Sang-Rae Lee

Monitoring of phytoplankton community structure using terminal restriction fragment length polymorphism (T-RFLP)

To establish molecular monitoring for the phytoplankton community in aquatic ecosystems, we analysed the terminal restriction fragment length polymorphism (T-RFLP) of small subunit ribosomal RNA gene (18S rDNA) sequences of nuclear genomes from the algal strains of culture collections and environmental samples of two freshwater reservoirs (Sangcheon reservoir and Seoho reservoir, Korea). Terminal restriction fragment (T-RF) length database was also constructed from twelve strains of algal culture collections to annotate and identify the phytoplankton species from T-RFLP profiles. Algal species in reservoirs were identified and monitored through the colony sequencing and T-RF length patterns of 18S rRNA. In this study, 41 unique clones were identified from two reservoirs including Chlorophyta, Cryptophyta, and Alveolata. In the case of Cryptomonas sp., we found significant linear relationships between T-RF peak areas and biovolumes by cell counting. Our results suggest that T-RFLP analysis can be a fast and quantitative monitoring tool for species changes in phytoplankton communities.

Effective molecular examination of eukaryotic plankton species diversity in environmental seawater using environmental PCR, PCR-RFLP, and sequencing

Phytoplankton are primary producers and can be important indicators of environmental change. To monitor the plankton species composition of environmental seawater samples, we developed a molecular method composed of colony polymerase chain reaction (PCR), polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and sequencing. A clone library of the ribosomal small subunit RNA gene (18S rDNA) in the nuclear genome was constructed by environmental PCR using a newly designed primer set and clones were directly amplified by colony PCR. To select unique putative clones, we choose a PCR-RFLP method that employed two restriction enzymes (MseI and Tsp509I). After the PCR-RFLP pattern was evaluated, selected clones were sequenced and analyzed. In this study, we revealed the hidden biodiversity in environmental seawater containing a wide range of taxonomic groups in the Alveolata (Ciliphora and Dinophyceae), Euglenozoa, Stramenopiles (Bacillariophyta), and Viridiplantae (Chlorophyta) without the need to conduct extensive colony isolation techniques. Moreover, we found species of fungi and Metazoa (Arthropoda, Annelida, and Mollusca). Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples and provide useful information regarding the dynamics of the eukaryotic plankton community structure.

Metagenomic examination of diversity within eukaryotic plankton from the ulleung basin in the East Sea of Korea

We constructed planktonic 18S rDNA clone libraries for micro-organisms in the euphotic zone of the Ulleung Basin in the East Sea of Korea. They revealed the cryptic biodiversity of eukaryotic planktons and demonstrated specific phylogenetic affiliations at certain water depths. Dinoflagellate clones dominated at the surface and 75-m depth. Members of the Syndiniales order (Groups I and II), containing wellknown parasitic dinoflagellates, as well as one their prominent hosts, Polycystinea, were found exclusively at 75 m. These observations suggest that the presence of species from Groups I and II is closely related to the specific water mass. Therefore, their metagenomics information might be useful for delineating both water-mass and physico-chemical properties.

Dynamic genetic features of eukaryotic plankton diversity in the Nakdong River estuary of Korea

Estuaries are environments where freshwater and seawater mix and they display various salinity profiles. The construction of river barrages and dams has rapidly changed these environments and has had a wide range of impacts on plankton communities. To understand the dynamics of such communities, researchers need accurate and rapid techniques for detecting plankton species. We evaluated the diversity of eukaryotic plankton over a salinity gradient by applying a metagenomics tool at the Nakdong River estuary in Korea. Environmental samples were collected on three dates during summer and autumn of 2011 at the Eulsukdo Bridge at the mouth of that river. Amplifying the 18S rDNA allowed us to analyze 456 clones and 122 phylotypes. Metagenomic sequences revealed various taxonomic groups and cryptic genetic variations at the intra- and inter-specific levels. By analyzing the same station at each sampling date, we observed that the phylotypes presented a salinity-related pattern of diversity in assemblages. The variety of species within freshwater samples reflected the rapid environmental changes caused by freshwater inputs. Dinophyceae phylotypes accounted for the highest proportion of overall diversity in the seawater samples. Euryhaline diatoms and dinoflagellates were observed in the freshwater, brackish and seawater samples. The biological data for species composition demonstrate the transitional state between freshwater and seawater. Therefore, this metagenomics information can serve as a biological indicator for tracking changes in aquatic environments.

Eukaryotic Plankton Species Diversity in the Western Channel of the Korea Strait using 18S rDNA Sequences and its Implications for Water Masses

Organisms entering the East Sea (Sea of Japan) through the Korea Strait, together with water, salt, and energy, affect the East Sea ecosystem. In this study, we report on the biodiversity of eukaryotic plankton found in the Western Channel of the Korea Strait for the first time using small subunit ribosomal RNA gene (18S rDNA) sequences. We also discuss the characteristics of water masses and their physicochemical factors. Diverse taxonomic groups were recovered from 18S rDNA clone libraries, including putative novel, higher taxonomic entities affiliated with Cercozoa, Raphidophyceae, Picozoa, and novel marine Stramenopiles. We also found that there was cryptic genetic variation at both the intraspecific and interspecific levels among arthropods, diatoms, and green algae. Specific plankton assemblages were identified at different sampling depths and they may provide useful information that could be used to interpret the origin and the subsequent mixing history of the water masses that contribute to the Tsushima Warm Current waters. Furthermore, the biological information highlighted in this study may help improve our understanding about the complex water mass interactions that were highlighted in the Korea Strait.

Wart-like spot formation on the fronds of Chondrus ocellatus (Gigartinales) by a brown alga, Mikrosyphar zosterae (Ectocarpales) in Korea

A brown filamentous endophyte, Mikrosyphar zosterae, occasionally appears as an epiendophyte of seagrass in Europe and North America. In the present study, M. zosterae was first isolated from wart-like spots of the red alga Chondrus ocellatus and identified by morphological features and molecular analysis. The production of wart-like spots M. zosterae on the host C. ocellatus fronds was confirmed using Koch’s postulates and in mixed culture of the two species. Neither hypertrophic nor hyperplastic cells of host C. ocellatus were found in the wart-like spots which differed from gall composition in other species. Wart-like spot formation by brown filamentous M. zosterae on the red macroalgal fronds is reported for the first time. In the culture, three morphological types of M. zosterae were observed, which were heterotrichous, pseudoparenchymatous and monostromatic prostrate thalli. Phaeophycean hairs and reproductive organs (both unilocular and plurilocular sporangia) of M. zosterae were also observed in the culture. In conclusion, M. zosterae is a causative agent producing wart-like spots on Chondrus fronds, which is a new record in red seaweeds and was confirmed in the laboratory culture for the first time.