INTRODUCTION

Dinoflagellates are the largest group of eukaryotic microalgae including approximately 2,000 species (Taylor et al. 2008). Most of them are planktonic, but about 10% of dinoflagellate species are known as benthic and/or epiphytic species (Taylor et al. 2008). Recently, interest in these benthic species has increased rapidly because of its harmful effect on other marine organisms and public health by producing serious toxic substances (Berdalet et al. 2017;Durán- Riveroll et al. 2019).

Genus Amphidinium Claparéde & Lachmann is naked dinoflagellates including approximately 100 species (Guiry and Guiry 2022). Most of them are autotroph and thrive worldwide in a wide variety of habitats (Dodge 1982;Murray and Patterson 2002;Jørgensen et al. 2004a;Dolapsakis and Economou-Amilli 2009). Especially, Amphidinium is known as most diverse and highly abundant in sandy intertidal zones around world (Hoppenrath 2000;Murray and Patterson 2002). In addition, it can produce toxins and make bioactive compounds (Durán-Riveroll et al. 2019). Several species, such as Amphidinium carterae and A. operculatum, have been reported to be associated with the production of hemolytic poison and a ciguatera fish poisoning (CFP), resulting in a mess fish mortality rate (Hallegraeff 1993;Nayak and Karunasagar 1997;Ge et al. 2009;Meng et al. 2010).

A. operculatum Claparéde & Lachmann is a type species of Amphidinium (Claparéde and Lachmann 1859). From the traditional description, Amphidinium was defined by small epicone compared to total cell size (Claparéde and Lachmann 1859), however, this morphological feature is insufficient to fully explain this genus taxonomically (Daugbjerg et al. 2000). A wide variety of morphological characteristics such as shape, location, presence and numbers of nuclei, chloroplasts, pyrenoids, pusules, eyespots and scales have been using to identify Amphidinium species, however, recent taxonomic studies using molecular tools indicate that Amphidinium is a polyphyletic taxon (Jørgensen et al. 2004a;Borchhardt et al. 2021). Therefore, nearly 100 species in this taxon group need to be re-classified according to more stringent criteria. Based on cladistic and molecular analyses, Amphidinium group was redefined by the species having minute left-deflected triangular or crescent-shaped epicone, and it constitutes the “true” genus Amphidinium sensu stricto ( Jørgensen et al. 2004a). The other species of Amphidinium belong to Amphidinium sensu lato group meaning a wide-range of Amphidinium. Therefore, subsequent studies have re-established new genus, such as Ankistrodinium Hoppenrath, Murray, Sparmann & Leander, Apicoporus Sparmann, Leander & Hoppenrath, Prosoaulax Calado & Moestrup, Togula Flø Jørgensen, Murray & Daugbjerg and Bindiferia Borchhardt, Chomérat, Murray & Hoppenrath, from the Amphidinium sensu lato group, and even A. testudo and A. corrugatum species belonging to group of Amphidinium sensu stricto have been re-established as a Testudodinium Horiguchi, Tamura, Katsumata & Yamaguchi species (as Testudodinium testudo and T. corrugatum) based on precise morphological and molecular phylogenetic analysis (Jørgensen et al. 2004b;Calado and Moestrup 2005;Sparmann et al. 2008;Hoppenrath et al. 2012;Horiguchi et al. 2012;Borchhardt et al. 2021).

During biodiversity investigations for benthic dinoflagellates in the intertidal zone of Jeju Island, Korea, the cells similar to Amphidinium species were observed, and a culture was established. The purpose of this study is to summarize the latest morphological taxonomic information of Amphidinium genus, and to describe the morphological details of the Amphidinium species and report on its molecular characterization based on ITS region and large subunit (LSU) rDNA sequences.

MATERIALS AND METHODS

1. Sample collection and establishment of strain culture

The sand samples including the surrounding pore waters were collected from Sinyang Beach located in Jeju Island (33°26′1.67″N, 126°55′21.88″E) in March 2019, using a trowel, and then placed into a plastic bottle of 250 mL. The collected samples were transported to the laboratory, immediately. Then 50 gram of sand sample was incubated with 100 mL of IMK media (Daigo IMK; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) at incubator of 16°C to isolate dinoflagellate cells. One mL of GeO₂ solution was added to prevent growing diatom cells (Marknam and Hagmeier 1982). Incubated sample was checked every two days of interval. After an incubation of 8 days, the cells similar to Amphidinium species were observed and isolated one-by-one by using glass capillary and inoculated into 24 well plate (JET BIOFIL; Guangzhou, China) containing 2 mL of IMK media. Well plate was incubated at 16°C with a photon flux of approximately 50 μmol m^-2 s^-1 under a 12 : 12 hr light : dark cycles. After sufficient growth, they were then sequentially transferred to 12 well plate ( JET BIOFIL; Guangzhou, China) and 50 mL culture flask (JET BIOFIL; Guangzhou, China) and incubated. Finally, an unknown Amphidinium culture strain AF-1903SY-04 was established.

2. Morphological observation

The Amphidinium cells at exponential phase were fixed by glutaraldehyde solution with 2% final concentration. Fixed cells were observed at a magnification of 400× using light microscope (LM; Axioplan; Cal Zeiss, Overkochen, Germany), and micrographs were taken with digital camera (AxioCam ERc 5c; Cal Zeiss, Overkochen, Germany). The cell sizes of length and width were measured (n=5) by taken LM images. For observations via scanning electron microscope (SEM), the cells were also fixed for 1 hr by glutaraldehyde solution with 2% final concentration. Then, it was post-fixed to Osmium tetroxide (OsO₄) for 10 mins. The fixed cells were filtered through diameter 2-μm of pore size polycarbonate membrane filter (25 mm diameter, Whatman, Floreham Park, NJ, USA) and washed with filtered seawater, 50% diluted filtered seawater and distilled water for each 15 mins, subsequently. Then, cells were dehydrated in the ethanol series (10, 30, 50, 70, 90, 100 and 100%) for each 10 mins. The cells were dried using critical point dryer (CPD; EMS 3000; Electron Microscopy Sciences, Hatfield, USA) and sputter-coated with platinum (Q150R; Quarum, Laughton, U.K.), and examined at 5 kV using SEM (MIRA 3; TESCAN, Brno, Czech Republic). After observation, the morphological characteristics were com pared to the other species of Amphidinium sensu stricto and summarized.

3. DNA extraction, PCR and sequencing

Two ml of mono-clonal culture of Amphidinium species was centrifuged at 3,000 rpm for 10 mins, and mixed with 0.8 mL of extraction buffer [100 mM Tris-HCl, 100 mM Na₂-EDTA, 100 mM sodium phosphate, 1.5 M NaCl, 1% acetyl trimethyl ammonium bromide (CTAB)], and stored at minus 80°C until DNA extraction. Total genomic DNA was extracted by the modified CTAB method by Faria et al. (2014).

Small subunit (SSU) to Large subunit (LSU) rDNA sequences were amplified by the long PCR technique with a dinoflagellate-specific forward and reverse primer set, 18F01 (forward; 5′-CAC CTG GTT GAT CCT GCC AGT AG-3′) and PM28-R1318 (reverse; 5′-TCG GCA GGT GAG TTG TTA CAC AC-3′) (Ki et al. 2011). The PCR was carried out in 20 μL reaction mixtures containing 11.8 μL of sterile distilled water, 2 μL of 10× Ex PCR buffer (TaKaRa; Shiga, Japan), 2 μL of a dNTP mixture (4 mM each), 1 μL of each primer solution (final concentrations of 500 nM), 0.2 μL of Ex Taq polymerase (2.5 U), and 2 μL of template DNA sample. PCR cycling was performed on a thermal iCycler (Bio-Rad, Hercules, CA) via the following program: 94°C for 3 mins; followed by 40 cycles of 94°C for 30 secs, 55°C for 40 secs, and 68°C for 5 mins; with a final extension at 72°C for 10 mins. The resulting PCR products were electrophoresed in a 1.0% agarose gel (Promega, Madison, WI), stained with Midori Green (Nippon Genetics Europe, GmbH, Germany), and visualized on a transilluminator (Gel Doc GDS-200T; Optinity, China) under ultraviolet light. The PCR amplicons were then purified with the QIAquick PCR Purification Kit (Qiagen GmbH, Germany), and DNA sequencing reactions were run with the ABI PRISM^® BigDye^TM Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA). After that, the remaining DNA sequences were determined by primer walking. Labeled DNA fragments were analyzed on an automated DNA sequencer (Model 3700; Applied Biosystems, Foster City, CA). Editing and contig assembly of the sequence fragments were carried out in the Sequencer 4.7 software (Gene Codes, Ann Arbor, MI).

4. BLAST search and phylogenetic analysis

Obtained sequence information was searches in Basic Local Alignment Search Tool (BLAST; https://blast.ncbi. nlm.nih.gov/Blast.cgi) of the National Center for Biotechnology Information (NCBI). Then genetic similarity was compared with the other same species already uploaded in NCBI. Then phylogenetic analyses were carried out using ITS region (ITS1+5.8S+ITS2) and LSU rDNA sequences of dinoflagellates that were retrieved from the NCBI database. A data matrix of the 28S rDNA sequences (11 genera and 33 species) and ITS sequences (3 genera and 17 species) was constructed considering the BLAST search results. The sequences of data matrix were aligned in the MAFFT software (Katoh et al. 2019), and ambiguous regions were removed by considering the Gblocks server (Castresana 2000). Maximum-Likelihood (ML) analysis was conducted on an LSU (564 alignment sites) and ITS (170 alignment sites) data matrix using the GTR+G+I nucleotide substitution model with RAxML 8.2.4 (Stamatakis 2014). In this analysis, total five thousand replicates for bootstrap analyses were conducted. The phylogenetic tree was visualized in MEGA X (Kumar et al. 2018).

RESULTS AND DISCUSSION

1. Morphological features of the Korean isolate of Amphidinium fijiense

Cell sizes of strain AF-1903SY-04 were 13.0-15.0 μm in length and 10.0-13.0 μm in width. The ratio length to width was 0.8-0.9. The cells were round to elliptical shape and dorsoventrally flattened in ventral view (Fig. 1). A nucleus was located posteriorly and a pyrenoid was located in the center of the cell (Fig. 1A and 1B), but pusules did not observe. Relatively thick flagellum was observed in LM (Fig. 1C). Dividing cells formed the temporary hyaline cyst without flagella (Fig. 1D). The epicone was curved to the left, crescent shaped, very small, and had a round tip (Fig. 2A-2C). The ventral ridge was short and straight, and connected two flagella insertion points (Fig. 2A and 2B). The longitudinal flagellum was inserted in the middle of the cell (Fig. 2A). The sulcus was shallow and didn’t reach the antapex of the cell (Fig. 2B). The hypocone was larger than epicone and had an oval or circular shape (Fig. 2B and 2C). The anterior part of hypocone is symmetrical (Figs. 1A and 2B). Vertical stripes were visible in a whole body of cell (Fig. 2B-2D). Body scales of small (near 80-90 nm) ring-shape were observed on the surface of cell, and it condensed at left-side of sulcus (Fig. 2B and 2D). The cell had faint polygonal amphiesmal vesicles (Fig. 2E). The cyst-like dividing cells was surrounded by organic substance (Fig. 2F).

Amphidinium fijienseKarafas & Tomas (2017) was described first in “live rock” imported from Korotoga, Fiji, Western South Pacific. The taxonomic features of this species belong to Amphidinium sensu stricto. It has a small left-facing epicone and round, elliptical and pear-shape of cells. The Amphidinium AF-1903SY-04 isolated from the Jeju Island, Korea was close to the original description of A. fijiense, such as small, rounded tip and left-deflecting epicone, middle 1/3 longitudinal flagellum insertion, central pyrenoid, posteriorly located nucleus and reproduction within temporary hyaline cysts. The maximum cell size of the isolate of A. fijiense was smaller, but it was included in the range of cell sizes of original description. The shape of A. fijiense from Fiji varied greatly from round to oval, elliptical, or pear-shaped, however, the pear-shaped cell was not observed in Korean strain. In addition, the small body scales were not observed in original descriptions, but it was observed in Korean strain (Fig. 2D). Few numbers of dinoflagellates, such as genus Heterocapsa Stein (Iwataki et al. 2003;Iwataki et al. 2009;Benico et al. 2021) and Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara (Watanabe et al. 1990), has organic body scales at outside of plasma membrane. In case of Heterocapsa species, it should be an important identification key because of its specific and distinct three-dimensional structure (Iwataki et al. 2002), however, it is difficult to be a major morphological key to identify Amphidinium species. Scale formation is a distinct trait in Amphidinium species, but many original taxonomic studies did not describe it. Moreover, this flat and simple rounded shape of scales are similar in several species, and it occasionally could not be visible because the cell surface is covered with a gelatinous matrix (Sekida et al. 2003). Additionally, strain AF-1903SY-04 had a smooth cell surface and very faint amphiesmal vesicles (Fig. 2B-2E), however, original A. fijiense strain had rougher surface and shown more distinct polygonal amphiesmal vesicles (Karafas et al. 2017). These morphological differences between strains suggest that more stringent and consistent criteria of pretreatment may be required when observing the cell surface of Amphidinium through electron microscopy.

2. Checklist of the Amphidinium sensu stricto species

To date, the list of species belonging to Amphidinium sensu stricto are as follows:

Class Dinophyceae West et Fritsch

Order Amphidiniales Moestrup et Calado

Family Amphidiniaceae Moestrup et Calado

Genus Amphidinium Claparéde et Lachmann

Amphidinium carterae Hulburt^#,B

Amphidinium cupulatisquama Tamura et Horiguchi^B

Amphidinium eilatiensis Lee

Amphidinium fijiense Karafas et Tomas^*,B

Amphidinium gibbosum (Maranda et Shimizu)

Flø Jørgensen et Murray^#,B

Amphidinium herdmanii Kofoid et Swezy^#,B

Amphidinium incoloratum Campbell^#,B

Amphidinium magnum Karafas et Tomas^B

Amphidinium massartii Biecheler^#,B

Amphidinium operculatum Claparéde et Lachmann^#,B

Amphidinium pagoense Phua et Wakeman^B

Amphidinium paucianulatum Karafas et Tomas

Amphidinium pseudomassartii Karafas et Tomas

Amphidinium steinii (Lemmermann) Kofoid et Swezy^#,B

Amphidinium stirisquamtum Luo, Wang et Gu^B

Amphidinium theodorei Tomas et Karafas

Amphidinium thermaeum Dolapsakis et Economou-Amilli^#

Amphidinium tomasii Karafas

Amphidinium trulla Murray, Rhodes et Flø Jørgensen^#,B

Amphidinium uduigamense Phau et Wakeman^B

Nine (marked by ^#) out of 20 species were discovered in Korea (Lee et al. 2013;Shah et al. 2013;Lee et al. 2017). The strain AF-1903SY-04 (marked by ^*) isolated in Jeju was identified as A. fijiense unrecorded species according to the morphological characteristics described above section. To date, 14 species have been reported in benthic environments of marine (marked by ^B) (Dolapsakis and Economou- Amilli 2009;Hoppenrath et al. 2014;Karafas et al. 2017;Luo et al. 2021;Phua et al. 2022), but sampling information of the other six species was not usable. According to many survey records, the populations of Amphidinium species are presumed to be more abundant in benthic environments rather than in pelagic zone (Hoppenrath 2000;Murray and Patterson 2002).

3. Key to the species of Amphidinium sensu stricto

1a. Antapical shape of hypocone is rounded··························2

1b. Antapical shape of hypocone is pointed···························9

1c. Antapical shape of hypocone varies·································13

1d. Antapical shape of hypocone is indented····· A. herdmanii

2a. Longitudinal flagellum insertion is middle of cell···········3

2b. Longitudinal flagellum insertion is posterior 1/3 of cell···8

2c. Longitudinal flagellum insertion is anterior 1/3 of cell··················································································A. theodorei

3a. Pyrenoid is present, central················································4

3b. Pyrenoid is present, lateral·····················A. pseudomassartii

3c. Pyrenoid varies······················································ A. tomasii

3d. Pyrenoid is absent········································ A. incoloratum

4a. Dividing cells are motile······················································5

4b. Dividing cells are non-motile (cyst form)·························7

5a. Hypocone is symmetrical···················································6

5b. Hypocone is asymmetrical····················· A. paucianulatum

6a. Cell has reticulated lobes·····································A. carterae

6b. Cell has 6 finger-like, lateral and peripheral ventrally projecting lobes························································A. eilatiensis

7a. Hypocone is symmetrical····································· A. fijiense

7b. Hypocone is asymmetrical··························· A. thermaeum

8a. Pyrenoids are present, central····················A. uduigamense

8b. Pyrenoid is absent·········································A. operculatum

9a. Longitudinal flagellum insertion is anterior 1/3 of cell···10

9b. Longitudinal flagellum insertion is middle of cell············· ··············································································A. massartii

9c. Longitudinal flagellum insertion is posterior 1/3 of cell·· ····································································· A. stirisquamtum

10a. Dividing cells are motile···················································11

10b. Dividing cells are non-motile (cyst form)············A. steinii

11a. Body scales is absent·························································12

11b. Body scale is present······························A. cupulatisquama

12a. Cell shape is hump-back···································A. gibbosum

12b. Cell shape is not hump-back··································A. trulla

13a. Longitudinal flagellum insertion is middle of cell············· ···············································································A. pagoense

13b. Longitudinal flagellum insertion is posterior 1/3 of cell·· ··············································································A. magnum

The 20 species belonging to Amphidinium sensu stricto can be identified through above summarized morphological taxonomic keys based on original descriptions of each species. Compared to A. carterae which is most similar in morphology, the shape of the cell, the insertion position of longitudinal flagellum, the presence and location of the pyrenoid and nucleus are similar (Table 1). The epicone shape also similar, but A. carterae has more sharpen epicone (crescent-like) (Murray et al. 2004). Most different feature is that the cells of A. fijiense produce the temporary hyaline cysts when cell divide, but A. carterae does not produce it (Murray et al. 2004). In addition, the body scales were not observed in A. carterae (Murray et al. 2004).

4. Phylogenetic position of the Korean Amphidinium fijiense

DNA sequence (from SSU to LSU rDNA regions) of the Korean Amphidinium fijiense (AF-1903SY-04) was deposited to GenBank (Accession No. OP555754). BLAST searches showed that the sequence of SSU rDNA (1,773 bp) was high similarity (99.8%) with the already-known Amphidinium fijiense (EU046336). In addition, ITS (463 bp) and LSU (1,247 bp) rDNA sequences were matched with A. fijiense (KY697937 and EU046329) isolated from Fiji, having 99.3% and 99.8% similarities, respectively.

Phylogenetic trees of separate ITS and LSU rDNA sequences showed close relationship between the strain AF-19 03SY-04 and the other A. fijiense strains (Figs. 3 and 4). In the ITS phylogenetic tree, A. fijiense, including AF-1903 SY-04 formed the same clade (93% bootstrap value), separating from other Amphidinium species (Fig. 3). Only A. uduigamense formed a different clade with Bindiferia boggay, of which genus was formerly classified within Amphidinium (Borchhardt et al. 2021). In addition, phylogenetic tree of LSU rDNA sequences showed similar branch patterns compared to those of the above ITS rDNA tree (Fig. 4). In the LSU phylogenetic tree, AF-1903SY-04 was clustered with A. fijiense, and was separated from other Amphidinium species with 100% bootstrap support. Overall, Amphidiniales was monophyletic, and was separated from Gymnodiniales. This represents that species of both Amphidiniales and Gymnodiniales are evolutionarily close, but they are highly complexed, especially in the Gymnodiniales. For example, Heterocapsa species which belonged to Peridiniales were closely located with Gymnodiniales, and Ankistrodinium group belong to the Gymnodiniales group were formerly classified as Amphidinium before 2012 (Hoppenrath et al. 2012).

Present study summarized the morphological characteristics and provided a table of identification key to species for Amphidinium sensu stricto group. As a result of the morphology and molecular analysis, Amphidinium strain AF-1903SY-04 isolated from Sinyang Beach of Jeju Island was identified as an A. fijiense which unrecorded species in Korea. Additionally, morphological characteristics of its body scale which have not been described were reported.