Phylogenetic diversity of the picocyanobacterial community from a novel winter bloom in Lake Biwa

In Lake Biwa, picocyanobacteria blooms usually occur during the summer-autumn thermal stratification period. Intriguingly, a novel bloom was detected in winter 2015-2016, in which picocyanobacterial cell density increased by one order of magnitude despite lower water temperature, suggesting the possibility that "cold-water-preference" species dominate in the picocyanobacterial community. In the present study, we investigated the phylogenetic diversity of picocyanobacteria in Lake Biwa by analyzing the 16S rRNA gene. We found that the picocyanobacterial community were highly diverse in Lake Biwa, with eight Synechococcus-related operational taxonomic units (OTUs) detected in different seasons. These OTUs fell into distinct phylogenetic groups, and the majority were closely related to clusters reported previously. Notably, OTU04, detected during the winter bloom, was highly affiliated with sequences found in a variety of lakes, such as Tibetan lakes and Lake Superior, where the water bodies generally have a low trophic state and temperature, and different concentrations of total dissolved solids. Thus, we inferred that the group containing OTU04 may be a psychrotolerant lineage that is widely distributed in oligotrophic water systems with low-intermediate salinity

Influence of potential grazers on picocyanobacterial abundance in Lake Biwa revealed with empirical dynamic modeling

Picocyanobacteria in lakes generally occur as single cells (single-celled picocyanobacteria; SPcy) or colonies (colonial picocyanobacteria; CPcy), and the latter form has been considered an adaptation to grazing pressure. In addition to direct effects of grazing, grazers may also have important indirect effects on picocyanobacteria, such as those from nutrient regeneration and trophic cascades. Interactions between picocyanobacteria and their grazers in lakes can thus be complex and difficult to predict. To evaluate the influence of various grazers on SPcy and CPcy in Lake Biwa, Japan, we followed seasonal changes in their abundances and potential grazers at 2-week intervals over 2 years. The data collected were analyzed using empirical dynamic modeling (EDM), a model-free, nonlinear time-series method. We found that heterotrophic nanoflagellates (HNF), rotifers (Keratella, Polyarthra, and Trichocerca), cladocerans, and copepods played important and differing roles in controlling the abundances of SPcy and CPcy. Notably, HNF had an apparent positive influence on SPcy abundance, despite being considered major consumers of SPcy. This result suggested that the enhancement of SPcy growth due to nutrient regeneration by HNF might exceed losses from mortality due to grazing by HNF. EDM also suggested that colony formation by picocyanobacteria may be unidirectional, with SPcy tending to form CPcy. Our findings show that the seasonal dynamics of SPcy and CPcy in Lake Biwa are influenced by a variety of grazers, which may play differing ecological roles in the aquatic food web

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 Lake Biwa, Japan. 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 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 marine 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 shows 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

The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa

<p>Abstract</p> <p>Background</p> <p>The cosmopolitan freshwater snail <it>Physa acuta </it>has recently found widespread use as a model organism for the study of mating systems and reproductive allocation. Mitochondrial DNA phylogenies suggest that <it>Physa carolinae</it>, recently described from the American southeast, is a sister species of <it>P. acuta</it>. The divergence of the <it>acuta/carolinae </it>ancestor from the more widespread <it>P. pomilia </it>appears to be somewhat older, and the split between a hypothetical <it>acuta/carolinae/pomilia </it>ancestor and <it>P. gyrina </it>appears older still.</p> <p>Results</p> <p>Here we report the results of no-choice mating experiments yielding no evidence of hybridization between <it>gyrina </it>and any of four other populations (<it>pomilia, carolinae</it>, Philadelphia <it>acuta</it>, or Charleston <it>acuta</it>), nor between <it>pomilia </it>and <it>carolinae</it>. Crosses between <it>pomilia </it>and both <it>acuta </it>populations yielded sterile F1 progeny with reduced viability, while crosses between <it>carolinae </it>and both <it>acuta </it>populations yielded sterile F1 hybrids of normal viability. A set of mate-choice tests also revealed significant sexual isolation between <it>gyrina </it>and all four of our other <it>Physa </it>populations, between <it>pomilia </it>and <it>carolinae</it>, and between <it>pomilia </it>and Charleston <it>acuta</it>, but not between <it>pomilia </it>and the <it>acuta </it>population from Philadelphia, nor between <it>carolinae </it>and either <it>acuta </it>population. These observations are consistent with the origin of hybrid sterility prior to hybrid inviability, and a hypothesis that speciation between <it>pomilia </it>and <it>acuta </it>may have been reinforced by selection for prezygotic reproductive isolation in sympatry.</p> <p>Conclusions</p> <p>We propose a two-factor model for the evolution of postzygotic reproductive incompatibility in this set of five <it>Physa </it>populations consistent with the Dobzhansky-Muller model of speciation, and a second two-factor model for the evolution of sexual incompatibility. Under these models, species trees may be said to correspond with gene trees in American populations of the freshwater snail, <it>Physa</it>.</p

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 [ 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 and identification of potentially toxic cyanobacteria: Ubiquitous distribution of Microcystis aeruginosa and Cuspidothrix issatschenkoi in Japanese lakes

We studied the frequency and composition of potentially toxic cyanobacteria in 30 samples from 25 Japanese lakes using automated rRNA intergenic spacer analysis (ARISA) and eight primer sets for nodularin, microcystin, cylindrospermopsin, anatoxin-a, and saxitoxin synthetase genes. Potential microcystin- and anatoxin-a-producers were detected in 25 and 7 samples, respectively. Cylindrospermopsin- or saxitoxin-producers were not detected. PCR and clone library analyses indicated that Microcystis was the sole microcystin-producing genus. Moreover, potential microcystin-producing Microcystis strains were detected in 25 of 26 samples which included Microcystis ARISA fragments, suggesting that toxic Microcystis is ubiquitous. Potential anatoxin-a-producers detected in the samples were estimated to be Cuspidothrix issatschenkoi (synonym: Aphanizomenon issatschenkoi) from clone library analyses of the anatoxin-a biosynthesis gene cluster and 16S–23S rDNA intergenic spacer region. Anatoxin-a-producing strains of C. issatschenkoi are known from two lakes in New Zealand and Germany. In contrast, C. issatschenkoi was the second most common toxic cyanobacteria in our study. This is the first report of potential anatoxin-a-producing C. issatschenkoi strains in Asia

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

Identification of species and genotypic compositions of Cryptomonas (Cryptophyceae) populations in the eutrophic Lake Hira, Japan, using single-cell PCR

Single-cell PCR and gene sequencing were conducted to evaluate species and genotypic compositions of Cryptomonas in the eutrophic Lake Hira, Japan. We determined the sequences of nuclear internal transcribed spacer 2 region from single Cryptomonas cells with a high success rate (83.3–97.9 %), excluding one case (56.3 %). A total of 325 sequences were obtained over eight sampling days from May 28, 2012, to October 3, 2012, and phylogenetic analysis indicated that all sequences were divided into six groups. Four groups were clustered together with known sequences of C. curvata, C. marssonii, C. pyrenoidifera or C. tetrapyrenoidosa, although the sequences of the other two groups did not show high similarity to known Cryptomonas species. Cryptomonas curvata dominated during the study period (45–98 %), and unidentified Cryptomonas species (group 2) became dominant at high water temperatures. The genotypic composition of C. curvata also varied temporarily, suggesting that the genotypic composition of Cryptomonas was susceptible to environmental changes. These results indicated that single-cell PCR can be used to analyze the species composition and ecology of Cryptomonas

Grazing impact on the cyanobacterium Microcystis aeruginosa by the heterotrophic flagellate Collodictyon triciliatum in an experimental pond

We estimated the grazing impact of the heterotrophic flagellate Collodictyon triciliatum on the harmful, bloom-forming cyanobacterium Microcystis aeruginosa in an experimental pond during a Microcystis bloom from summer to winter in 2010. For these experiments, we calculated the grazing rates from the digestion rate of C. triciliatum and its food vacuole contents. During the study period, M. aeruginosa exhibited one bloom event with a maximum density of 1.1 × 10[5] cells ml[−1]. The cell density of C. triciliatum fluctuated from below the detection limit to 291 cells ml[−1]. The number of M. aeruginosa cells ingested by C. triciliatum food vacuoles ranged between 0.4 and 10.8 cells flagellate[−1], and the digestion rate of C. triciliatum at 25 °C was 0.73 % cell contents min[−1]. The grazing rate of C. triciliatum on the M. aeruginosa prey was 0.2–6.9 cells flagellate[−1] h[−1], and its grazing impact was 0.0–25.3 % standing stock day[−1]. The functional response of C. triciliatum to the M. aeruginosa prey followed the Michaelis–Menten model of significance (r[2] = 0.873, p < 0.001) in our experimental systems, in which the prey concentration varied from 1.0 × 10[4] to 2.1 × 10[6] cells ml[−1]. The maximum grazing rate was 6.2 prey cells grazer[−1] h[−1], and the half-saturation constant was 1.2 × 10[5] cells ml[−1]. We present evidence that C. triciliatum grazing explained the remarkable decrease in M. aeruginosa cell density in the pond. The present study is the first demonstration of the high potential of protistan grazing on M. aeruginosa to reduce cyanobacterial blooms