over a wide area revealed that MG1 is a dominant genotype in Japan.

Widespread Dominance of Kinetoplastids and Unexpected Presence of Diplonemids in Deep Freshwater Lakes.

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.

Adaptation of the Freshwater Bloom-Forming Cyanobacterium Microcystis aeruginosa to Brackish Water Is Driven by Recent Horizontal Transfer of Sucrose Genes.

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.

A massive tsunami promoted gene flow and increased genetic diversity in a near threatened plant species.

The magnitude and frequency of disturbances affect species diversity and spatial distributions, but the direct effects of large-scale disturbances on genetic diversity are poorly understood. On March 11, 2011, the Great Tohoku Earthquake in Japan caused a massive tsunami that resulted in substantial alteration of community compositions. Populations of a near-threatened tidal marsh Carex rugulosa inhabiting brackish sandbars was also affected. We found four out of six remnant C. rugulosa populations along the Pacific Ocean had become completely extinct. Newly emergent post-tsunami populations, however, had higher allelic numbers than pre-tsunami populations, indicating higher genetic diversity after the tsunami. In addition, genetic differentiation (Fst) between post-tsunami populations was significantly lower than that of pre-tsunami populations. We therefore conclude that the tsunami enhanced gene flow. Seeds of many Carex species persist for long periods in soil, which suggests that seed banks are important genetic resources for post-disturbance recovery of genetic diversity. When its brackish sandbar habitat is no longer subject to disturbance and changes to the land, C. rugulosa is outcompeted by terrestrial plant competitors and eliminated. Disturbance is a driving force for the recovery and maintenance of populations of species such as C. rugulosa-even after near-complete eradication.

Kinetoplastid flagellates overlooked by universal primers dominate in the oxygenated hypolimnion of Lake Biwa, Japan.

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.

Genotypic composition and the relationship between genotypic composition and geographical proximity of the cyanobacterium Microcystis aeruginosa in western Japan.

Microcystis aeruginosa is one of the bloom-forming harmful algae in freshwater ecosystems. We genetically characterized Microcystis populations during bloom-forming periods in various reservoirs, lakes, and ponds in Japan during 2009. Using phylogenetic analysis, we evaluated the relationship between current genotype expansions and geographic location within western Japan and intraspecific variation. Microcystis aeruginosa colonies were isolated at 15 sites and were analyzed by sequencing the 16S-23S internal transcribed spacer (ITS) region of the ribosomal operon, and the potential to produce toxins was assessed by PCR-based detection of the microcystin synthetase gene mcyG. In total, 171 colonies were separated into 41 genotypes. The highest genotypic composition was detected in the south basin of Lake Biwa and the lowest in Lagoon Iba. Cluster analysis indicated no obvious association between genotypic composition and geographic distance. Thus, clear genetic differentiation accompanied by geographic origins was not found in western Japan. The resulting neighbor-joining tree revealed 3 clusters, 2 of which contained strains that showed both nonamplification and amplification of the mcyG gene.

Seasonal dominance of CL500-11 bacterioplankton (phylum Chloroflexi) in the oxygenated hypolimnion of Lake Biwa, Japan.

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.

Detection of UVBR-sensitive and -tolerant bacteria in surface waters of the western North Pacific.

In order to evaluate the effects of solar ultraviolet radiation (UVR) on eubacterial community composition, we examined the tolerance of eubacterial phylotypes to solar UV radiation in surface waters of the western North Pacific during September 2005. Bromodeoxyuridine (BrdU), a halogenated thymine analogue, was used for labeling newly synthesized DNA in proliferating cells. Thymine dimers (TD), which are specifically formed in DNA by biologically harmful ultraviolet B radiation (UVBR; 280-315nm), were also applied to detect UVB damaged genomes selectively. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) on the labeled samples revealed that UVBR-resistant cells showing active synthesis of DNA without accumulating TD, varied among phylotypes. In addition, UVBR-sensitive band positions with TD indicated inter-specific variations in sensitivity to UVBR. Phylogenetic analysis revealed that 12 DNA sequences were classified into eight phylogenetic groups: three Roseobacter, one Sphingomonas, two Gammaproteobacteria, one Actinobacteria, one Synechococcus, two Prochlorococcus, one plastid and one another group. A UVBR-resistant phylotype was affiliated to Erythrobacter sp. (previously designated as Sphingomonas sp.), which was distributed in warmer waters from the south of Oyashio to Kuroshio regions. A UVBR-sensitive phylotype was affiliated to Pseudoalteromonas sp. in Gammaproteobacteria. Dominant heterotrophic eubacteria were composed of both sensitive and resistant phylotypes. This is the first report on TD accumulated eubacterial phylotypes in oceanic surface waters.