EUKARYOME: the rRNA gene reference database for identification of all eukaryotes.

Molecular identification of micro- and macroorganisms based on nuclear markers has revolutionized our understanding of their taxonomy, phylogeny and ecology. Today, research on the diversity of eukaryotes in global ecosystems heavily relies on nuclear ribosomal RNA (rRNA) markers. Here, we present the research community-curated reference database EUKARYOME for nuclear ribosomal 18S rRNA, internal transcribed spacer (ITS) and 28S rRNA markers for all eukaryotes, including metazoans (animals), protists, fungi and plants. It is particularly useful for the identification of arbuscular mycorrhizal fungi as it bridges the four commonly used molecular markers-ITS1, ITS2, 18S V4-V5 and 28S D1-D2 subregions. The key benefits of this database over other annotated reference sequence databases are that it is not restricted to certain taxonomic groups and it includes all rRNA markers. EUKARYOME also offers a number of reference long-read sequences that are derived from (meta)genomic and (meta)barcoding-a unique feature that can be used for taxonomic identification and chimera control of third-generation, long-read, high-throughput sequencing data. Taxonomic assignments of rRNA genes in the database are verified based on phylogenetic approaches. The reference datasets are available in multiple formats from the project homepage, http://www.eukaryome.org.

Improving taxonomic classification of marine zooplankton by molecular approach: registration of taxonomically verified 18S and 28S rRNA gene sequences.

Background: Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods: rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results: A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion: The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems.

Growth, Toxin Content and Production of Dinophysis Norvegica in Cultured Strains Isolated from Funka Bay (Japan).

The successful cultivation of Dinophysis norvegica Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL-1) for more than 20 months was achieved by feeding them with the ciliate Mesodinium rubrum Lohmann, 1908, along with the addition of the cryptophyte Teleaulax amphioxeia (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL-1 (n = 7), and 0.7 and 3.6 ng per mL-1 (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell-1 (n = 7) and 0.5 to 1.2 pg per cell-1 (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, D. norvegica exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, D. norvegica grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24-27), reaching a maximum concentration of 3000 cells per mL-1 at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL-1 and 154.7 ng per mL-1 of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL-1) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of D. norvegica, as well as insights into the maintenance and culturing of this species.

Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR.

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.

Monitoring of the toxic dinoflagellate Alexandrium catenella in Osaka Bay, Japan using a massively parallel sequencing (MPS)-based technique.

Since 2002, blooms of Alexandrium catenella sensu Fraga et al. (2015) and paralytic shellfish toxicity events have occurred almost yearly in Osaka Bay, Japan. To better understand the triggers for reoccurring A. catenella blooms in Osaka Bay, phytoplankton community was monitored during the spring seasons of 2012-2015. Monitoring was performed using massively parallel sequencing (MPS)-based technique on amplicon sequences of the 18S rRNA gene. Dense blooms of A. catenella occurred every year except in 2012, however, there was no significant correlation with the environmental parameters investigated. Plankton community diversity decreased before and middle of the A. catenella blooms, suggesting that the decline in diversity could be an indicator for the bloom occurrence. The yearly abundance pattern of A. catenella cells obtained by morphology-based counting coincided with the relative sequence abundances, which supports the effectiveness of MPS-based phytoplankton monitoring.

Toxic HAB species from the Sea of Okhotsk detected by a metagenetic approach, seasonality and environmental drivers.

During recent decades, the distribution of harmful algal bloom (HAB) species has expanded worldwide together with the increase of blooms and toxicity events. In this study, the presence of toxic HAB species in the Sea of Okhotsk was investigated based on metagenetic data collected during 6 years of weekly monitoring. Operational taxonomic units (OTUs) associated with the toxic HAB species were detected based on amplifying 18S V7-V9 and 28S D1 rRNA gene regions. In total, 43 unique OTUs associated with toxic HAB species were revealed, with 26 of those previously not reported from the Sea of Okhotsk. More OTUs belonging to dinoflagellates were detected by 18S, whereas a similar number of OTUs associated with dinoflagellates and diatoms were detected by targeting the 28S region. Species belonging to genera Alexandrium, Karenia and Karlodinium were mainly associated with OTUs under Dinophyceae, whereas Bacillariophyceae was represented by the species belonging to genus Pseudo-nitzschia. From the detected OTUs, 22 showed a clear seasonal pattern with the majority of those appearing during summer-autumn. For Alexandrium pacificum, Aureococcus anophagefferens, and Pseudo-nitzschia pungens, the seasonal pattern was detected based on both rRNA regions. Additionally, 14 OTUs were detected during all seasons and two OTUs appeared sporadically. OTUs associated with the toxic species had low relative read abundances, which together with other factors such as similar and variable morphology as well as usage of fixatives, may explain why those species have previously not been detected by light microscopy. Environmental parameters, especially water temperature, significantly (<0.05) influenced the variability in OTU relative abundances and displayed significant (<0.05) correlations with the unique OTUs. The results of this study demonstrate the usefulness of the metagenetic approach for phytoplankton monitoring, which is especially relevant for detecting toxic HAB species.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates.

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Spatio-Temporal Interdependence of Bacteria and Phytoplankton during a Baltic Sea Spring Bloom.

In temperate systems, phytoplankton spring blooms deplete inorganic nutrients and are major sources of organic matter for the microbial loop. In response to phytoplankton exudates and environmental factors, heterotrophic microbial communities are highly dynamic and change their abundance and composition both on spatial and temporal scales. Yet, most of our understanding about these processes comes from laboratory model organism studies, mesocosm experiments or single temporal transects. Spatial-temporal studies examining interactions of phytoplankton blooms and bacterioplankton community composition and function, though being highly informative, are scarce. In this study, pelagic microbial community dynamics (bacteria and phytoplankton) and environmental variables were monitored during a spring bloom across the Baltic Proper (two cruises between North Germany to Gulf of Finland). To test to what extent bacterioplankton community composition relates to the spring bloom, we used next generation amplicon sequencing of the 16S rRNA gene, phytoplankton diversity analysis based on microscopy counts and population genotyping of the dominating diatom Skeletonema marinoi. Several phytoplankton bloom related and environmental variables were identified to influence bacterial community composition. Members of Bacteroidetes and Alphaproteobacteria dominated the bacterial community composition but the bacterial groups showed no apparent correlation with direct bloom related variables. The less abundant bacterial phyla Actinobacteria, Planctomycetes, and Verrucomicrobia, on the other hand, were strongly associated with phytoplankton biomass, diatom:dinoflagellate ratio, and colored dissolved organic matter (cDOM). Many bacterial operational taxonomic units (OTUs) showed high niche specificities. For example, particular Bacteroidetes OTUs were associated with two distinct genetic clusters of S. marinoi. Our study revealed the complexity of interactions of bacterial taxa with inter- and intraspecific genetic variation in phytoplankton. Overall, our findings imply that biotic and abiotic factors during spring bloom influence bacterial community dynamics in a hierarchical manner.

Competitive advantage and higher fitness in native populations of genetically structured planktonic diatoms.

It has been shown that the planktonic diatom Skeletonema from neighbouring areas are genetically differentiated despite absence of physical dispersal barriers. We revisited two sites, Mariager Fjord and Kattegat, NE Atlantic, and isolated new strains. Microsatellite genotyping and F-statistics revealed that the populations were genetically differentiated. An experiment was designed to investigate if populations are locally adapted and have a native competitive advantage. Ten strains from each location were grown individually in native and foreign water to investigate differences in produced biomass. Additionally, we mixed six pairs, one strain from each site, and let them grow together in native and foreign water. Strains from Mariager Fjord and Kattegat produced higher biomass in native water. In the competition experiment, strains from both sites displayed higher relative abundance and demonstrated competitive advantage in their native water. The cause of the differentiated growth is unknown, but could possibly be attributed to differences in silica concentration or viruses in the two water types. Our data show that dispersal potential does not influence the genetic structure of the populations. We conclude that genetic adaptation has not been overruled by gene flow, but instead the responses to different selection conditions are enforcing the observed genetic structure.