The raphidophyte Heterosigma akashiwo is well known to be a fish-killing red tide species, and the distribution of this species in Japan has reported from Otaru (43.2°N), Hokkaido to Okinawa (26.1°N). Occurrences of the red tide of H. akashiwo have been mainly reported in the Pacific coastal areas from Honshu to Kyushu, such as Sanriku coast, Tokyo Bay, Seto Inland Sea and Shikoku. However, there is a paucity of information on dynamics of this species in northern Japan. This is the first report to reveal the seasonal distribution of H. akashiwo in Funka Bay, Hokkaido, northern Japan. Monthly surveys were conducted at two fixed stations in the inner part of the bay during the period from April 2011 to July 2013, and the cell density of H. akashiwo and environmental factors (water temperature, salinity, Chl. a, DIN, PO₄-P, and SiO₂-Si) were measured. H. akashiwo was detected every summer with the maximum cell density reaching 1.9×10⁴ cells L^-1 from 0 m depth in August 2012. At the same time, H. akashiwo also accounted for 74.4% of the total cell density of phytoplankton. On the other hand, H. akashiwo was not detected from every autumn to next spring. H. akashiwo cell density and water temperature showed a significantly positive correlation (r = 0.53), while salinity or DIN showed significantly negative correlation (r = -0.33, -0.25, respectively). It is concluded that H. akashiwo commonly appears in summer with a rise of water temperature and overwinters in cyst form. Furthermore, it is suggested that this species sometimes occupies a potential important primary producer in the bay during summer when the primary produce changes relatively at a low level.

Genetic information (GI) has been commonly used to confirm divergence between closely related species in α-taxonomy. However, there are some difficulties in the usage of GI, especially when a new species that has been formerly identified as a different species is described. We present three examples of our recent α-taxonomic studies on estuarine copepods, in which GI was used to evaluate divergence, and discuss the advantage and difficulties of applying GI in α-taxonomy. The first example is a description of the new species Pseudodiaptomus nansei, which was formerly reported as P. inopinus. Although the longest caudal seta differs between them (generally swollen in P. inopinus, but thin in P. nansei), the difference had been considered as intra-specific variation. However, we concluded that P. nansei is a distinct species by clear differences in mitochondrial DNA (mtCOI) and other morphological characters. In this example, the GI was critical for the conclusions drawn and led us to conduct a detailed morphological comparison. The second example is for two forms of P. inopinus. Our intensive faunal surveys for estuarine copepods in western Japan revealed two morphological forms of P. inopinus, for which the distributions are strictly separated into the Japan Sea coast and the Pacific-East China Sea coasts, with a boundary at northwest Kyushu. We concluded that the two forms are distinct species by morphological variations and our unpublished GI. The Japan Sea form should be identifiable to P. japonicus Kikuchi, 1928, which was described from a brackish lake on the Japan Sea coast and has been regarded as a junior synonym of P. inopinus. However, some doubt remains that the other form is really P. inopinus s. str., because there are minor morphological differences between our specimens and the original illustrations of P. inopinus. The third example is two different size forms of Oithona dissimilis in the Nansei Islands. Both mitochondrial and nuclear DNAs showed distinct divergences between the forms, indicating that they are different species. The small and large forms are similar to the two subspecies from India, O. d. dissimilis and O. d. oceanica, respectively, in regard to their habitat conditions and body size. However, it is doubtful that the two forms in the Nansei Islands are identical to these subspecies, because of minor morphological differences and a far-distant geological distribution between them. In the latter two examples, the species names of our specimens could not be completely determined, because of the difficulty in obtaining the type specimens to be compared with our specimens, especially regarding genetic analyses. Therefore, if genetic comparisons with the type material are necessary for the creation of a new species, α-taxonomy would be more difficult to practice. In previous studies, mitochondrial GI of estuarine copepods sometimes showed large intra-specific variations. If it is unsure whether the mitochondrial GI indicates intra- or inter-specific variations, a genealogical concordance approach using both mitochondrial and nuclear GI is recommended.

Molecular identification, such as with DNA barcoding, of freshwater copepods has huge potential. It may enable us to identify specimens at species level, even though the copepods in a sample lack diagnostic morphological characters (e.g., eggs and juveniles). It may also provide us with information on the prehistory of copepod species in the geographical area considered. Similarly, it may enable us to specify the donor area(s) of non-indigenous copepod species. Thus, molecular identification of freshwater copepods may be of great importance in terms of biodiversity conservation; however, the development of sequence libraries needs to be hurried as copepods are currently one of the worst represented groups in genetic databases.

Dinoflagellates are a large microalgal group, widely distributed both in marine and freshwater environments. The taxonomy of dinoflagellates has been studied on the basis of their morphology. Since many morphological variations, overlapping with other taxa, have been observed for some dinoflagellate taxa, the classification has been recognized as artificial for a long time. Recent molecular phylogenetic analyses have revealed the phylogeny of dinoflagellates. Even though the phylogeny of higher taxa has not yet been clarified, the presences of several supported clades have at least been verified. Molecular phylogeny showed the pass of evolutionary acquisition of some characteristic morphological features of dinoflagellates, so far unveiled only by morphological observations, e.g., the nematocyst obtained in the clade of Gymnodinium sensu stricto, and several different eyespot types acquired in different phylogenetic clades. Molecular phylogeny has also revealed species related to the type species in several genera, e.g., Amphidinium, Gymnodinium and Gyrodinium. Based on these results, the type and related species have been re-characterized and the diagnoses of the genera emended, while other species not related to the type species have been transferred to newly established genera, e.g., Apicoporus, Testudodinium and Togula from Amphidinium, Akashiwo, Karenia and Takayama from Gymnodinium, and Levanderina and Moestrupia from Gyrodinium. For several genera the classifications were revised based on their molecular phylogeny, however, taxonomic problems related to their phylogeny still remain. For example, in the case of the taxa for which their diagnoses were revised based on their phylogeny, e.g., Amphidinium sensu stricto, new isolates having identical morphological characters and different phylogenetic positions to previously reported Amphidinium species, are difficult to identify to any particular species. Moreover, even discrepancies in phylogenetic positions of some newly isolated thecate dinoflagellates have been demonstrated, making it hard to assign them to genera having identical thecal plate numbers due to their different phylogenetic positions. Importance of the type species and specimens collected from the type locality were therefore recognized as important for molecular and morphological characterization of genera, species and intraspecific populations of dinoflagellates.

We have studied the intra- and inter-species phylogeography of chaetognaths by molecular biological methods to understand their speciation processes in the open sea. An outline of part of our study is described here. Firstly, we sequenced and analyzed part of the mitochondrial cytochrome c oxidase subunit I gene for various chaetoganths species to clarify the genetic distances indicative of intra- and inter-species relationships. The interspecies genetic distance between Eukrohnia bathyantarctica and E. fowleri was the smallest among the analyzed species. Also, monophyly for clades that are comprised by a single morphological species was supported by high statistical values, except for in the case of E. hamata and E. bathypelagica, for which the morphological classification was not supported by our molecular analysis. On the other hand, the present study showed that morphological species often comprised more than two genetic groups where the genetic distance was larger between these groups than within that morphological species clade, and this was supported by a high bootstrap values. In some groups of several species, differences in the morphology and distribution between groups were discovered by detailed observations on the meso- and bathypelagic species. These results suggest that both classification by molecular methods and new observations on of morphology are needed to understand the real species diversity of chaetoganths.

Coral reefs have amongst richest biodiversity of all the shallow marine ecosystems. However, coral reefs are severely threatened by global warming as well as local anthropogenic stresses world-wide. Establishing proper Marine Protected Areas (MPAs) is considered as the most feasible and effective approach for the conservation of marine ecosystems. Although adults are sessile, many coral reef invertebrate species have a pelagic larval stage in their early life history; therefore the estimation of larval dispersal is essential for planning MPAs. Because planktonic larvae are very small and morphologically indistinguishable, direct tracking of larval dispersal in the vast ocean is almost impossible. In this paper, we shortly review genetic methods for estimation of larval dispersal and species delimitation, exemplifying three starfish taxa with relatively long pelagic larval duration (PLD) of 2–7 weeks as well as blue coral with a relatively short PLD (a few hours–2 weeks). Population genetic analysis of crown-of-thorns starfish, Acanthaster planci, across the Indo-Pacific Ocean using highly polymorphic microsatellite loci revealed genetic homogeneity along strong western boundary current such as the Kuroshio (over 2,600 km from the Philippines to Japan) and the Eastern Australian Current (along the Great Barrier Reef) which partly supports the secondary outbreak hypothesis via larval dispersal. Indeed, successive population outbreaks of A. planci have been chronically reported especially a few years after the approach of the Kuroshio to the Ryukyu Islands. On the other hand, significant genetic differentiation was found among remote Pacific Islands, rejecting the secondary outbreak hypothesis for these Pacific islands. Further intensive sampling together with the genotyping of multiple loci (nuclear microsatellite and mitochondrial DNA) revealed that strong gene flow occurs via larval dispersal at sites up to 200 km apart along the Society Islands in French Polynesia, implying possible secondary outbreak without strong ocean currents. Mitochondrial analysis of crown-of-thorns starfish, as well as other coral reef starfish species such as pincushion starfish and blue starfish, showed genetic discontinuity between the Indian and Pacific Oceans, implying the possible effect of sea level fluctuations during the Pleistocene. Despite distinct differences in morphology and ecology between the sibling Linckia species (Linckia laevigata and Linckia multifora), the two species shared the same haplotypes of mitochondrial DNA and could not be distinguished species by mtDNA sequences. However, population genetic analysis of the two species using microsatellite loci across Indo and Pacific Ocean indicate that morphologically different populations are weakly differentiated (Yasuda et al. in prep). While mitochondrial DNA is useful for species delimitation due to its higher mutation and sorting rate than nuclear DNA, this result highlighted the fact that caution is warranted when we are to delimit recently speciated taxa and a population-based approach covering the species-range is robust enough to examine speciation history. Population genetic analysis of blue coral, Heliopora coerulea, which has a relatively short PLD, indicated that there are at least two evolutionary different clades along the Kuroshio. Each of the two cryptic clades (possible cryptic species) distributed along the Kuroshio and tend to appear in different environments, implying some environmental and/or biological factors are associated with their distributions. Significant isolation by distance patterns were found in each genetic clade of H. coerulea, suggesting the split of the two genetic clades occurred a relatively long time ago and that each genetic clade thereafter spread out into their preferred habitats along the Kuroshio.

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A review of the current status of DNA barcoding in pelagic cnidarians is given. Most current studies tend towards using the 16S mitochondrial ribosomal RNA gene for barcoding purposes in pelagic cnidarians, judged more appropriate for this group than the mitochondrial COI gene. Although further studies on mitochondrial genome structure and the prevalence of nuclear insertions of mitochondrial sequences (NUMTs) are advised, empirically it seems that the sequence fragment of the 16S gene that is currently being used is robust enough to apply DNA barcoding to a range of outstanding questions concerning the taxonomy, ecology and biology of pelagic cnidarians.

DNA barcoding is a method, which use a portion of mitochondrial CO1 gene sequence to identify it as belonging to a particular species. More than a decade had been passed since the introduction of DNA barcoding. Active debates over its pros and cons continue up until the present. In the present manuscript, I have reviewed its advantages and disadvantage and further discussed the future perspective of the method.

Most of the protistan microplankton can be identified to species level based on the 18S rRNA gene sequences generated by previous primer sets. However, these primer sets amplify the 18S rRNA genes of not only protistan microplankton but also metazoan species, which can sometimes be abundant in field samples and thereby hampering a comprehensive analysis of the protistan microplankton. Recently, we introduced newly designed PCR primer sets and a protocol that is able to amplify a broad range of protistan microplankton taxa without any amplification of metazoan species. These primers and the protocol were evaluated a community analysis of protistan microplankton in Sagami-Bay and in the Kuroshio Current. In total, 55 genera of protistan microplankton were detected. This method could be beneficial for rapid analyses of large portion of the protistan microplankton community in various aquatic environments.

In recent days, DNA metabarcoding has enabled us to figure out the ecological community at low cost. Massively parallel sequencers extremely reinforce such ability of metabarcoding. We can also apply metabarcoding technique to elucidating predator-prey relationshps, exploring habitat of larvae, food inspection, pathogen testing and rapid exploration of unknown organisms. A variety of algorithms are used for sequence clustering/assembly, denoising, chimera removal, and taxonomic assignment in metabarcoding. A framework and standard procedure of metabarcoding are described in this review. I also provide theoretical background, brief overview and known problems of those algorithms, and future direction of metabarcoding.

All the biological information of a cell resides in its genes. Analyses of genes make it possible to clarify what kind of functions they potentially have and when those functions are turned on depending on the environmental conditions. Recent developments and applications of molecular techniques, especially genetic ones, make it possible to determine gene sequences rather easily. The introduction of “Next generation sequencers” (NGS) in particular have greatly enhanced our ability to obtain massive sequence datasets during a short period of time.

The application of molecular techniques to study on marine organisms began with microbes. There are a couple of major reasons for this. First, most marine prokaryotes are difficult or impossible to be cultured by ordinary microbiological methods. Usually less than 1％ of the total population are recovered by such methods. Therefore, techniques that do not depend on culturing methods are indispensable. Second, even cultured microbes require considerable work to identify to species, partly due to the limited morphological characters. Therefore, it takes a lot of time and laborious work to determine what types of microbes are present in nature even for culturable species. Finally, the extraction of nucleic acids is relatively easy for microbes compared with multicellular organisms. Cells can be collected by simple filtration of seawater. Since the introduction of molecular techniques in the 1980^th, molecular techniques became commonly used by marine microbiologists because of the reasons stated above.

The introduction of culture-independent molecular techniques clarified the following. First, there were many formerly unknown phylogenetic groups in the ocean. Also some groups, such as Archaea, for which their presence in marine environments had previously been unknown were found to commonly occur. Second, cultured microbes do not always represent the entirety of the population. For instance, microbes belonging to the genus Vibrio are often isolated and thoroughly investigated, but this group comprises only a very minor portion of the total population. Third, in marine environments, Bacteroidetes (phylum), alphaproteobacteria (class), gammaproteobacteria (class) usually comprise the major part of populations. In the upper water column, cyanobacteria (phylum) are also commonly found. One particular group, SAR11, that belongs to the alphaproteobacteria, is distributed quite widely over almost all part of the ocean. Fourth, the introduction of NGS clarified that in addition to some dominant phylogenetic groups, there are many minor groups existing at a low concentration of individuals. They are referred to as the, “rare biosphere”. Apparent species richness or the total number of unique sequences are dependent on those groups. It should be pointed out that it is now technically possible to recover mRNAs from the environment and analyze the sequences of such genes. This enables us to detect possible microbial functions that cannot be elucidated by DNA analyses.

The application of metagenomic approaches to investigating marine environments has revealed the presence of many formerly unknown or overlooked sequences, such as those related to nitrogen fixation, anoxygenic photosynthesis, and proteorhodopsin (PR). For example, PR was recognized to be a rhodopsin-like gene flanked with SAR86 16S rDNA. Because prokaryotic rhodopsin was believed to be present only among the Archaea, this finding quickly stimulated further analyses of its actual function and distribution among different phylogenetic groups in aquatic environments. It is now evident that PR is a proton pump powered by solar energy, and is quite widely spread among marine prokaryotes, possibly being present in more than 50％ of surface dwelling populations.

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New sequencing technologies such as the Roche 454 pyrosequencing platform have made it possible to obtain millions of sequence reads in a single experiment, and massively parallel sequencing (MPS) is currently revolutionizing the survey of eukaryotic diversity, as this technology enables the detection of thousands of operational taxonomic units (OTUs) of eukaryotes from various ecosystems and facilitates the detection of low-abundance populations in complex eukaryote communities. However, this new technology brings with it different sources of sequencing error to traditional dideoxy capillary sequencing, such as ambiguity of homopolymer length, particularly for long homopolymers. MPS of PCR amplicon libraries can lead to overestimates of microbial diversity through the generation of low-frequency, error-prone reads. For this reason, empirical studies to evaluate per-base accuracy of MPS and to identify factors that can be used to eliminate low-quality reads have been employed. The nuclear 18S-rRNA gene has been used as a metabarcoding marker in MPS-based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations of 18S-rRNA were carried out, namely, the number of sequences deposited in international nucleotide sequence databases (INSDs), and the amplicon sequence variability among the three variable regions, V1–3, V4–5, and V7–9, using in silico PCRs based on INSDs. We also examined the taxonomic identification power, using MPS-based environmental surveys of a planktonic eukaryote community, to determine which region is more useful for MPS-based monitoring. We succeeded in concluding that the V1‒3 region will be the best one to apply to MPS-based monitoring of natural eukaryote communities in future, as the registration number of sequences in INSDs increases.

In this study, we compared the eukaryote biodiversity between Hiroshima Bay and Ishigaki Island in Japanese coastal waters by using the MPS-based technique to collect preliminary data. The relative abundance of Alveolata was highest in both localities, and the second highest groups were Stramenopiles, Opisthokonta, or Hacrobia, which varied depending on the samples considered. For microalgal phyla, the relative abundance of operational taxonomic units (OTUs) and MPSs was highest for Dinophyceae in both localities, followed by Bacillariophyceae in Hiroshima Bay, and by Bacillariophyceae or Chlorophyceae in Ishigaki Island. The number of detected OTUs in Hiroshima Bay and Ishigaki Island was 290 and 236, respectively, and ca. 40％ of the OTUs were common between the two localities. In the non-metric multidimensional scaling analysis, the samples from the two localities were plotted in different positions, reflecting geographic differences in biodiversity. Thus, we succeeded in demonstrating biodiversity differences between the two localities, although the read numbers of the MPSs were generally low. The MPS-based technique shows a great advantage of high performance by detecting several hundreds to thousands of OTUs simultaneously.

Marine planktonic copepods are an ecologically important group with high species richness and abundance. Here, we propose a metagenetic approach for revealing the community structure of marine planktonic copepods using 454 pyrosequencing of nuclear large subunit ribosomal DNA. We determined an appropriate similarity threshold for clustering pyrosequencing data into molecular operational taxonomic units (MOTUs) using an artificial community containing 33 morphologically identified species. The artificial community was appropriately clustered into MOTUs at 97％ similarity, with little inflation in MOTU numbers and with relatively high species-level resolution. Next, we applied the method to field-collected samples, and the results corresponded reasonably well with morphological analysis of these communities. Numbers of MOTUs were well correlated with species richness at 97％ similarity, and large numbers of sequence reads were generally observed in MOTUs derived from species with large biomass. Further, MOTUs were successfully classified into taxonomic groups at the family level at 97％ similarity; similar patterns of species richness and biomass were revealed within families with metagenetic and morphological analyses. The metagenetic approach reported here can be an effective tool for rapid and comprehensive assessment of copepod community structure. In this paper, we also introduce an example of metagenetic community study of copepods in the tropical and subtropical Pacific.

A large number of metagenomic studies, targeting microbial communities have been published over the last decade. In contrast, only two community-based studies targeting zooplankton assemblages have been published. In the present manuscript, those two community-based zooplankton genetic studies were reviewed. Potential reasons for the lack of progress in the discipline and future perspectives on community-based zooplankton studies are discussed.