1. INTRODUCTION

In Chinese coastal areas, dense blooms caused by an unknown dinoflagellate species occurred in April of 2006, September and October of 2009 and August of 2011, and the outbreaks of blooms caused serious economic losses in fisheries industries (Hu et al. 2021 and references therein). The organism responsible for the dense blooms was initially identified as Cochlodinium germinatum (Schütt) Schütt by Qi et al. (2009), and then this species was independently named as Cochlodinium sp. (Wang et al. 2011), and subsequently transferred to the genus Polykrikos as Po. germinatus (Schütt) Qui et Lin (Qiu et al. 2013). Recently, Hu et al. (2021) proposed this organism as a new genus and species, Pseudocochlodinium profundisulcus Zhang Hu, N. Xu, H. Gu, M. Iwataki, T. Takahashi, Y. Z. Tang & K. Matsuoka, based on detailed morphological observations and phylogenetic data of strains and resting cysts collected in China and Japan.

Many harmful dinoflagellates produce resistant resting cysts as part of their life cycle, and the resting cyst stage can play an important role in both the initiation and decline of harmful algal blooms (HABs) (e.g. Anderson 2012;Li et al. 2015;Hu et al. 2021). The blooms or harmful effects caused by P. profundisulcus have not yet been recorded in Korean coastal area, however morphologically similar resting cysts (described as Cochlodinium cf. polykrikoides-like resting cyst) to that of P. profundisulcus described by Hu et al. (2021) have been reported (Park 2007;Shin et al. 2011). This indicates that P. profundisulcus may be considered as being potentially harmful in Korean coastal area.

Hence, the resting cysts were isolated from Korean coastal sediments, and the germling cells were identified by morphological observation and phylogenetic analysis. Based on the obtained results, this study documents for the first time the occurrence of harmful dinoflagellate P. profundisulcus in Korean coastal area.

2. MATERIALS AND METHODS

2.1. Sediment collection and culture establishment

Surface sediment samples were collected at a station in the Tongyeong coastal area, Korea (34°45ʹ99ʺN, 128°12ʹ36.00ʺE), using a gravity corer. The top 2 cm of the core samples were sliced and preserved immediately in dark and cool conditions at 4°C prior to further analysis. The samples were analyzed basically in accordance with the panning methods of Matsuoka and Fukuyo (2000); however, the sediment suspension in the present study was treated using screens with mesh sizes of 125 and 10 μm. Cochlodinium cf. polykrikoides-like resting cyst described by Matsuoka and Fukuyo (2000) were isolated using an inverted microscope (ECLIPSE Ti; Nikon, Tokyo, Japan) and its morphological features were recorded using a digital camera. The isolated cysts were inoculated into individual wells of 96 well tissue culture plates (SPL, Seoul, South Korea) filled with f/2-Si culture medium (salinity of 35) and were cultured at 20°C and ca 100 μmol photons m^-2 s^-1 cool-white illumination under a 14L : 10D photo-cycle. The germinated cells were transferred to individual wells of six well culture plates, and after sufficient growth the cells were transferred to a culture flask (70025; SPL Life Science, Korea) containing 25 mL sterile f/2-Si culture medium. A strain (LIMSPS- 3456) was established successfully and deposited in the Library of Marine Samples, Korea Institute of Ocean Science and Technology, and a specimen (MABIK PD00002407) was deposited in the National Marine Biodiversity Institute of Korea (MABIK).

2.2. Light microscopy (LM)

Living cells of the strains were photographed at ×1,000 magnification using an ultra-high resolution digital camera (DS-Ri2; Nikon, Japan) on an upright microscope (ECLIPSE Ni; Nikon, Japan). Cell size was measured based on LM images. For fluorescence microscopy, approximately 2 mL of culture was transferred to a 5 mL microcentrifuge tube, and SYTOX^® Green Nucleic Acid Stain (Molecular Probes, Eugene, OR, USA) was added at a final concentration of 1.0 μM. The cells were incubated in the dark at room temperature for 10 min. The cells were observed through a Dual excitation filter block FITC-TRITC specifications (excitation: BP 483-553, emission: BP 517-600) and photographed using a Digital Sight 10 digital camera on an upright microscope (ECLIPSE Ci; Nikon, Japan).

2.3. Scanning electron microscopy (SEM)

For SEM, 15 mL of mid-exponential batch culture was fixed by the addition of an equal volume of 1% osmium tetroxide (Sigma, St Louis, MO, USA) prepared in deionized water for 30 min at room temperature. The fixed cells were rinsed twice with deionized water and dehydrated in a graded ethanol series (10-99% in six steps) and a chemical preparation method using hexamethyldisilazane (HMDS) was employed to dry the germlings. The filters were mounted on stubs, coated with platinum-palladium, and were examined with a field emission scanning electron microscope (JSM 7600F; JEOL, Tokyo, Japan).

2.4. DNA extraction and sequencing

Genomic DNA was extracted from 2 mL of the culture using the DNeasy^® Plant Mini Kit (QIAGEN Inc., Valencia, California USA), following the manufacturer’s instructions. The partial LSU rRNA gene sequence was amplified using the primer pairs 25F1 and R2 (Yamaguchi and Horiguchi 2005;Takano and Horiguchi 2006). The PCR was conducted using a Thermal Cycler (Mastercycler^® nexus; Eppendorf, Hamburg, Germany) at 95℃ for 2 min, followed by 30 cycles of denaturation at 95℃ for 20 sec, annealing at 55℃ for 1 min, and extension at 72℃ for 1 min. The reaction was completed with a final elongation at 72℃ for 5 min. The PCR products were confirmed by 1% agarose gel electrophoresis. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN). The cycle sequencing reaction was performed using the ABI PRISM BigDye^TM Terminator Ver. 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA).

2.5. Sequence alignment and phylogenetic analysis

Sequences were viewed and assembled in DNABaser version 5.0 (http://www.dnabaser.com). Contigs were imported into Mafft online version 7.0 (http://mafft. cbrc.jp/alignment/server) and aligned using MEGA version 7.0 with default settings, excluding poorly aligned positions. The separate alignments were then checked using sequenceMatrix version 1.8 (Vaidya et al. 2011). For the analysis of LSU rRNA gene sequences the data set contained 49 taxa and consisted of 1,338 characters (including gaps introduced for alignment). Alexandrium minutum (GenBank accession number JF521634) was used as the out group. The GTR+G model of nucleotide substitution, with a gamma-distributed rate of variation across sites, was chosen. The assumed nucleotide frequencies were 0.2336 for A, 0.2147 for C, 0.2932 for G, and 0.2584 for T. The proportion of sites assumed to be invariable was 0.1660, and the rates for the variable sites were assumed to follow a gamma distribution with a shape parameter of 0.5480.

Phylogenetic trees for both datasets were constructed using maximum likelihood (ML) analyses and Bayesian inference. The ML analyses were performed using PhyML ver. 3.1 (Guindon et al. 2010). The starting tree was generated using BIONJ using optimization of the topology, branch lengths, and selected rate parameters. Bayesian inference was conducted on the LSU rRNA gene sequence alignments using MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003) under the TrN+G model taking into account six-class gamma and invariant sites. Four Markov chain Monte Carlo chains were run for 10 million generations, with sampling every 100 generations. A majority rule consensus tree was created in order to examine the posterior probabilities of each clade.

3. RESULTS AND DISCUSSION

3.1. Morphological features of a resting cyst and germling cell

Cochlodinium cf. polykrikoides-like resting cysts described by Matsuoka and Fukuyo (2000) were collected and germinated, however only one strain (LIMSPS- 3456) was established. The morphological features of a resting cyst and germling are shown in Figs. 1-3. The resting cyst in this study was ellipsoidal and brown in color, and size is 55.6 μm in length and 43.2 μm in width (Fig. 1). A large dark red body and granular contents were observed at the margin of the resting cyst (Fig. 1A-C, F and G). Cyst surface was characterized by fibrous lobed ornaments (Fig. 1A-C and E). These lobed ornaments were variable in size and arranged spirally on the cyst surface (Fig. 1E). Archeopyle was chasmic and occupied one-third of the cyst and its shape was unclear (Fig. 1D and E).

In this study, the morphological characteristics of the resting cyst were consistent with those of resting cysts from Korean, Japanese, Chinese and Russian coastal areas (Matsuoka and Fukuyo 2000;Orlova et al. 2004;Park 2007;Hu et al. 2021), and from sediments of ballast tanks (Shang et al. 2022). This cyst was previously regarded as the resting cyst of Cochlodinium polykrikoides (=Margalefidinium polykrikoides) or Cochlodinium cf. polykrikoides (e.g. Matsuoka and Fukuyo 2000;Orlova et al. 2004;Park 2007), and since then different morphological features of the resting cysts of C. polykrikoides was provided based on results of laboratory experiments and sediment sample analysis (Tang and Gobler 2012;Li et al. 2015). Recently, it has been clear that the previously described cysts belong to Pseudocochlodinium profundisulcus that was initially described as C. germinatum Schütt (Hu et al. 2021). According to Hu et al. (2021), the resting cysts of P. fundisulcus are morphologically characterized by ellipsoidal to ovoidal, reddish to dark brownish in color and irregular verrucate lobed ornaments. These morphological characteristics were also observed in the resting cyst isolated from Korean coastal sediments.

Germinated cells (strain LIMS-PS-3456) were ellipsoidal in shape, with irregular cell outline (Figs. 2, 3). Cells were yellowish in color, and the average size is 48.5±7.76 μm in length and 40.2±6.0 μm (n=50) in width. Only single cells were observed in culture. In the sells, the apex is flattened (Fig. 2B, D). The cingulum and sulcus were deep and originated at one third of the length of the of the cell from the anterior end (Fig. 2A, E-G). The shape of the apical structure complex (ASC) was unclear however the line of the ASC was observed in LM (Fig. 2D, F, G). The nucleus was spherical and located near the center or slightly above center of the cell (Fig. 2B-D, H). Chloroplasts were distributed, peripherally (Fig. 2H).

SEM photographs showed small amphiesmal vesicles on the cell surface and the sulcus deepening towards the antapical and the ASC (Fig. 3A-H). The sulcal extension on the epicone and the tranverse flagellum on the transverse furrow was clearly visible (Fig. 3A-C, E-H). The cingulum circled 1.5 times around the cell (Fig. 3B, E-H). The sulcus is narrow and deep, and circled 0.5 times, immediately diverging from the cingulum at the intersection of two furrows (Fig. 3A, B) and joining the cingulum near the antapical (Fig. 3G, H). A comma-shaped ASC that looped anticlockwise on the apex was observed, and the ASC started from the anterior end of the sulcal intrusion and ended almost at the right edge of the sulcal intrusion but did not contact it (Fig. 3A-D, F).

The morphological features of the germinated cells were completely consistent with those of Chinese isolates of P. profundisulcus described by Hu et al. (2021); this species has an open comma-shaped ASC, a wide and deep cingulum, and a deep sulcus. However, Hu et al. (2021) observed two-celled chains of P. profundisulcus both in culture and field samples, whereas in our culture only single cells were observed. In addition, average cell sizes (48.5±7.76 μm in length and 40.2±6.0 μm in width) of Korean specimen were slightly larger than those of Chinese isolates (34.3±3.1 μm in length and 31.8±2.7 μm in width) (Hu et al. 2021), indicating that the size of P. profundisulcus can be variable among specimens.

Based on the historical descriptions, Hu et al. (2021) reviewed morphological similarities and differences of Cochlodinium species (including Magalefidinium species), Polykrikos and Pheopolykrikos species with P. profundisulcus, and concluded that the Chinses specimens of P. profundisulcus are distinguished from Cochlodinium species by an open comma-shaped ASC, a wide and deep cingulum and a deep sulcus, and from Polykrikos and Pheopolykrikos species by morphology of ASC, cingular displacement, sulcal torsion and cyst morphology. However, as recent studies have revealed that the traditional criteria for identification of unarmoured dinoflagellates, such as characteristics of cingulum and sulcus, are problematic and the cells with abnormal shapes were frequently observed (e.g. Shin et al. 2019a), the cingulum and sulcus to distinguish species should be treated with caution. Actually, both Korean and Chinese specimens have irregular cell outline.

3.2. Phylogenetic positions of germling cells

The accession numbers of LSU rRNA gene sequences for strain LIMS-PS-3456 and a germling were obtained and deposited in NCBI. Maximum-likelihood (ML) and Bayesian inference (BI) analyses yield similar phylogenetic trees. The ML tree based on LSU rRNA gene sequences showed that the germlings (accession numbers OR855909 and OR855908 for LIMS-PS-3456) form a larger clade with Pseudocochodinium profundisucus (MW811440, MW811439, MG874046, MG87 4047, MG874048 and MG874049), Polykrikos germinatum (JX967270, MF445291 and KF878934), and Cochlodinium cf. germinatum (EF616462) (ML bootstrap support 99, BI posterior probability 1.0) (Fig. 4). In the phylogenetic tree, the larger clade exhibited a sister relationship with a clade consisting isolates of Polykrikos species (Po. schwartzii, Po. kofoidii and Po. cf. schwartzii) and Pheopolykrikos hartmanii. Po. tanit and Ph. hartmanii was distant from other Polykrikos species (Fig. 4).

The molecular phylogeny indicates that the Korean and Chinese specimens can be conspecific. These specimens also formed a clade with an Australian specimen of Cochlodinium cf. germinatum (EF616462). Although clear morphological descriptions of Australian specimen were not recorded, this specimen is probably conspecific with P. profundisucus. This clade is clearly distinct from the clade consisting isolates of Polykrikos species and Ph. hartmanii. This is supported by the morphological differences between those species. In particular, an open comma-shaped ASC and morphology of resting cyst are considered as key features to differentiate P. profundisucus from other allied species.

According to Hu et al. (2021), P. profundisucus has been regarded as harmful species and have a global distribution. Shang et al. (2022) suggested that P. profundisucus could be transported as resting cyst via ship’s ballast water and sediments. This is widely regarded as a very important external cause for the dispersal of harmful species. In addition, Shin et al. (2019b, 2020) suggested that in Korean, Japanese and Chinese coastal areas the harmful species such as Margalefidinium polykrikoides and Prorocentrum obtusidens can be transported by Tsushima Current that is the branch of the Kuroshio Current (see Fig. 1 in Shin et al. 2019b). This may also apply to intrusion of P. profundisucus to Korean coastal area. Consequently, monitoring studies for P. profundisucus are necessary, although blooms or harmful effects caused by this species have not yet been recorded in Korean coastal area.