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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.42 No.4 pp.616-632
DOI : https://doi.org/10.11626/KJEB.2024.42.4.616

First report on epiphytic and toxic dinoflagellates in the seawaters of Ulleungdo and Dokdo islands, Korea

Seung Joo Moon, Hangy Lee, Jun-Ho Hyung*, Jaeyeon Park*
Environment and Resource Convergence Center, Advanced Institute of Convergence Technology, Suwon 16229, Republic of Korea
*Co-corresponding authors Jaeyeon Park Tel. 031-888-9042 E-mail. bada0@snu.ac.kr
Jun-Ho Hyung Tel. 031-888-9063 E-mail. hjh1120@snu.ac.kr

Contribution to Environmental Biology


▪ First report on the presence of epiphytic and toxic dinoflagellates in the seawaters of Ulleungdo and Dokdo islands.


▪ This study highlights information on the expansion of subtropical dinoflagellates to the central part of the East Sea.


07/12/2024 15/12/2024 20/12/2024

Abstract


Strains of epiphytic and toxic dinoflagellates were collected from the seawaters surrounding Ulleungdo and Dokdo islands in Korea, and their morphology, molecular phylogeny, and toxicity were analyzed. Each dinoflagellate strain was examined under a microscope for genus-level identification, while species-level confirmation was achieved for Amphidinium operculatum, Ostreopsis sp. (type 1), Protoceratium reticulatum, Coolia canariensis, and C. malayensis through molecular phylogenetic analysis. The genera Gambierdiscus, Heterocapsa, and Prorocentrum were identified based on morphological traits, with Gambierdiscus characterized by a round-flattened shape with distinct thecal plates, Heterocapsa exhibiting a fusiform to oval shape, and Prorocentrum showing an oblong-to-ovate shape. Toxicity assessments for five species involved exposing Artemia salina nauplii to concentration-dependent extracts of the dinoflagellates. At a concentration of 100 ppm, P. reticulatum, A. operculatum, C. canariensis, and Ostreopsis sp. resulted in significant mortality among the nauplii, with survival rates dropping to as low as 0% over a 24-hour period. These findings underscore the potential ecological and toxicological impacts of these species and highlight the necessity for further research to evaluate their behavior under varying environmental conditions. This study marks the first documentation of subtropical epiphytic dinoflagellates in the waters around Ulleungdo and Dokdo islands, encompassing twelve strains from eight epiphytic species. Additionally, we investigated the toxicity of five species, including a toxic planktonic dinoflagellate. The results suggest a potential northeastward shift in their distribution, likely originating from Jeju Island, and being transported by the Tsushima Warm Current through the East Sea. This shift has enabled the successful establishment of populations in these areas under favorable conditions influenced by changes in the oceanic climate.


epiphytic dinoflagellate , geographic distributions , East Sea , phylogeny , toxicity

초록

    1. INTRODUCTION

    Dinoflagellates are major contributors to the marine food web, playing crucial ecological roles and often being associated with harmful algal blooms and toxin events (Lessard 1991;Jeong 1999;Taylor et al. 2008). They inhabit diverse environments, ranging from tropical and subtropical to temperate and even polar regions, and exhibit remarkable ecological diversity. Dinoflagellates can be found in various habitats, including planktonic, benthic, epiphytic, and parasitic environments. Among them, many epiphytic or benthic dinoflagellates often exhibit toxigenic characteristics, with certain species such as Ostreopsis, producing palytoxin, particularly under conditions of dense cell aggregations (Usami et al. 1995;Durán-Riveroll et al. 2019). Toxic dinoflagellates were once believed to be confined to tropical waters; however, due to climate change and rising seawater temperatures, these species are increasingly found in temperate regions at higher latitudes (Wells et al. 2020;Accoroni et al. 2024).

    Diversity of epiphytic or benthic dinoflagellate in Korean coastal water has been extensively studied (Kim et al. 2011;Baek 2012;Jeong et al. 2012a, 2012b;Shah et al. 2013). Most studies have focused on the coastal waters of Jejudo Island and the eastern coast of Korea, where rising seawater temperatures are observed due to the influence of warm currents and global warming (Han and Lee 2020). Various species of benthic and epiphytic dinoflagellate have been reported, based on phylogenetic studies. Notable species include Coolia canariensis and C. malayensis (Jeong et al. 2012b), Gambierdiscus jejuensis (Jeong et al. 2012a;Jang et al. 2018), Ostreopsis cf. ovata (Kang et al. 2013;Park et al. 2020), Ostreopsis sp. type 1 (Kim and Seo 2019), Ostreopsis sp. type 6 (Lee and Park 2020), Amphidinium massartii (Lee et al. 2013), and Prorocentrum mexicanum (Lim et al. 2013). Recently, the tropical species C. palmyrensis was observed for the first time in Korean coastal waters (Hyung et al. 2024).

    The seawaters surrounding Ulleungdo and Dokdo, volcanic islands located in the East Sea of Korea, present a unique marine environment and ecosystem, making this region of significant importance for marine biological research (Lee et al. 2019, 2020a;Park et al. 2024). Previous studies have examined the distribution of dominant dinoflagellate communities across surface and deep-water layers in the Ulleung Basin using metagenomic analysis (Lee et al. 2012). Additionally, it has shown that vertical mixing during episodic strong winds increases nutrient concentrations in surface waters, resulting in spring phytoplankton blooms in oligotrophic waters (Baek et al. 2018). However, the introduction or species diversity of epiphytic and toxic dinoflagellates in the seawaters around Ulleungdo and Dokdo islands area has not been investigated so far. Recently, tropical sea urchin Lovenia elongata, which is widely distributed in the subtropical and tropical regions of the Indo- Pacific Ocean, was found in the seawater of Ulleugdo island (Lee and Lee 2023). Therefore, this study was initiated based on the high possibility of the introduction of subtropical plankton.

    Hence, we investigated the species diversity of epiphytic and toxic dinoflagellates in the seawaters of Ulleungdo and Dokdo islands, Korea. Based on morphological and molecular analyses, we identified eight dinoflagellate species, including one planktonic and seven epiphytic species, although three species were identified based on only morphological observations under light microscopy. In addition, their potential toxic effects were assessed to investigate the ecological and toxicological impacts in study regions, by exposing Artemia salina nauplii to concentration-dependent dinoflagellate extracts and measuring their survival rates. This research provides insights into the potential northeastward distributional shifts of subtropical epiphytic dinoflagellates and their implications for marine ecosystems in the middle of East Sea.

    2. MATERIALS AND METHODS

    2.1. Sampling and strain setup

    Field sampling was conducted two times, in June and September 2024 from ten sampling sites across the coastal waters, comprising eight sites in Ulleungdo (U1-U8) island and two in Dokdo (D1, D2) island (Fig. 1, Table 1). Five liters of surface seawater samples were collected: 500 mL was retained as a live sample for genus-level identification using an optical microscope (Axioskop; Zeiss, Oberkochen, Germany) and singlecell isolation, and the remaining volume was used for measuring environmental factors. Water temperature and salinity at each location were measured using a YSI Professional Plus model (Yellow Spring Instrument Co., Yellow Springs, OH, USA). Phytoplankton samples were collected by gently towing a plankton net with a mesh size of 80 μm. The collected samples were transferred to 300 mL PE bottles, mixed with 220 mL of surface seawater, and transported to the laboratory for single-cell isolation and microscopic analysis. At each site, macroalgae samples, which were dominant during the sampling period, were collected by scuba diving using clean sealable plastic bags. These samples were transferred to 1-L of polycarbonate (PC) bottles (Hardy Diagnostics, Santa Maria, CA, USA), and gently shaken over 100 times to detach epiphytic dinoflagellate cells from the macroalgal substrates. The samples were filtered through an 80 μm mesh to separate macroalgae and remove zooplankton. A total of 300 mL of the filtered samples was transported to the laboratory for the isolation and establishment of monocultured strains of epiphytic dinoflagellates, as well as for light microscopic analysis.

    The live sample transported to the laboratory was placed in a six-well plate, and sing-cell isolation was performed under a dissecting microscope (SZX10; Olympus, Tokyo, Japan). After single-cell isolations with 5 washings were established, the cells were cultured at 21°C with continuous illumination of 65 μmol m-2 s-1 photon irradiance in autoclaved, filtered seawater supplemented with diluted f/2-Si media (Guillard and Ryther 1962). After sufficient growth, the dense cultures were then transferred to 50, 250, and 500 mL polycarbonate (PC) bottles (Nalgene, USA), established as monocultured dinoflagellate strains, and subcultured monthly.

    2.2. Microscopy

    Images of the cells from strains established by singlecell isolation were captured using a light microscope (Olympus BX53; Tokyo, Japan) equipped with a digital camera (Olympus DP73) under Differential Interference Contrast (DIC) settings. Live cells from each strain were placed on slide glass for imaging, and high-magnification images were acquired at ×400.

    2.3. DNA extraction, PCR, and sequencing

    Genomic DNAs (gDNAs) were extracted from nine monocultured dinoflagellate isolates, representing the genera Amphidnium, Coolia, Ostreopsis, Protoceratium, identified through light microscopy. The isolates were collected on GF/C glass microfiber filters with a 1.2 μm pore size (Whatman® Inc., Maidstone, United Kingdom), and the filtered samples were frozen at -80°C until DNA extraction. The preserved samples were homogenized using Bel-Art® Disposable Micro Pestles, and gDNA extraction was performed using GeneAll® ExgeneTM Plant SV mini kit (GeneAll Biotechnology Co., Ltd., Korea), following the manufacturer’s instructions. For taxonomic identification based on ribosomal DNA (rDNA) sequence analysis, SSU rDNA sequences were amplified using primer pairs EukA-G18R, and 570F-EukB (Medlin et al. 1988;Weekers et al. 1994;Litaker et al. 2003). LSU rDNA regions were amplified with the primer pairs ITSF2-LSUB (Litaker et al. 2003), D1R-D3B (Nunn et al. 1996), and D2CF-1256R (Momigliano et al. 2013). The polymerase chain reaction (PCR) mixture (50 μL total volume) contained 5 μL of 10× F-Star Taq Reaction Buffer, 38.75 μL of UltraPureTM DNAse/RNAse-Free Distilled Water (Invitrogen, Carlsbad, CA, USA), 1 μL of 10 mM dNTPs, 0.25 μL of 5 U μL-1 BioFACTTM F-Star Taq DNA Polymerase (BioFACT Co., Ltd., Daejeon, Korea), 0.02 μM of each forward and reverse primer, and 3 μL of template DNA. PCR was conducted using an Eppendorf Master Cycler (Eppendorf, Hamburg, Germany) under the following thermocycling conditions: for EukAG18R, 570F-EukB, and ITSF2-LSUB, an initial denaturation at 94°C for 5 min; 40 cycles of 95°C for 30 s (denaturation), 56°C for 30 s (annealing), and 72°C for 1 min (extension); and a final extension at 72°C for 10 min. For ITSF2-ITSR2, D1R-D3B, and D2CF-1256R, the conditions included an initial denaturation at 94°C for 5 min; 40 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were purified using the AccuPrep PCR Purification Kit (BIONEER) and sent to Bionics Co., Ltd. (Daejeon, Korea) for Sanger sequencing. Sequence results were aligned and analyzed using BLAST (Basic Local Alignment Search Tool) provided by the National Center for Biotechnology Information (NCBI). Unfortunately, we were unable to obtain rDNA sequences for two strains of Gambierdiscus sp., one strain of Heterocapsa sp., and one strain of Prorocentrum sp. due to technical limitations.

    2.4. Phylogenetic analysis

    A total of 35 SSU rDNA sequences, including nine isolates from this study, were analyzed alongside publicly available sequences of A. operculatum, Ostreopsis sp., P. reticulatum, C. canariensis, and C. malayensis, as well as one outgroup sequence of Trichomonas vaginalis obtained from the GenBank database (http://www. ncbi.nlm.nih.gov). The SSU phylogenetic analysis was conducted to showcase the overall genetic diversity of dinoflagellates identified in the studied region. However, while SSU rDNA analysis provides a general overview, it is less suitable for accurate species-level identification due to the limited availability of SSU database references for epiphytic dinoflagellates. To address this limitation, we analyzed the LSU rDNA sequences separately for each genus to refine species identification and confirm their molecular relationships with closely related taxa. To confirm species identification, the D1-D3 region of LSU rDNA was analyzed for one strain each of A. operculatum, P. reticulatum, C. malayensis, along with three strains of C. canariensis and Ostreopsis sp., in comparison with closely related sequences from the respective genera. Sequence alignment was performed using the ClustalW algorithm, with all data manually trimmed and inspected for misalignments using the SILVA alignment tool and ARB software package (version 6.0.6; Pruesse et al. 2012). Phylogenetic reconstruction was conducted via Bayesian inference (BI) using BEAST 1.10.4 (Drummond et al. 2012), with evolutionary distances estimated. Consensus trees were generated and visualized using the TreeAnnotator 1.10.4 (Rambaut and Drummond 2013), and Figtree 1.4.5 (Rambaut 2009). Bayesian posterior probability values indicating statistical support were displayed on the BI tree.

    2.5. Preparation of extracts of dinoflagellates and toxicity assessment

    To investigate the toxicological impacts of species isolated from the study area, toxicity tests were conducted on five monocultured dinoflagellate species, A. operculatum, Ostreopsis sp., C. malayensis, C. canariensis, and P. reticulatum, followed by Solis et al. (1993) and Phua et al. (2021). Two liters of each monocultured dinoflagellate culture were filtered using GF/C glass microfiber filters (1.2 μm pore size, 47 mm diameter; Whatman® Inc., Maidstone, UK) to separate the cells from the seawater. The cells retained on the filters were extracted with 100% methanol (99.5% purity; Samchun Pure Chemical Co., Ltd., Korea) at room temperature (20°C) for 24 hours to prepare crude extracts of dinoflagellates. The methanol extracts were filtered through cotton wool to remove debris and concentrated using a rotary evaporator (EYELA N-1000, DPE-1220-1220 series) to remove methanol. To eliminate salts and other impurities that are abundant in marine organisms and could interfere with toxicity evaluation, the following steps were performed: (1) the concentrated extract was mixed with distilled water and methylene chloride (99.8% purity; Samchun Pure Chemical Co., Ltd., Korea) in a 1 : 1 volume ratio, separating the mixture into aqueous and organic layers; (2) the aqueous layer was partitioned with 1-butanol (99% purity; Samchun Pure Chemical Co., Ltd., Korea) in a 1 : 1 volume ratio to isolate the 1-butanol layer; and (3) the organic layer was partitioned with an 85% methanol aqueous solution and n-hexane (95% purity; Samchun Pure Chemical Co., Ltd., Korea) in a 1 : 1 volume ratio, following the Kupchan method (Kupchan et al. 1967). Finally, the 1-butanol layer from the aqueous phase and the 85% methanol aqueous layer from the organic phase were combined, concentrated, and weighed to determine the final extract mass. Artemia salina nauplii were used for potential toxicity assessment, and cultured for 48 hours were utilized in the experiments. The total volume of the experimental solution was set to 5 mL, using fresh seawater with a salinity of 30 and maintained at a room temperature of 20°C. Crude extracts of dinoflagellate were dissolved in fresh seawater with dimethyl sulfoxide (DMSO, 99.8% purity; Samchun Pure Chemical Co., Ltd., Korea) as the solvent, ensuring that DMSO did not exceed 1% of the total volume (Solis et al. 1993). For the potential toxicity assessment, the crude extracts of dinoflagellate were added to the experimental solutions at concentrations of 100 ppm, 10 ppm, and 0 ppm (control group). Each concentration was tested in duplicate, and toxicity was determined by counting the surviving A. salina nauplii after 12-hour and 24-hour exposure periods.

    3. RESULTS

    3.1. Isolated epiphytic and toxic dinoflagellate

    Twelve epiphytic dinoflagellate strains were isolated by detaching cells from macroalgae. Light micrographs of dinoflagellates isolated from the seawaters of Ulleungdo and Dokdo islands, Korea, illustrated the morphological diversity of the identified strains, representing seven genera (Fig. 2). All strains of dinoflagellates were examined under a microscope for genus-level identification, and species-level confirmation of Amphidinium operculatum, Ostreopsis sp. (type 1), Protoceratium reticulatum, Coolia canariensis, and C. malayensis was achieved through molecular phylogenetic analysis. Gambierdiscus, Heterocapsa species and Prorocentrum were identified to the genus level based on morphological characteristics, with Gambierdiscus displaying a round-flattened shape with distinct thecal plates, Heterocapsa exhibiting a fusiform to oval shape and Prorocentrum displaying oblong-to-ovate shape.

    In June 2024, several dinoflagellate species were isolated from Ulleungdo island under seawater conditions with temperatures ranging from 20.4 to 21.1°C and salinity between 33.3 and 34.9 (Table 2). A. operculatum strain ULCB2406 was collected from Cheonbu, Ulleungdo (Fig. 2A, B), while C. malayensis ULNM2406 (Fig. 2E, F) was identified at Namyang (U7). Gambierdiscus sp. ULNS2406 (Fig. 2G, H) was isolated from Naesujeon (U5), and Ostreopsis sp. was found at multiple sites, including Cheonbu (U2; ULCB2406), Namyang (U7; ULNM2406), and Naesujeon (U5; ULNS 2406) (Fig. 2K, L). Additionally, Heterocapsa sp. strain ULNM2409 was collected from Namyang (U7; Fig. 2I, J). In September 2024, additional dinoflagellate species were isolated from Dokdo and Ulleungdo islands under seawater conditions with temperatures ranging from 22.2 to 27.5°C and salinity between 32.1 and 34.7. C. canariensis DD22409-B9 (Fig. 2C, D), Gambierdiscus sp. strain DD2409 was collected from Dokdo island (Fig. 2G, H) and Prorocentrum sp. strain ULMB2409 (Fig. 2M, N) was isolated from Malbawichu, Ulleungdo (U8). Additionally, one planktonic toxic dinoflatellate strain, P. reticulatum (strain ULWD2409) (Fig. 2O, P), was isolated from phytoplankton net sample collected at Wadalri, Ulleungdo (U4).

    3.2. Phylogeny

    Among the thirteen dinoflagellates strains isolated from study area, eight strains of epiphytic strains and one planktonic strain were identified through molecular taxonomic validation using Bayesian phylogenies based on SSU and D1-D3 LSU rDNA sequences, with reference sequences obtained from GenBank. The SSU rDNA phylogenetic tree revealed that the Ulleungdo strains of Ostreopsis sp. and P. reticulatum shared higher genetic similarity with strains from Jejudo Island and Jinhae Bay, Korea, then with those from Russia and the Adriatic Sea, respectively (Fig. 3). However, slight genetic divergence was observed between the Ulleungdo strains and the Jejudo Island and Jinhae Bay strains in the LSU rDNA phylogenetic analysis (Figs. 5, 7). Despite this divergence, both strains consistently clustered within the same clade as those from the southern coastal waters of Korea. For Coolia species, the SSU phylogenetic analysis showed that the Dokdo strains of C. canariensis were more closely related to the sequence from Jejudo Island than to those from Australia (Fig. 3). The Ulleungdo strain of C. malayensis exhibited slight genetic divergence from strains in Jejudo Island and China. In the LSU rDNA analysis, however, the Ulleungdo strain of C. malayensis clustered with strains from Pohang and Jejudo Island, Korea (Fig. 6). Similarly, the Dokdo strains of C. canariensis displayed slight genetic divergence from strains in Jejudo Island and Jinhae Bay, yet clustered within the same clade as Jejudo Island strains in the LSU rDNA tree.

    3.3. Toxicity

    After 12 hours, the survival rate of Artemia salina nauplii exposed to 100 ppm of cell extracts decreased significantly for most species compared to the control group, which consistently maintained a 100% survival rate throughout the experiment (Fig. 8A). Among the tested species, A. operculatum exhibited the lowest survival rate in A. salina nauplii at 10%, followed by P. reticulatum (50%) and C. canariensis (80%). At 10 ppm, most species displayed high survival rates exceeding 70%, indicating relatively low toxicity at this concentration. At 24 hours, the toxicity at 100 ppm became more evident, as both Ostreopsis sp. and A. operculatum exhibited complete mortality, resulting in survival rates of 0% in A. salina nauplii (Fig. 8B). Similarly, C. canariensis and P. reticulatum displayed significant toxicity, with survival rates of 30%. Conversely, at 10 ppm, no toxic responses were observed in most species. In the control group (0 ppm), all species consistently maintained a 100% survival rate at both 12-hour and 24-hour intervals, confirming the stability and reliability of the experimental conditions.

    4. DISCUSSION

    Understanding changes in phytoplankton community in the East Sea is crucial for assessing the impacts of climate-induced variations on marine ecosystems. A long-term study conducted from 2001 to 2009 in the northwest Pacific, Chiba et al. (2012) demonstrated that large-scale climatic forces, such as the Pacific Decadal Oscillation, significantly influence phytoplankton community structure, highlighting the connection between climate variability and lower trophic levels. In the East Sea, encompassing our study area of Ulleungdo and Dokdo islands, previous research has primarily focused on phytoplankton responses to environmental factors, including oceanographic complexity, windstorms, and nutrient dynamics (Baek et al. 2016, 2018, 2023;Lee et al. 2020b). Recent metagenomic analyses have also revealed the cryptic biodiversity of eukaryotic plankton in this region (Lee et al. 2012). However, the identification of epiphytic dinoflagellate species inhabiting subtropical region, particularly in Ulleungdo and Dokdo islands remains largely unexplored. Since the first report of five epiphytic dinoflagellate genera, Amphidinium, Coolia, Gambierdiscus, Ostreopsis, and Prorocentrum, in Korean coastal waters (Kim et al. 2011), subsequent studies on their discovery, distribution, and ecological characteristics have predominantly focused on Jejudo Island, Korea (Jeong et al. 2012a;Kang et al. 2013;Lim et al. 2013;Shah et al. 2013;Jang et al. 2018;Kim and Seo 2019;Park et al. 2020;Kang et al. 2024). Baek (2012) confirmed the distribution and adaptations of epiphytic dinoflagellates, including Gambierdiscus spp., Prorocentrum lima, and Ostreopsis spp., in the East Sea through analyses of macroalgal samples collected from coastal waters of Korea. Park et al. (2020) examined the temporal and spatial distribution of the toxic epiphytic dinoflagellate Ostreopsis cf. ovata around Jejudo Island, Korea, including its seasonal abundance and substrate preference based on attachment rates to macroalgae, during a study spanning from 2016 to 2019.

    Recently, the northeastward migration of benthic dinoflagellates along the Korean Peninsula has been observed, with their distribution expansion potentially driven by the Tsushima Current and its branches (Lim and Jeong 2021). Notably, Coolia palmyrensis, a tropical species, was reported in Korea for the first time, with overwintering populations observed (Hyung et al. 2024). Additionally, shifting distribution patterns of C. malayensis, previously observed in Jejudo island, have now been validated along the northeastward coastal areas of the Korean Peninsula, including seawaters of Ulsan and Pohang (Hyung et al. 2024). While many studies on subtropical epiphytic dinoflagellates have focused on Jejudo island or the coastal waters of Korea Peninsula, research on marine ecological changes around Ulleungdo and Dokdo islands-located in the central East Sea and holding a critical position in understanding marine environmental changes-has been limited due to difficulties in access. This study represents the first report on the presence of subtropical epiphytic dinoflagellates in study area and extends these findings by demonstrating the presence of epiphytic dinoflagellates, including A. reticulatum, Ostreopsis sp., C. canariensis, C. malayensis, Gambierdiscus sp., Heterocapsa sp. and Prorocentrum sp., at higher latitudes in the seawaters of Ulleungdo and Dokdo islands, Korea. Furthermore, we successfully established monoclonal cultures for these species, providing valuable resources for further research into their ecology and potential impacts.

    This study revealed that the subtropical benthic epiphytic dinoflagellate strains isolated from Ulleungdo and Dokdo islands exhibit significant genetic similarity to strains from the southern coastal waters of Korea, specifically Jinhae Bay and Jejudo Island, as demonstrated through both SSU and LSU rDNA phylogenetic analysis. Notably, in the LSU rDNA phylogenetic tree, C. malayensis strains from Pohang, Jejudo Island, and Ulleungdo clustered closely, indicating a high degree of genetic similarity despite being isolated from different locations in Korea (Fig. 6). This clustering suggests the potential northeastward expansion of this species into the East Sea, likely facilitated by the Tsushima Warm Current. Notably, the genetic differences observed among the strains between Ulleungdo-Dokdo islands and those from southeastern coastal waters, including Jejudo Island, Jinhae Bay, and Pohang, are likely the result of adaptive responses during their northward migration. These adaptations appear to have been driven by environmental factors such as habitat changes, variations in seawater temperature, and salinity along their migration pathway. This highlights the dynamics of subtropical epiphytic dinoflagellates and their remarkable ability to adapt environmental shifts, potentially driven by oceanic climate change.

    Gambierdiscus jejuensis (previously reported as G. caribaeus in Jeong et al. 2012a) is the only Gambierdiscus species identified in Korea based on both morphological and molecular analysis (Jeong et al. 2012a;Jang et al. 2018). However, the Gambierdiscus species found in this study was not detected using the species-specific primers of G. jejuensis, suggesting the possibility of a novel species not yet reported in Korea.

    The planktonic dinoflagellate P. reticulatum has been reported as cosmopolitan, with occurrences documented in Japan (Satake et al. 1999), Norway (Ramstad et al. 2001), Italy (Ciminiello et al. 2003), White Sea of Russia (Vershinin et al. 2006), USA (Howard et al. 2008), China (Li et al. 2012), and Argentina (Akselman et al. 2015). In Korea, P. reticulatum has been previously reported only in the form of the resting cyst in the Saemangeum area and as DNA fragments identified through metagenomic analysis in the Jeju Strait (Park et al. 2004;Min and Kim 2023). In this study, monocultures of P. reticulatum isolated from Ulleungdo island were established and identified using morphological and molecular analysis.

    Most benthic dinoflagellates have garnered attention for their ability to produce toxins that bioaccumulate in marine organism, leading to seafood contamination (Yasumoto et al. 1987;Accoroni and Totti 2016). Among the benthic dinoflagellates identified in this study, the genera Amphidinium, Ostreopsis, and Coolia, have been extensively studied for their association with harmful algal blooms and toxicity. A. operculatum is known for producing amphidinols, potent cytotoxic properties (Mejía-Camacho et al. 2021). Ostreopsis species, such as Ostreopsis sp. type 1, are well known for producing palytoxins, which are linked to severe human intoxications (Tanimoto et al. 2013). Ostreol A and B are novel polyhydroxy compounds isolated from the dinoflagellate O. cf. ovata, specifically from strains collected in the coastal waters of Jejudo Island, Korea. These compounds exhibit significant cytotoxicity, as demonstrated in brine shrimp lethality assays (Hwang et al. 2013, 2018). C. canariensis and C. malayensis have been reported to produce neurotoxins, including cooliatoxins and yessotoxin analogs, with documented lethal effects on A. salina nauplii (Li et al. 2020;Phua et al. 2021;Miralha et al. 2023). Additionally, the planktonic dinoflagellate P. reticulatum exhibits potential toxicity through the production of yessotoxins (YTXs) and adriatoxins, although no cases of human illness associated with YTXs have been reported to date (Murata et al. 1987;Satake et al. 1997;Domínguez et al. 2010).

    The result on the toxic effect of monoclonal cultures isolated in this study showed that the five tested dinoflagellate species produce toxic substances. Furthermore, P. reticulatum, A. operculatum, C. canariensis, and Ostreopsis sp. exhibited high mortality rates in A. salina nauplii, indicating a significant toxic impact. Since Lee et al. (2014) reported the toxicity of Gambierdiscus sp. isolated from Jejudo island, it has high possibility that Gambierdiscus strain from Ulleungdo and Dokdo islands also exhibit toxicity. Further research is required to confirm this. The expansion and proliferation of toxic subtropical dinoflagellates may lead to increased toxin production, suggesting the potential impact on predators within the marine food web or affect humans through the accumulation of toxins in filter-feeding organisms.

    5. CONCLUSION

    Based on the morphological and molecular analyses, we identified eight dinoflagellate species, including one planktonic and seven epiphytic species, from the waters around Ulleungdo and Dokdo islands, Korea. The phylogenetic analyses confirmed the taxonomic placements of A. operculatum, Ostreopsis sp., P. reticulatum, C. canariensis, and C. malayensis, while the Gambierdiscus and Heterocapsa isolates were identified to the genus level based solely on morphological observations. Notably, this study represents the first documentation of subtropical epiphytic dinoflagellates occurring in Ulleungdo and Dokdo islands, Korea, highlighting that these species, carried by the Tsushima Warm Current, have migrated into the East Sea and successfully established populations in these regions as well as potential toxic impacts on the marine ecosystems. The toxicity evaluation revealed that extracts from the multiple dinoflagellates exhibited concentration-dependent lethal effects on A. salina nauplii, with A. operculatum, Ostreopsis sp., C. canariensis, and P. reticulatum showing significant toxicity at 100 ppm. These findings highlight the ecological and toxicological significance of epiphytic dinoflagellate blooms in the seawaters of Ulleungdo and Dokdo islands in the East Sea, Korea, emphasizing the first report of subtropical epibenthic dinoflagellates adapted to higher latitude than 36° at which their occurrence have been previously reported in Korea. This research enhances our understanding of the biodiversity, distribution, and ecological impact of subtropical epiphytic dinoflagellates in the East Sea, highlighting the implications of their expansion into temperate regions under climate-induced changes.

    ACKNOWLEDGEMENTS

    This study was supported by a grant (RS-2024-00 436373) awarded to J Park and funded by KIMST/Ministry of Oceans and Fisheries, Korea. We extend our gratitude to the crew members of the research vessel DOKDONURI and the researchers at the Ulleungdo- Dokdo Ocean Science Station of Korea Institute of Ocean Science and Technology for their support.

    CRediT authorship contribution statement

    SJ Moon: Writing, Methodology, Morphology, Data curation. H Lee: Writing, Methodology, Toxin analysis. JH Hyung: Writing-Review & editing, Methodology, Phylogeny. J Park: Administration, Writing-Review & editing, Resource, Investigation.

    Declaration of Competing Interest

    The authors declare no conflicts of interest.

    Figure

    KJEB-42-4-616_F1.gif

    Location of Ulleungdo and Dokdo islands in East Sea and sampling sites in Ulleungdo and Dokdo islands, Korea.

    KJEB-42-4-616_F2.gif

    Light micrographs of epiphytic dinoflagellates isolated from the seawaters of Ulleungdo and Dokdo islands, Korea: (A, B) Amphidinium operculatum ULCB2406; (C, D) Coolia canariensis DD2409-B9; (E, F) Coolia malayensis ULNM2406; (G, H) Gambierdiscus sp. DD2409; (I, J) Heterocapsa sp. ULNM2409; (K & L) Ostreopsis sp. ULNM2406; (M & N) Prorocentrum sp. ULMB2409; and (O, P) Protoceratium reticulatum ULWD2409. Scale bars represent 5 μm in all images.

    KJEB-42-4-616_F3.gif

    Bayesian phylogenetic tree based on SSU rDNA sequences, illustrating the molecular taxonomic placement of nine dinoflagellate strains isolated from Ulleungdo and Dokdo islands, Korea. Strains isolated in this study are highlighted in bold and color-coded: Amphidinium operculatum (pink), Ostreopsis sp. (green), Protoceratium reticulatum (neon green), Coolia malayensis (light green), and Coolia canariensis (cyan blue). Geographic origins of closely related sequences from other regions are labeled in parentheses. Bayesian posterior probability values are shown at the nodes, indicating statistical support for each clade. The scale bar represents 0.03 substitutions per site.

    KJEB-42-4-616_F4.gif

    Bayesian phylogenetic tree based on D1-D3 LSU rDNA sequences, illustrating the taxonomic placement of Amphidinium operculatum AICT isolated from Ulleungdo island, Korea. This strain clustered within the A. operculatum clade strong posterior probability support. Geographic origins of closely related sequences from other regions are labeled in parentheses. Posterior probability values at each node represent the statistical confidence of the clades. The scale bar indicates 0.05 substitutions per site.

    KJEB-42-4-616_F5.gif

    Bayesian phylogenetic tree based on D1-D3 LSU rDNA sequences, illustrating the taxonomic placement of Protoceratium reticulatum AICT isolated from Ulleungdo Island, Korea. This strain is grouped within Clade I, alongside other P. reticulatum sequences, supported by high posterior probability values. Geographic origins of closely related sequences from other regions are indicated in parentheses. Posterior probability values at each node represent the statistical confidence for the clades. The scale bar represents 0.05 substitutions per site.

    KJEB-42-4-616_F6.gif

    Bayesian phylogenetic tree based on D1-D3 LSU rDNA sequences, illustrating the taxonomic placement of Coolia malayensis isolated from Ulleungdo Island, Korea, and C. canariensis AICT strains isolated from Dokdo island, Korea. The isolates clustered within the C. malayensis and C. canariensis clades, respectively, with high posterior probability values. Bayesian posterior probability values are displayed at each node to indicate the statistical confidence for each clade. The scale bar represents 0.05 substitutions per site.

    KJEB-42-4-616_F7.gif

    Bayesian phylogenetic tree based on D1-D3 LSU rDNA sequences, showing the taxonomic placement of Ostreopsis sp. strains (ULNM2406, ULCB2406, and ULNS2406) isolated from Ulleungdo Island, Korea. These strains clustered within the Ostreopsis sp. type 1 clade with strong posterior probability values, distinct from other Ostreopsis species such as Ostreopsis cf. ovata. Geographic origins of closely related sequences from other regions are in parentheses. Posterior probability values are displayed at each node, indicating the statistical support for each clade. The scale bar represents 0.05 substitutions per site.

    KJEB-42-4-616_F8.gif

    Survival rates of Artemia salina nauplii exposed to dinoflagellate extracts after (A) 12 hours and (B) 24 hours of exposure at concentrations of 100 ppm (black bars) and 10 ppm (gray bars). Data are presented for six dinoflagellate species: Protoceratium reticulatum, Amphidinium operculatum, Coolia canariensis, Coolia malayensis, and Ostreopsis sp., along with the control group (0 ppm).

    Table

    Sampling locations of epiphytic dinoflagellates collected from ten sites in the seawaters of Ulleungdo (U1-U8) and Dokdo (D1, D2) islands, Korea

    Epiphytic dinoflagellates collected from the seawaters of Ulleungdo and Dokdo islands, Korea

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    Vol. 40 No. 4 (2022.12)

    Journal Abbreviation 'Korean J. Environ. Biol.'
    Frequency quarterly
    Doi Prefix 10.11626/KJEB.
    Year of Launching 1983
    Publisher Korean Society of Environmental Biology
    Indexed/Tracked/Covered By

    Contact info

    Any inquiries concerning Journal (all manuscripts, reviews, and notes) should be addressed to the managing editor of the Korean Society of Environmental Biology. Yongeun Kim,
    Korea University, Seoul 02841, Korea.
    E-mail: kyezzz@korea.ac.kr /
    Tel: +82-2-3290-3496 / +82-10-9516-1611