Journal of Oceanology and Limnology   2022, Vol. 40 issue(3): 1191-1219     PDF       
http://dx.doi.org/10.1007/s00343-021-1049-2
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

LUO Zhaohe, ZHANG Hua, LI Qun, WANG Lei, MOHAMED Hala F, LÜ Songhui, GU Haifeng
Characterization of Amphidinium (Amphidiniales, Dinophyceae) species from the China Sea based on morphological, molecular, and pigment data
Journal of Oceanology and Limnology, 40(3): 1191-1219
http://dx.doi.org/10.1007/s00343-021-1049-2

Article History

Received Feb. 6, 2021
accepted in principle Jun. 2, 2021
accepted for publication Jul. 22, 2022
Characterization of Amphidinium (Amphidiniales, Dinophyceae) species from the China Sea based on morphological, molecular, and pigment data
Zhaohe LUO1, Hua ZHANG2,3, Qun LI2, Lei WANG1, Hala F MOHAMED1,4, Songhui LÜ2,5, Haifeng GU1,6     
1 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China;
2 Research Center of Harmful Algae and Marine Biology, College of Life Science and Technology, Jinan University, Guangzhou 510362, China;
3 Shenzhen Academy of Environmental Science, Shenzhen 510008, China;
4 Botany & Microbiology Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo 11751, Egypt;
5 Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai 519000, China;
6 Observation and Research Station of Coastal Wetland Ecosystem in Beibu Gulf, Ministry of Natural Resources, Beihai 536015, China
Abstract: Amphidinium species are amongst the most abundant benthic dinoflagellates in marine intertidal sandy ecosystems. Some of them are able to produce a variety of bioactive compounds that can have both harmful effects and pharmaceutical potentials. The diversity of Amphidinium in shallow waters along the Chinese coast was investigated by isolating single cells from sand,coral,and macroalgal samples collected from 2012 to 2020. Their morphologies were subjected to examination using light microscopy (LM) and scanning electron microscopy (SEM). A total of 74 Amphidinium strains were morphologically identified,belonging to 11 species: A. carterae,A. gibbosum,A. operculatum,A. massartii,A. cf. massartii,A. fijiensis,A. pseudomassartii,A. steinii,A. thermaeum,A. theodori,A. tomasii,as well as an undefined species. The last seven species have not been previously reported in Chinese waters. Amphidinium carterae subclades I,II,and IV were found in the South China Sea,while subclade III was only found in the Yellow Sea. Threadlike body scales were observed on the surface of subclades III and V,supporting the idea that A. carterae might contain several different species. Large subunit ribosomal RNA (LSU rRNA) sequences-based phylogeny revealed two groups (Groups I and II) within Amphidinium,which is consistent with the relative position of sulcus (in touch with cingulum or not). In addition,large differences in morphology and molecular phylogeny between A. operculatum (the type species of Amphidinium) and other species,suggest that a subdivision of Amphidinium might be needed. The pigment profiles of all available strains were analyzed by high performance liquid chromatography (HPLC). Eleven pigments,including peridinin,diadinoxanthin,diatoxanthin,pheophorbide (and pheophorbide a),antheraxanthin,β-carotene,and four different chlorophylls were detected. The high pheophorbide/pheophorbide a ratio in Amphidinium implies that it may be a good candidate as a natural source of photosensitizers,a well-known anticancer drug.
Keywords: harmful algae    benthic-epiphytic dinoflagellate    phylogeny    geographic distribution    pheophorbide    photosensitizers    
1 INTRODUCTION

Dinoflagellates are an integral component of marine ecosystems. Some of them are also responsible for harmful algal blooms in the coastal zone, which have adverse effects on the marine ecosystem and public health. About 10% of the 2 500 known dinoflagellate species show benthic-epiphytic behavior through their life cycles (Hoppenrath et al., 2014). The genus Amphidinium Claparède & Lachmann is one of the most abundant members of the benthic dinoflagellates in marine intertidal and neritic sandy ecosystems (Dodge, 1982; Daugbjerg et al., 2000; Murray and Patterson, 2002). Some Amphidinium species are a source of ichthyotoxins in shoal reefs and form harmful blooms (Kobayashi et al., 1991; Lee et al., 2003; Murray et al., 2015; Gárate-Lizárraga et al., 2019). On the other hand, some Amphidinium species are able to produce a variety of bioactive compounds that exhibit antifungal or antimicrobial properties (Bauer et al., 1995; Echigoya et al., 2005; Kong et al., 2013; Nuzzo et al., 2014). Most Amphidinium species grow easily in culture, and are therefore candidates for the mass production of bioactive compounds.

Amphidinium was traditionally defined by its small epicone size (Claparède and Lachmann, 1859), which does not exceed one-third of the total cell length (Kofoid and Swezy, 1921; Steidinger and Tangen, 1997). About 200 marine and freshwater species have been described, which are either autotrophic, mixotrophic, or heterotrophic in terms of their nutrition modes, and can be pelagic or benthic (Taylor, 1971; Calado et al., 1998; Guiry and Guiry, 2021). It has long been suspected that the definition does not mirror its phylogeny (Daugbjerg et al., 2000; Saldarriaga et al., 2001), but a later emendation of the genus definition was performed after reinvestigation of A. operculatum Claparède & Lachmann, the type species of Amphidinium, as well as putative relatives of dozens of Amphidinium taxa units (Jørgensen et al., 2004; Murray et al., 2004). The genus Amphidinium sensu stricto now only includes those athecate benthic or endosymbiotic dinoflagellates with minute irregular triangular-or crescent-shaped epicones that are deflected towards the left (Jørgensen et al., 2004). Species that do not fit the criteria for the genus as redefined (Jørgensen et al., 2004; Murray et al., 2004), but have not yet been investigated to determine the generic affinities, are classified as Amphidinium sensu lato (Hoppenrath et al., 2014). A variety of morphological features have been used to identify Amphidinium species, including the cell size and shape, the nucleus location and shape, the presence, location, and number of pusules, pyrenoids, chloroplasts, eyespots, scales, as well as life cycle stages (Claparède and Lachmann, 1859; Taylor, 1971; Maranda and Shimizu, 1996; Steidinger and Tangen, 1997; Sekida et al., 2003). However, there are no unambiguous features that can be used to differentiate Amphidinium species, and some characters can even overlap among species (Murray and Patterson, 2002; Jørgensen et al., 2004; Murray et al., 2004). Therefore, a combination of characters seems to be the best approach to differentiate Amphidinium species (Karafas et al., 2017). The genus of Amphidinium sensu stricto now harbor two major clades, i.e., the Herdmanii and Operculatum clades, which are sisters to one another in large subunit ribosomal RNA (LSU rRNA) sequences-based phylogeny (Jørgensen et al., 2004; Karafas et al., 2017), although this is not reflected by their morphology. For example, within the Operculatum clade, A. operculatum has a distinctively large cell size, with a longitudinal flagellum inserted in the lower third of the cell (Claparède and Lachmann, 1859; Murray et al., 2004), while A. gibbosum (Maranda & Shimizu) Flø Jørgensen & Murray and A. trulla Murray, Rhodes & Flø Jørgensen also have a large cell size but with a longitudinal flagellum inserted in the anterior third of the cell (Maranda and Shimizu, 1996; Murray et al., 2004). The rest of the Operculatum clade have a small cell size and a longitudinal flagellum inserted in the middle third of the cell (Jørgensen et al., 2004; Karafas et al., 2017).

There are several reports of Amphidinium sensu stricto along the Chinese coast. Amphidinium strains isolated from Lingshui Bay, Hainan, have been reported to produce polyhydroxyl compounds, with strong cytotoxic activity (Huang et al., 2004a, b ), but their taxonomic identity has not been determined. The toluene extract of a strain of A. carterae Hulburt isolated from Sanya, Hainan, was found to be toxic to pearl oysters (Wu et al., 2005). Four Amphidinium species, i.e., A. carterae, A. massartii Biecheler, A. gibbosum, and A. operculatum were recorded in the subtidal zone during a year-long survey around Hainan, China (Zhang, 2015), and their distribution pattern were well discussed. To fully understand the diversity of epibenthic Amphidinium sensu stricto species in the China Sea, we collected samples from tropical to temperate areas and isolated single cells to establish strains. Their morphology was examined in detail using light microscopy (LM) and scanning electron microscopy (SEM). Moreover, both LSU rRNA and internal transcribed spacer (ITS, including ITS1, 5.8S rRNA, and ITS2) sequences were obtained. The molecular phylogeny was inferred based on ITS and LSU rRNA sequences and pigment profiles were determined from available strains.

2 MATERIAL AND METHOD 2.1 Sample collection and treatment

A total of 21 sites were sampled from the China Sea between 2012 and 2020 (Supplementary Fig.S1; Table 1). The macroalgal, seagrass, dead coral reef and upper centimeter of sandy sediments were collected from the seabed by scuba divers, and deposited into a 1-L plastic bottles containing seawater collected at the same location. The samples were vigorously stirred to detach the epibenthic cells and the suspension settled in a composite settling chamber. The settled materials were subsequently sieved through 120-μm and 10-μm filters. The 10– 120-μm fractions were rinsed with filtered seawater and transferred into a polycarbonate bottle and brought back to laboratory. Single cells were isolated from this material with a micropipette with an inverted microscope Eclipse TS100 (Nikon, Tokyo, Japan) into a 96-well tissue culture plate containing 330-μL f/2-Si (Guillard and Ryther, 1962) or L1 medium (Guillard and Hargraves, 1993). The plate was placed at 20 ℃ or 25 ℃, 90 μmol photons/(m2·s) from cool-white tubes, and under a light꞉dark cycle of 12 h꞉12 h and examined daily with the inverted microscope. The cultures were then transferred to 50-mL polystyrene tissue culture flasks.

Table 1 Strains of Amphidinium examined in the present study, including collection data, locations, and sample sources
2.2 Light microscopy (LM)

Living cells were examined and photographed using a Zeiss Axio Imager microscope (Carl Zeiss, Göttingen, Germany) equipped with a Zeiss Axiocam HRc digital camera. More than 30 cells were measured using Axiovision (4.8.2 version). To observe the shape and location of the nucleus, cells were stained with 1꞉100 000 Sybr Green (Sigma Aldrich, St. Louis, USA) for 1 min, and photographed using the Zeiss fluorescence microscope equipped with a Zeiss-38 filter set (excitation BP 470/40, beam splitter FT 495, emission BP 525/50). Chloroplast auto-fluorescence microscopy was carried out on live cells using a Leica DM6000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a G filter cube (emission filter BP495/15, dichromatic mirror 510, suppression filter BP530/30), and digitally photographed using a Leica DFC300 FX digital camera.

2.3 Scanning electron microscopy (SEM)

Mid-exponential batch cultures were fixed overnight at 4 ℃ with 1% OsO4 made up with 0.2-μm filtered seawater. The supernatant was removed and the cell pellet was allowed to adhere to a coverslip coated with poly-L-lysine (molecular weight 70 000-150 000). Subsequently, cells were washed in Milli-Q water for 10 min and dehydrated through a graded ethanol series (10%, 30%, 50%, 70%, 90% and 3 times in 100%) for 10 min at each step. The samples were then critical point dried in a K850 Critical Point Dryer (Quorum/Emitech, West Sussex, UK), sputter-coated with gold, and examined with a Zeiss Sigma FE (Carl Zeiss Inc., Oberkochen, Germany) scanning electron microscope. Images were presented on a black background using Photopea online program (https://www.photopea.com/). The standard terminology proposed by Jørgensen et al. (2004) was applied for the description of morphological features, and cell orientation.

2.4 Molecular analysis

Total genomic DNA was extracted from 15 mL of exponentially growing cultures using a MiniBEST Universal DNA Extraction Kit (TaKaRa, Japan) according to the manufacturer's protocol. PCR amplifications were carried out using 1×PCR buffer, 50-μmol/L dNTP mixture, 0.2 μmol/L of each primer, 10 ng of template genomic DNA, and 1 U of ExTaq DNA Polymerase (TaKaRa, Japan) in 50-μL total volume reactions. The total ITS1-5.8S-ITS2 was amplified using ITSA/ITSB (Adachi et al., 1996) primers. The LSU rRNA was amplified using the primers of D1R/28-1483R (Daugbjerg et al., 2000). The thermal cycle procedure was 4 min at 94 ℃, followed by 30 cycles of 1 min at 94 ℃, 1 min at 47 ℃, 1 min at 72 ℃, and a final extension of 7 min at 72 ℃ with a Mastercycler (Eppendorf, Hamburg, Germany). The PCR product was purified using a DNA purification kit (Sangon Biotech, Shanghai, China) and sequenced directly in both directions on an ABI PRISM 3730XL (Applied Biosystems, Foster City, CA) following the manufacturer's instructions.

2.5 Sequence alignment and phylogenetic analyses

Newly obtained LSU rRNA (D1-D6) and ITS region sequences were incorporated into those of Amphidinium sensu stricto available in GenBank (Supplementary Table S1 & S2). Sequences were aligned using MAFFT v7.110 (Katoh and Standley, 2013) online program (http://mafft.cbrc.jp/alignment/server/) under L-INS-Ⅰ (Carroll et al., 2007). Alignments were manually checked with BioEdit v. 7.2.5 (Hall, 1999). Completed alignments of ITS sequences were saved as NEXUS files and imported into PAUP*4b10 software (Swofford, 2002) to estimate divergence rates using simple uncorrected pairwise (p) distance matrices. Karlodinium armiger were used as the outgroup in both ITS and LSU rRNA sequences-based phylogeny. For Bayesian inference (BI), the program jModelTest 2 v2.1. 4 (Darriba et al., 2012) was used to select the most appropriate model of molecular evolution with Akaike Information Criterion (AIC). Bayesian reconstruction of the data matrix was performed using MrBayes 3.2 (Ronquist et al., 2012) with the best-fitting substitution model (GTR+G for LSU, TVM+G for ITS). Four Markov chain Monte Carlo (MCMC) chains ran for 5 000 000 generations, sampling every 100 generations. Convergence diagnostics were graphically estimated using Tracer ver. 1.7.1 (http://tree.bio.ed.ac.uk/software/tracer/) and the first 10% of burn-in trees were discarded. A majority rule consensus tree was created in order to examine the posterior probabilities of each clade. Maximum likelihood (ML) analyses were conducted with RaxML v7.2.6 (Stamatakis, 2006) on the T-REX web server (Boc et al., 2012) using the model GTR+G. Node support was assessed with 500 bootstrap replicates.

2.6 Pigment analysis

Five milliliters of culture were filtered onto a 25-92 diameter Whatman GF/F filter (Whatman International Ltd., Kent, UK) under gentle vacuum (< 100 mmHg). The filter was then soaked in 1-mL N, N-dimethylformamide (DMF) and extracted in a freezer (-20 ℃) in the dark for 1 h. Whatman GF/F filters of 13 mm diameter (Swinnex® filter holder, Millipore, Bedford, Massachusetts, USA) were used to clean the debris in the extractions. The filtrate was mixed 1 1 with 1-mol/L ammonium acetate solution. A quarter of each mixture was injected into a Shimadzu LC20A-DAD HPLC system fitted with a 3.5-μm Eclipse XDB C8 column (100 mm×4.6 mm; Agilent Technologies, Palo Alto, California, USA). The gradient elution was performed according to the standard method (Zapata et al., 2000). Pigment quantification was confirmed using standards manufactured by the Danish Hydraulic Institute Water and Environment (DHI), Hørsholm, Denmark.

3 RESULT

Totally 74 Amphidinium strains were established from the Chinese waters. Among them, 22 strains were identified based on morphology and phylogeny as A. carterae (including four ribotypes), ten as A. fijiensis, three as A. gibbosum, three as A. massartii, three as A. cf. massartii, six as A. pseudomassartii, nine as A. operculatum, three as A. steinii, nine as A. thermaeum, one as A. theodori Tomas and Karafas, two as A. tomasii, and one strain as undefined species (Table 1; Supplementary Table S3; Supplementary Fig.S1). In addition, two strains were attributed to A. trulla based on LSU rRNA sequences only, as the morphological features could not be examined due to the loss of cultures.

3.1 Morphology 3.1.1 Amphidinium carterae

Cells of A. carterae strain WZD09 (subclade Ⅰ in the phylogeny) appeared as oval to elliptical shape from the ventral side and dorsoventrally slightly flattened (Fig. 1a–g). Cells were 14.2–19.4-μm long (16.4±1.4 μm, n=41) and 9.4–12.9-μm wide (11.2±1.2 μm, n=41), with the length/width ratio varying from 1.3 to 1.7 (1.5±0.1, n=41). Cells were actively swimming in the water column, and asexual division was by binary fission in the motile cell (Fig. 1b). A pyrenoid was visible in the center of the cell (Fig. 1a–c). The nucleus was round and located in the posterior end of the cell (Fig. 1c). A single and reticulate chloroplast was present (Fig. 1d). No pusule could be observed by LM. The epicone was crescent shaped, left-deflecting, and 6.5±1.2-μm (n=10) long (Fig. 1e). The ventral ridge was short and straight (3.6±0.7 μm, n=13), and connected the two flagella insertion points (Fig. 1e). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 1e). The longitudinal flagellum was inserted in the middle third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus (Fig. 1e). The sulcus was shallow and did not reach the antapex of the cell (Fig. 1e). The anterior shoulders of the hypocone were symmetrical or slightly higher in the left, and the posterior of the hypocone was rounded to elliptical (Fig. 1e–g). Amphidinium carterae subclade Ⅰ was encountered in Guangdong, Guangxi, and Hainan (Xisha Islands), South China Sea (stations 4, 11, 12, and 21; Supplementary Fig.S1; Table 1). Amphidinium carterae subclade Ⅱ (strain TIO16, Fig. 1h & Supplementary Fig.S2) showed no obvious differences from subclade Ⅰ, and was encountered in Hainan (including Nansha island), South China Sea (station 16; Supplementary Fig.S1; Table 1). Amphidinium carterae subclade Ⅲ (strain TIO574, Fig. 1i & Supplementary Fig.S3) and subclade Ⅳ (strain TIO81, Fig. 1j & Supplementary Fig.S4) had obvious pentagonal or hexagonal amphiesmal vesicles, which were covered by threadlike body scales. Amphidinium carterae subclade Ⅲ was encountered in Shandong, Yellow Sea (station 1; Supplementary Fig.S1; Table 1), while A. carterae subclade Ⅳ was encountered in Guangxi and Hainan, South China Sea (stations 9, 13, and 16; Supplementary Fig.S1; Table 1).

Fig.1 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium carterae a–g. Amphidinium carterae subclade Ⅰ; a. LM image of the dorsal view showing the cell shape and location of the pyrenoid (Py); b. LM image of the lateral view showing the dividing cells; c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the ventral view showing reticulate chloroplasts (Chl); e–g. SEM images of motile cells showing the epicone shape, ventral ridge (arrow), points of flagellar insertion and smooth cell surface with amphiesmal vesicles; h. SEM image of the ventral view of A. carterae subclade Ⅱ, showing the epicone shape, ventral ridge (arrow), points of flagellar insertion and smooth cell surface with amphiesmal vesicles; i. SEM image of the ventral view of A. carterae subclade Ⅲ, showing the epicone shape, ventral ridge (arrow), points of flagellar insertion and amphiesmal vesicles covered with threadlike body scales (arrowhead); j. SEM image of the ventral view of A. carterae subclade Ⅳ, showing the epicone shape, ventral ridge (arrow), points of flagellar insertion and amphiesmal vesicles covered with threadlike body scales (arrowhead).
3.1.2 Amphidinium fijiensis Karafas & Tomas

Cells of A. fi jiensis strain WZD19 varied from round, oval to elliptical and dorsoventrally slightly flattened (Fig. 2). Cells were 12.8–20.2-μm long (15.9±2.1 μm, n=31) and 9.4–15.7-μm wide (11.3±1.4 μm, n=31), with the length/width ratio varying from 1.0 to 2.2 (1.4±0.3, n=31). A pyrenoid was visible in the center of the cell (Fig. 2a–b). The nucleus was round, located in the posterior end of the cell (Fig. 2c). A single and reticulate chloroplast was present (Fig. 2d). No pusule could be observed by LM. Asexual reproduction occurred within temporary hyaline cysts (Fig. 2e–h). The metabolic movements were not observed. The epicone was minute, left-deflecting and 9.4±0.6-μm long (n=6), and protruded minimally on the ventral side (Fig. 2i & k). Hexagonal amphiesmal vesicles were observed on the surface of cell (Fig. 2j). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 2i–k). The ventral ridge was short and straight (4.7±0.3-μm long, n=6), and connected the two flagella insertion points (Fig. 2i & k). The longitudinal flagellum was inserted in the middle third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus (Fig. 2i & k). The sulcus was shallow and did not reach the antapex of the cell (Fig. 2i & k). The anterior shoulders of the hypocone were symmetrical or slightly higher in the left, and the posterior of the hypocone was rounded (Fig. 2i–k). Amphidinium fi jiensis was encountered in Guangxi, South China Sea (station 10; Supplementary Fig.S1; Table 1).

Fig.2 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium fijiensis a–b. LM images of the ventral and right view showing the cell shape and location of the pyrenoid (Py); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the dorsal view showing reticulate chloroplasts (Chl); e–h. LM images showing the dividing encysted cells; i–k. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion and pattern of amphiesmal vesicles.
3.1.3 Amphidinium gibbosum

Cells of A. gibbosum strain 3XS19 were asymmetrically ellipsoid and dorsoventrally flattened (Fig. 3). Cells were 31.8–43.9-μm (36.2±2.4 μm; n=61) long and 14.5–22.5-μm (18.8±1.5 μm; n=61) wide, with a length/width ratio of 1.6–2.6 (1.9±0.2; n=61). A pyrenoid was visible in the center of the cell (Fig. 3a–b). Lipid globules were observed in the center and periphery of cells (Fig. 3a). The nucleus was round, located in the posterior end of the cell (Fig. 3c). The chloroplast comprised of several slender lobes (Fig. 3c & d). No pusule was observed by LM. The epicone was triangle, left-deflecting and 6.0±0.9-μm long (n=3, Fig. 3a & e). The hypocone varied from oval to heart shaped with a strongly convex right side (Fig. 3a & e), giving it a hump-backed shape. A ventral ridge (5.3±0.2-μm long; n=5) ran between the two points of the flagellar insertion (Fig. 3e). The cingulum was deeply incised and the end was displaced (Fig. 3a & e). The proximal and distal ends of the cingulum nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 3a & e). The sulcus originated from just posteriorly to the cingulum. It was very shallow, short and did not run to the antapex (Fig. 3e). Pentagonal or hexagonal plate-like structure was observed on the cell surface (Fig. 3e–g). Small round pores were scattered randomly on the cell surface (Fig. 3f–g; arrows). Amphidinium gibbosum was encountered in Hainan (including Xisha Islands), South China Sea (stations 17, 18, and 21; Supplementary Fig.S1; Table 1).

Fig.3 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium gibbosum a–b. LM images of motile cells showing the cell shape and location of the pyrenoid (Py), lipid (lip); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the dorsal view showing slender lobes of chloroplast (Chl); e–f. SEM images showing the epicone shape, ventral ridge, points of flagellar insertion, plate and pores on the cell surface (arrows); g. SEM image showing a close-up view of the cell surface.
3.1.4 Amphidinium massartii & A. cf. massartii

Cells of A. massartii strain TIO12 were oval to elliptical, and dorsoventrally flattened (Fig. 4a–g). Cells were 13.8–19.1-μm long (17.2±2.0 μm, n=30) and 10.8–12.6-μm wide (11.5±0.7 μm, n=30), with the length/width ratio varying from 1.3 to 1.7 (1.5±0.1, n=30). A pyrenoid was visible in the upper half of the cell (Fig. 4a & c). Cells were actively swimming in the water column, and asexual divisions occurred by binary fission in the motile cells (Fig. 4b). The nucleus was round, located in the posterior end of the cell (Fig. 4c). A single and reticulate chloroplast was present (Fig. 4d). No pusule could be observed by LM. The epicone was crescent shaped, left-deflecting and 9.4±1.5-μm long (n=7, Fig. 4e–f). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 4e–f). The ventral ridge was short and straight (4.9±0.5-μm long, n=7), and connected the two flagella insertion points (Fig. 4e). The longitudinal flagellum was inserted in the middle third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus (Fig. 4e). The sulcus was shallow and did not reach the antapex of the cell (Fig. 4e). Round doughnut-like body scales were observed on the surface of the cell body and flagella (Fig. 4g). The anterior shoulders of the hypocone were symmetrical or slightly higher in the left, and the posterior of the hypocone was rounded (Fig. 4e–f). Amphidinium massartii was encountered in Hainan (including Nansha islands), South China Sea (stations 10 and 15; Supplementary Fig.S1; Table 1).

Fig.4 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium massartii and A. cf. massartii a–g. Amphidinium massartii; a. LM image of the lateral view showing the cell shape and location of the pyrenoid (Py); b. LM image of the lateral view showing the dividing cells. c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the ventral view showing reticulate chloroplasts (Chl); e–f. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion and smooth cell surface with amphiesmal vesicles; g. SEM image showing a close-up view of round doughnut-like body scales on cell surface; h–j. Amphidinium cf. massartii; h–i. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion and smooth cell surface with amphiesmal vesicles; j. SEM image showing a close-up view of round doughnut-like body scales on cell surface.

Cells of A. cf. massartii strain WZD23 were oval to elliptical, and dorsoventrally flattened (Fig. 4h–j). Cells were 12.1–18.1-μm long (15.4±1.8 μm, n=30) and 10.4–15.5-μm wide (13.1±1.8 μm, n=30), with the length/width ratio varying from 1.0 to 1.3 (1.2±0.1, n=30). The epicone was crescent shaped, left-deflecting, and 7.3±0.8-μm long (n=7, Fig. 4h). The ventral ridge was short and straight (3.8±0.4-μm long, n=7), and connected the two flagella insertion points (Fig. 4h). Round doughnut-like body scales were observed on the surface of both the cell body and flagella (Fig. 4j). Amphidinium cf. massartii was encountered in Guangxi and Hainan, South China Sea (stations 10, 12, and 16; Supplementary Fig.S1; Table 1).

3.1.5 Amphidinium pseudomassartii Karafas & Tomas

Cells of A. pseudomassartii strain TIO555 were round to elliptical (Fig. 5). Cells were 17.7–23.3-μm long (20.7±1.8 μm, n=50) and 12.9–18.7-μm wide (14.7±1.9 μm, n=50), with the length/width ratio varying from 1.2 to 1.6 (1.4±0.1, n=50). A pyrenoid was visible in the center of the cell (Fig. 5b). The nucleus was round, located in the posterior end of the cell (Fig. 5c). A single and reticulate chloroplast was present (Fig. 5d). No pusule was observed by LM. The epicone was crescent shaped, left-deflecting with a rounded tip, and 11.8±1.8-μm long (n=10, Fig. 5e–f). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 5e–f). The ventral ridge was short and straight (6.2±0.8-μm long, n=10), and connected the two flagella insertion points (Fig. 5e). The longitudinal flagellum was inserted in the middle third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus (Fig. 5e). The sulcus was shallow and did not reach the antapex of the cell (Fig. 5e). Body scales were not observed on the surface of the cell (Fig. 5g). The anterior shoulders of the hypocone were asymmetrical with the left side slightly higher, and the posterior of the hypocone was rounded (Fig. 5e–f). Cells were actively swimming in the water column, and asexual division took place by binary fission in the motile cell (Fig. 5h–k). Amphidinium pseudomassartii was encountered in Fujian, East China Sea (station 2; Supplementary Fig.S1; Table 1).

Fig.5 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium pseudomassartii a–b. LM images of motile cells showing the cell shape and location of the pyrenoid (Py); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the dorsal view showing reticulate chloroplasts (Chl); e–f. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion and smooth cell surface with amphiesmal vesicles; g. SEM image showing a close-up view of cell surface; h–k. LM image showing the dividing cells.
3.1.6 Amphidinium operculatum

Cells of A. operculatum strain TIO40 were oval to elliptical (Fig. 6). The right side of the hypocone was convex, whereas the left was almost straight (Fig. 6a). Cells were 28.7–36.3-μm long (32.9±2.4 μm, n=50) and 20.9–25.8-μm wide (23.9±1.7 μm, n=50), with the length/width ratio varying from 1.3 to 1.5 (1.4±0.1, n=50). Many lipid globules were observed in the periphery of cells (Fig. 6a), and one big "dark spot", globular inclusions, was located in the center of the cell (Fig. 6b & c). No pyrenoid was observed by LM (Fig. 6b–c). The nucleus was elongated, located in the posterior end of the cell (Fig. 6c). A single and reticulate chloroplast was present (Fig. 6d). The epicone was quite minute compare to the hypocone, left-deflecting, and 8.7±0.7-μm long (n=6, Fig. 6a–b, & e). The ventral ridge was 17.8±1.9-μm long (n=6), and connected the two flagella insertion points (Fig. 6e–f). The cingulum was displaced and the proximal end is distant from the sulcus (Fig. 6e). The longitudinal flagellum was inserted in the lower third of the cell and at the beginning of the sulcus (Fig. 6e). The sulcus was shallow and did not reach the antapex of the cell (Fig. 6e). The anterior shoulders of the hypocone were symmetrical, and the posterior of the hypocone were rounded (Fig. 6a–e). Cell were actively swimming in the water column and temporary hyaline cysts were not found. Amphidinium operculatum was encountered in Guangdong, Guangxi, and Hainan, South China Sea (stations 6, 9, 14, 17, 19, and 20; Supplementary Fig.S1; Table 1).

Fig.6 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium operculatum a–b. LM images of motile cells showing the cell shape, location of the nucleus (N), and the big "dark spot" globular inclusions (arrow); c. fluorescence LM images of a Sybr Green stained cell showing the shape and location of the nucleus (N), and the big "dark spot" globular inclusions (arrow); d. fluorescence LM image of the dorsal view showing reticulate chloroplasts (Chl); e. SEM image of the ventral view showing the epicone shape, ventral ridge, points of flagellar insertion; f. SEM image showing a close-up view of the ventral ridge (arrow); g. SEM image of the dorsal view.
3.1.7 Amphidinium steinii Lemmermann

Cells of A. steinii strain TIO311 appeared oval to elliptical (Fig. 7). Cells were 33.3–47.7-μm long (39.1±5.3 μm, n=30) and 17.2–29.6-μm wide (23.8±3.1 μm, n=30), with the length/width ratio varying from 1.5 to 2.1 (1.7±0.2, n=30). Lipid or starch droplets were often found in cultures (Fig. 7a–b), and a pyrenoid was observed under LM (Fig. 7b). The nucleus was round to oval, located in the posterior part of the hypocone (Fig. 7a & c). A potential pusule was observed by LM (Fig. 7b & c). A single and reticulate chloroplast was present (Fig. 7d). The epicone was quite minute compared to the hypocone, triangular, left-deflecting, and 13.4±1.1-μm long (n=9, Fig. 7e–g). The ventral ridge was short and straight (6.9±0.6-μm long, n=9), and connected the two flagella insertion points (Fig. 7e & g). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 7e & g). The longitudinal flagellum was inserted in the upper third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus, which was narrow and short (Fig. 7e & g). Hexagonal amphiesmal vesicles were present on the cell surface, but body scales were not observed (Fig. 7g). The anterior shoulders of the hypocone were asymmetrical with the left side slightly higher and the posterior of the hypocone was elliptical (Fig. 7e–g). Asexual divisions occurred in hyaline-covered cysts (Fig. 7h–l). Metabolic movement was observed in strain TIO311. Amphidinium steinii was encountered in Fujian, East China Sea and Guangxi, South China Sea (stations 3, 8, and 9; Supplementary Fig.S1; Table 1).

Fig.7 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium steinii a–b. LM images of motile cells showing the cell shape, location of the nucleus (N), pyrenoid (Py), and pusules (Pu); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the dorsal view showing reticulate chloroplasts (Chl); e–f. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion; g. SEM image showing a close-up view of amphiesmal vesicles; h–l. LM and SEM images, showing the dividing cells.
3.1.8 Amphidinium thermaeum Dolapsakis & Economou-Amilli

Cells of A. thermaeum strain WZD07 were rounded, oval to oblong, and dorsoventrally slightly flattened (Fig. 8). Cells were 16.3–22.1-μm long (18.9±2.2 μm, n=30) and 14.9–18.1-μm wide (16.1±1.4 μm, n=30), with the length/width ratio varying from 1.0 to 1.3 (1.7±0.2, n=30). A pyrenoid was observed by LM (Fig. 8a & c). A potential pusule with radiating vesicles was present (Fig. 8b). The nucleus was round, located in the posterior end of the cell (Fig. 8c). A single and reticulate chloroplast was present (Fig. 8d). Metabolic movement was observed in a few cells, and cell shapes changed sharply from oblong to oval (Fig. 8e–g). Asexual divisions occurred in hyaline-covered cysts (Fig. 8h). The epicone was triangular, left-deflecting and 10.2±0.9-μm long (n=6, Fig. 8i–k). The ventral ridge was short and straight (6.1±0.5-μm long, n=6), and connected the two flagella insertion points (Fig. 8i–j). The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 8i–j). The longitudinal flagellum was inserted in the middle third of the cell just below the proximal end of the cingulum and at the beginning of the sulcus (Fig. 8i–j). The sulcus was wide and shallow and reached the antapex of the cell (Fig. 8i–j). The anterior shoulders of the hypocone were symmetrical or slightly higher in the left, and the posterior of the hypocone were rounded (Fig. 8i & k). Amphidinium thermaeum was encountered in Guangxi and Hainan (including Nansha Islands), South China Sea (stations 9, 11, 16, and 20; Supplementary Fig.S1; Table 1).

Fig.8 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium thermaeum a. LM image of the ventral view showing the cell shape and location of pyrenoid (Py); b. LM image of the ventral view showing the pusules (dotted circle); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. Fluorescence LM image of the dorsal view showing reticulate chloroplasts (Chl); e–g. LM images of the same cell showing different cell shapes attributed to cell plasticity; h. LM image of the four dividing encysted cells; i–j. SEM images of the ventral view showing the epicone shape, ventral ridge, points of flagellar insertion; k. SEM image of the dorsal view.
3.1.9 Amphidinium theodori Tomas & Karafas

Cells of A. theodori strain 3XS55 appeared oval to elliptical with a range of 21.1–35.8-μm (28.0±2.5 μm; n=35) long and 19.4–33.0-μm (24.9±2.7 μm; n=35) wide (Fig. 9). The length/width ratio was 1.0–1.3 (1.1±0.1; n=35). A central pyrenoid was observed, but no pusule was found under LM (Fig. 9a–b). The nucleus was oval and located in the posterior end of the cell (Fig. 9c). Chloroplast lobes appeared to radiate from the central pyrenoid and spread to the periphery of the cell (Fig. 9d). Asexual divisions occurred in hyaline-covered cysts (Fig. 9e). The epicone was triangular, left-deflecting, and the ventral ridge (6.6±0.4-μm long; n=4) was straight (Fig. 9f). The anterior shoulders of the hypocone were symmetrical or slightly higher at the left, and the posterior of the hypocone was rounded (Fig. 9a–b, f–g). Metabolic movement was observed. Amphidinium theodori was encountered in Hainan (Xisha Islands), South China Sea (station 21; Supplementary Fig.S1; Table 1).

Fig.9 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium theodori a–b. LM images of motile cells showing the cell shape and location of the pyrenoid (Py); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the ventral view showing radiate chloroplast (Chl); e. LM image of the four dividing encysted cells; f–g. SEM images of motile cells showing the epicone shape, ventral ridge and smooth cell surface.
3.1.10 Amphidinium tomasii Karafas

Cells of A. tomasii strain TIO31 were oval to elliptical and slightly flattened dorsoventrally (Fig. 10). Cells were 22.1–33.2-μm long (27.5±4.2 μm, n=30) and 16.7–25.6-μm wide (21.2±3.8 μm, n=30), with the length/width ratio varying from 1.1 to 1.6 (1.3±0.2, n=30). The epicone was curved and left-deflecting and 13±1.7-μm long (n=6, Fig. 10a–b). The nucleus was round, located in the posterior end of the cell (Fig. 10b & c). A single and reticulate chloroplast was present (Fig. 10d–e). No pusule could be observed by LM. Cell were actively swimming in the water column, and asexual divisions took place by binary fission in the motile cell (Fig. 10e–h), No immobile or encysted cells were observed. The cingulum was displaced and the proximal and distal ends nearly contacted the ventral ridge and sulcus to form a "v" shape (Fig. 10i). The sulcus originated around the ventral midpoint of the cell and widened gradually toward posteriorly (Fig. 10i). The ventral ridge was short and straight (8.5±1.6-μm long, n=6), and connected the two flagella insertion points (Fig. 10i). The anterior shoulders of the hypocone were asymmetrical with a higher left side, and the posterior of the hypocone were rounded (Fig. 10i). The left side of the hypocone varied from straight to convex, while the right side of the hypocone was always convex. Longitudinal cleave was not observed on the dorsal hypocone (Fig. 10j–k). The longitudinal flagellum was inserted in the middle third of the cell at the beginning of the sulcus, which was shallow and did not reach the antapex of the cell (Fig. 10i). Hexagonal amphiesmal vesicles were observed on the cell surface, but body scales were not observed (Fig. 10i). Amphidinium tomasii was encountered in Hainan, South China Sea (stations 14 and 16; Supplementary Fig.S1; Table 1).

Fig.10 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium tomasii a–b. LM images of motile cells showing the cell shape, points of flagellar insertion and location of the nucleus (N); c. fluorescence LM image of a Sybr Green stained cell showing the shape and location of the nucleus (N); d. fluorescence LM image of the ventral view showing reticulate chloroplasts (Chl); e–h. fluorescence LM image showing the dividing cells; i–j. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion; k. SEM image showing the dividing cells.
3.1.11 Amphidinium sp.

Cells of Amphidinium sp. strain 5XS22 were oval to elliptical and dorsoventrally flattened (Fig. 11). The epicone was minute and triangular, left-deflecting (Fig. 11). Cells were 20.3–30.9-μm long (26.3±5.0 μm, n=30) and 15.9–24.8-μm wide (21.3±2.5 μm, n=30), with the length/width ratio varying from 1.1 to 1.5 (1.2±0.3, n=30). Many lipid globules were observed in the periphery of the cells, but no pyrenoid or pusule were observed by LM (Fig. 11a–b). Chloroplast appeared to radiate from the center of the cell and extend to the periphery (Fig. 11c). The ventral ridge (8.97±1.31-μm long, n=4) was long and straight and connected the two flagella insertion points (Fig. 11d–e). The cingulum was displaced and the proximal and distal ends were distant from the sulcus (Fig. 11d–e). The cingulum ends nearly contacted the ventral ridge and formed a "v" shape (Fig. 11e). The longitudinal flagellum was inserted in the lower third of the cell and at the beginning of the sulcus (Fig. 11d–e). The sulcus was wide and reached to the antapex of the cell (Fig. 11d–e). The anterior shoulders of the hypocone were nearly symmetrical (Fig. 11d). The posterior of the hypocone were rounded (Fig. 11). Pentagonal or hexagonal plate-like structure was observed on the cell surface (Fig. 11d–e). Amphidinium sp. strain 5XS22 was encountered in Hainan (Xisha Islands), South China Sea (station 21; Supplementary Fig.S1; Table 1).

Fig.11 Light microscopy (LM) and scanning electron microscopy (SEM) of Amphidinium sp. a–b. LM images of motile cells showing the cell shape and lipid globules (Lip); c. fluorescence LM image of the dorsal view showing radiate chloroplast (Chl); d–e. SEM images of motile cells showing the epicone shape, ventral ridge, points of flagellar insertion and cell surface.
3.2 Molecular phylogeny

From a comparison of LSU rRNA gene sequences (Fig. 12), it was found that 22 strains of Chinese A. carterae differed from each other in 24–56 positions (95.4%–98.1% similarity). Ten strains of Chinese A. fijiensis shared identical sequences, and they differed from the type strain Amfi0508-1 of A. fijiensis in 34 positions (97.4% similarity). Three strains of Chinese A. gibbosum shared identical sequences with the USVI strain SI-36–50 of A. gibbosum. Three strains of Chinese A. massartii differed from each other in 6–23 positions (97.1%–99.2% similarity). The Chinese A. massartii strain TIO12 differed from the USA strain Amma0607-1 and Japanese strain HG115 in 31 and 35 positions (97.6% and 96.9% similarity), respectively, and differed from A. cf. massartii strain WZD23 isolated from Guangxi, South China Sea, in 50 positions (96.1% similarity). The Chinese A. cf. massartii strains WZD23 and 3WZD7 differed from one another in 7 positions (99.2% similarity). Six strains of Chinese A. pseudomassartii shared identical sequences, and they differed from the type strain AKLV01 of A. pseudomassartii in 18 positions (98.6% similarity). Nine strains of Chinese A. operculatum differed from each other in 0–17 positions (97.9–100% similarity). Three strains of A. operculatum (TIO175b, TIO175d, and TIO175e) isolated from the South China Sea shared identical sequences, and they differed from the strain TIO40 from the South China Sea, Australian strain K-0663, and New Zealand strain CAWD42 in 2, 41, and 19 positions (99.2%, 96.8%, and 97.9% similarity), respectively. Three strains of Chinese A. steinii differed from each other in 53–66 positions (93.1%– 94.4% similarity). Chinese A. steinii strain TIO311 differed from Australian strain CS-741 and Brazilian strain SM12 in 2 and 19 positions (99.2% and 97.9% similarity), respectively. Nine strains of Chinese A. thermaeum shared identical sequences, and they differed from the type strain UoABM-Atherm1 of A. thermaeum in a single position (99.8% similarity). The Chinese A. tomasii strain TIO31 differed from the type strain Amth1412-1 of A. tomasii in three positions (99.4% similarity). Two strains of Chinese A. trulla shared identical sequences, and they differed from the type strain CAWD68 of A. trulla in 11 positions (99.1% similarity). The Chinese A. theodori strain 3XS55 differed from the type strain Amth0702-1 of A. theodori in ten positions (98.7% similarity).

Fig.12 Molecular phylogeny of Amphidinium inferred from partial large subunit rRNA sequences based on Maximum likelihood (ML) Karlodinium armiger was used as the outgroup. Numbers at nodes represent the result of the ML bootstrap analysis and Bayesian posterior probabilities (left: Bayesian posterior probabilities; right: ML bootstrap support values); filled circles indicate the maximal support in Bayesian inference (BI) and ML (1.0 and 100%, respectively). Bootstrap values > 50% and posterior probabilities above 0.5 are shown. Newly obtained sequences were indicated in blue.

The maximum likelihood (ML) and Bayesian inference (BI) analyses based on partial LSU rRNA sequences yielded similar phylogenetic trees. The ML tree is shown in Fig. 12. The A. incoloratum Campbell and Amphidinium sp. strain 5XS22 were the earliest branch in the Amphidinum sensu stricto but with low support. Amphidinium operculatum was the second earliest branch followed by A. steinii, A. cupulatisquama M. Tamura & T. Horiguchi, A. herdmanii Kofoid & Swezy, and A. mootonorum Murray & Patterson clade. Two groups (Ⅰ and Ⅱ) were resolved by representing the sulcus connect with the cingulum or not (Fig. 12). All A. carterae strains were grouped together in a fully supported clade that was the sister to a clade containing A. massartii, A. fijiensis, A. pseudomassartii, A. tomasii, A. theodori, A. magnum Karafas & Tomas, and A. thermaeum. Amphidinium carterae from various parts of the world that comprised four well-supported subclades, referred as subclades Ⅰ, Ⅱ, Ⅲ, and Ⅳ. Three A. carterae subclades (Ⅰ, Ⅱ, and Ⅳ) were found in the South China Sea and subclade Ⅲ was found in the Yellow Sea. Strains of A. massartii and A. cf. massartii were sister clades with each other, and both were encountered in the South China Sea. Strains of A. pseudomassartii were grouped together in a fully supported clade, which consisted of two clades from the East China Sea and Australia, and were referred as subclades Ⅰ and Ⅱ, respectively. The A. fijiensis from the South China Sea were grouped together and formed a well-supported clade (subclade Ⅱ) with west coast of India-open sea strains, which was a sister clade to the type strain of A. fijiensis (subclade Ⅰ). Amphidinium thermaeum from various parts of the world were grouped together in a well-supported clade, which was positioned at the end of the branch of the phylogenetic tree.

Sequence similarity and genetic distances based on ITS sequences among Amphidinium species are listed in Table 2. The genetic distances between Amphidinium species ranged from 0.215 to 0.519, with an average of 0.352. The largest interspecific distances were found between A. steinii and the other species of Amphidinium (0.472–0.519). The ML and BI analysis based on ITS sequences generated similar trees, and the ML tree is shown in Supplementary Fig.S5. Amphidinum species were resolved in accordance with traditional morphometrics based taxa units, which is consistent with the LSU rRNA sequences-based phylogeny. Amphidinium steinii diverged early, followed by the rest of the Amphidinium sensu stricto formed a well resolved clade, with strong support (1.00, 100).

Table 2 Internal transcribed spacer (ITS) region sequences compared between Amphidinium species
3.3 Pigment profile

Eleven pigments, including peridinin, pheophorbide a, diadinoxanthin, antheraxanthin, diatoxanthin, pheophorbide, and β-carotene, and four different chlorophylls were detected in all tested strains (Table 3). Peridinin, pheophorbide, chlorophyll a, antheraxanthin, and chlorophyll c2 were the dominant pigments in all five species. The content ratio of pheophorbide ranged from 19.24% (A. carterae subclade Ⅱ) to 35.46% (A. carterae subclade Ⅳ) (Table 3).

Table 3 Pigment profiles of selected Amphidinium strains, including pigment cell quotas (pg/cell) and percentage in brackets
4 DISCUSSION 4.1 Morphology and phylogeny

There is a history of complication and confusion over the identification and placement of Amphidinium species due to their high morphological and genetic variability (Steidinger and Tangen, 1997; Daugbjerg et al., 2000; Saldarriaga et al., 2001). Jørgensen et al. (2004) and Murray et al. (2004) made notable strides in redefining the morphological characters of the type species of the genus and emended the genus definition. Presently, the Amphidinium sensu stricto includes both heterotrophic and autotrophic forms, possessing a characteristically minute epicone that is deflected towards the left (Jørgensen et al., 2004; Murray et al., 2004). About 20 species have been confirmed to fit the revised definition of the genus (Jørgensen et al., 2004; Murray et al., 2004; Dolapsakis and Economou-Amilli, 2009; Tamura et al., 2009; Karafas et al., 2017). Two major clades in the autotrophic Amphidinium have been reported based on partial LSU rRNA sequences, i.e., the Herdmanii and Operculatum clades (Jørgensen et al., 2004; Karafas et al., 2017), but the morphological features delimiting these clades are not clear. Our study identified two groups within the Amphidinium (Groups Ⅰ and Ⅱ) and highlighted the taxonomic significance of the position of sulcus in Amphidinium (Fig. 12). Group Ⅰ was characterized by the separation between sulcus and cingulum, and only included A. operculatum, A. incoloratum, and Amphidinium sp. strain 5XS22 so far. Group Ⅱ was characterized by the connection between sulcus and cingulum, and included other 14 species.

4.1.1 Amphidinium carterae

Amphidinium carterae is a well-known worldwide species that was first described as A. klebsii Kofoid and Swezy (Carter, 1937), but was later revised as a new species, based on its smaller size and unlike A. klebsii, it has a single chloroplast (Hulburt, 1957). Phylogenetic analyses based on partial LSU rRNA sequences have revealed a high genetic diversity within the A. carterae strains from around the world, and have shown many subclades (Murray et al., 2004, 2012; Lee et al., 2013). Karafas et al. (2017) proposed a division into four subclades based on the similarities and differences of the secondary structure of the ITS2 region among the different subclades. Our phylogeny based on ITS and partial LSU rRNA sequences seems to support this proposal (Fig. 12 and Supplementary Fig.S5). Amphiesmal vesicles were reported on the surface of A. carterae subclade Ⅰ (culture CCMP124, genotype 2 in Murray et al. (2004); Fig. 1e–g in the present study), and were also found in A. carterae subclade Ⅱ here (Fig. 1h). However, threadlike body scales were only found on the surface of Chinese strains of A. carterae subclades Ⅲ and Ⅳ, and are reported here for the first time (Fig. 1i–j; arrows). The morphology and phylogeny results seem to suggest that A. carterae might contain several different species (Karafas et al., 2017; this study).

Strains of A. carterae subclade Ⅲ were encountered in the Yellow Sea, but not from the East and South China Seas (Table 1). Strains of A. carterae subclade Ⅲ have also been reported from Italy, Canada, and Tasmania in Australia and were also reported as an "endosymbiont" in the jellyfish Cassiopeia xamachana from the Caribbean Sea. It may suggest that the free-living A. carterae subclade Ⅲ strains are distributed only in the higher latitude of the temperate zone. In the case of A. carterae subclades Ⅰ, Ⅱ, and Ⅳ no biogeographical distribution was found that was related to the molecular result (Fig. 12).

4.1.2 Amphidinium fijiensis

Amphidinium fijiensis was first described from a fish tank containing "live rock" imported from Korotoga Fiji (Karafas et al., 2017). Chinese A. fijiensis is consistent with the original description in terms of the small size, the insertion of the longitudinal flagellum in the middle third, the central pyrenoid, the posteriorly located nucleus, and reproduction within temporary hyaline cysts (Karafas et al., 2017). Hexagonal amphiesmal vesicles were seen on the surface of cells of strain WZD19 (Fig. 2j), but whether they were also present in the type strain was not clear. The phylogeny based on ITS and partial LSU rRNA sequences revealed two well supported subclades between the type strains and South China Sea strains. The genetic distances between the type strain Amfi0508 and strain WZD19 from Guangxi, South China Sea, was 0.235, which was below the average value for different Amphidinium species (0.352). Until a stable character distinct from the type strain is clarified, the identity of Chinese specimens is retained as A. fijiensis.

4.1.3 Amphidinium gibbosum

Amphidinium gibbosum was originally identified under the name, A. operculatum var. gibbosum (Maranda and Shimizu, 1996), and later changed to A. gibbosum due to their hump-backed cell shape and different molecular sequences from A. operculatum (Murray et al., 2004). The Chinese A. gibbosum strain 3XS19 fits the original description (Murray et al., 2004), in addition to round pores recorded on the cell surface of A. gibbosum here for the first time. The phylogenetic position of A. gibbosum was ambiguous (Karafas et al., 2017), and the adding Amphidinium sequences clarified its phylogenetic status. The LSU rRNA based phylogeny represented here revealed that A. gibbosum is located as an independent taxon between A. paucianulatum and A. trulla (Fig. 12). In the ITS phylogeny (Supplementary Fig.S5), A. gibbosum is shown to be separated from A. massartii and A. cf. massartii. Both the morphology and phylogeny results represented here confirm that A. gibbosum composes its own unique species as an independent taxon (Murray et al., 2004; this study).

4.1.4 Amphidinium massartii, A. cf. massartii, and A. pseudomassartii

Amphidinium massartii was originally described from France (Biecheler, 1952). Later A. massartii or A. cf. massartii were reported in the Mediterranean, and the coastal waters off Australia, USA, Japan, Korea, and Canada (Murray and Patterson, 2002; Murray et al., 2004; Lee et al., 2013; Karafas et al., 2017). However, three distinctly different molecular clades have been reported among them (Murray et al., 2004, 2012; Lee et al., 2013; this study). The first and second were sister clades, and were traditionally identified as A. massartii, characterized by possessing round doughnut-like body scales on the cell surface (Murray et al., 2012; Lee et al., 2013; this study). Round doughnut-like scales were observed on the surface of the flagella of the second A. massartii clade (Lee et al., 2013, Fig. 4j in the present study), although they were not specifically reported in the originally described French strain. Here, we found that round doughnut-like scales were present on the surface of the flagella of the first A. massartii clade for the first time (Fig. 4g), and there were no morphological differences between the two subclades. The ITS sequences-based distance between Chinese strains TIO12 (the first A. massartii clade) and WZD23 (the second A. massartii clade) was 0.260, which was below the average (0.352) of the different Amphidinium species, but higher than the smallest one (0.215). In summary, a notable genetic diversity was confirmed, but morphological differences were not observed between the first and second A. massartii clade of the Chinese strains, as also reported in previous studies (Murray et al., 2012; Karafas et al., 2017). More morphological and ultrastructure details are needed before their systematic status can be determined, and for the time being the first clade can be referred to as A. massartii and the second clade as A. cf. massartii (Karafas et al., 2017). The third clade included two Australian strains that were genetically distinct from the previous two clades (Murray et al., 2004, 2012; Lee et al., 2013). Karafas et al. (2017) assigned the third clade to a novel species, A. pseudomassartii, based on the lack of the round doughnut-like scales and wider epicones than A. massartii. The discovery of A. pseudomassartii from the East China Sea is the first record outside its type locality.

4.1.5 Amphidinium operculatum and Amphidinium sp.

Amphidinium operculatum is the type species of the genus Amphidinium, but its morphology was very different to the other Amphidinium sensu stricto species. The longitudinal flagellum of A. opeculatum is inserted in the lower third of the cell at the beginning of the sulcus, and did not connect with the cingulum. A similar location of flagellum insertion was only found in A. incoloratum, the only heterotrophic Amphidinium sensu stricto species. Here we reported a third one, Amphidinium sp. 5XS22 with the sulcus not connected to the cingulum. Amphidinium sp. 5XS22 was phylogenetically close to A. incoloratum (Fig. 12), but is an autotrophic species which is distinguishable from A. incoloratum. Morphological details of strain 5XS22 are needed before confirming its taxonomic status. The separation between sulcus and cingulum distinguishes A. opeculatum, A. incoloratum, and Amphidinium sp. 5XS22 from the other Amphidinium sensu stricto, and they comprised the Amphidinium Group Ⅰ (Fig. 12). The divergent nature of A. operculatum was also supported by having the greatest number of specific nucleotide changes in the partial LSU rRNA (Jørgensen et al., 2004; Murray et al., 2004). The long branch length in both the maximum likelihood and Bayesian inference analysis suggested a large divergence of this species from the others (Murray et al., 2004; Karafas et al., 2017). In addition, the ITS sequence of A. operculatum was not available, although we tried the primers ITSA/ITSB (Adachi et al., 1996), ITS1 and ITS4 (White et al., 1990; Gottschling and Plötner, 2004), as well as newly designed primers at the 3ʹ end of SSU and the 5ʹ end of LSU, which also indicated the divergence of this species from the other Amphidinium. Considering the divergent morphology and molecular sequences of A. operculatum, the other species currently included in Amphidinium may warrant the establishment of new genera.

4.1.6 Amphidinium steinii

Amphidinium steinii was first identified as A. operculatum; however, unlike the original description of A. operculatum (Stein, 1883), the insertion of the transverse flagellum is in an anterior position and division cysts are produced. We confirm that the most important characters distinguishing A. steinii from A. operculatum are asexual division by means of a division cyst, as well as the insertion of the transverse flagellum in the anterior third of the cell (Murray et al., 2004). In addition, more differences were revealed, i.e., 1) the nucleus of A. steinii is round to oval, but in A. operculatum it is elongated (Figs. 6c & 7c); 2) the chloroplast of both A. steinii and A. operculatum radiates from the center, but the former has more bulky and sparse lobes than the latter (Figs. 6d & 7d). The morphology of A. steinii is also similar to that of A. trulla in terms of cell size, epicone shape, the anterior position of insertion of the transverse flagellum, and the possession of a central pyrenoid from which the chloroplast lobes radiate. The asexual divisions of A. trulla occur in the motile cell (Murray et al., 2004), but in A. steinii they occur in division cysts (Fig. 7).

The LSU rRNA sequences of A. steinii has been previously reported for strains in Australia and Brazil (Murray et al., 2004). Here, we found a large genetic differentiation of LSU rRNA sequences in A. steinii (Fig. 12). Amphidinium steinii was also recorded in Wismar, Germany (Stein, 1883), and New Jersey and Maryland, USA (Thompson, 1951; Barlow and Triemer, 1988), but molecular sequences are not yet available. Additional strains of A. steinii from different locations are needed for a thorough examination of the genetic variation within this species.

4.1.7 Amphidinium thermaeum

The Chinese A. thermaeum strain WZD07 fits the original description regarding the plastic cell shape (metabolic movement), a pusule surrounded by small radiating vesicles, and asexual division within hyaline cysts (Dolapsakis and Economou-Amilli, 2009). Two pusules were reported in the original description of A. thermaeum (Dolapsakis and Economou-Amilli, 2009), but only one pusule was observed in Chinese strain WZD07. No pusules were observed in Dominican Republic strains (Karafas et al., 2017), suggesting that the number of pusules is also plastic. Very rarely, distorted cells may appear with furrows (striations or longitudinal cleave) on the hypocone in the original description (Dolapsakis and Economou-Amilli, 2009), but they were not observed in Chinese strains. Such furrows were also recorded in dividing cells of A. tomasii (Karafas et al., 2017). This indicates that such furrows in Amphidinium species are probably not common and may be restricted to certain life cycle stages. Metabolic movement has been reported in A. theodori, A. fijiensis, A. steinii, and A. thermaeum (Murray et al., 2004; Dolapsakis and Economou-Amilli, 2009; Karafas et al., 2017). Metabolic movement was observed in only a few cells in Chinese A. thermaeum, suggesting that this characteristic may only occur during certain life stages, such as the sexual stage in the life cycle (Murray et al., 2004; Karafas et al., 2017). Dinoflagellates display organellar evidence of secondary endosymbiotic events (Gould, 2012), as well as providing the basal position for Amphidinium in the dinoflagellate evolutionary lineage (Zhang et al., 2007). The metabolic movement ability of Amphidinium possibly indicates a constant relation between the ancestors of dinoflagellates and other cell plastic protist lineages.

4.1.8 Amphidinium theodori

The Chinese A. theodori strain 3XS55 fits the original description regarding the anterior 1/3 longitudinal flagellum insertion, a central pyrenoid, the poster located nucleus, plastic shape (metabolic movement), and asexual division within hyaline cysts (Karafas et al., 2017). One noticeable difference is related to the cell size, as cells of Chinese A. theodori strain 3XS55 were about twice larger than the type species (length: 28.0 μm vs. 16.6 μm, width: 24.9 μm vs. 12.6 μm) (Karafas et al., 2017). In addition, ring-like scales were not observed on the surface of the Chinese A. theodori strain 3XS55, possibly due to improper fixation. Amphidinium theodori was previously reported in Korotoga Fiji (the South Pacific), and U.S. Virgin Islands (the North Atlantic) (Karafas et al., 2017), here we extend its distribution to Xisha Islands (the North Pacific). All of these three spots are located between the tropic of cancer, implying that the distribution of A. theodori may be limited to the tropical zone.

4.1.9 Amphidinium tomasii

The Chinese strain of A. tomasii was characterized by an oval to elliptical shape, curved and rounded epicone, the insertion of the longitudinal flagellum in the middle third of the cell, and binary fission in the motile cell, which fits the original description (Karafas et al., 2017). A longitudinal cleave was observed on the dorsal hypocone of the division cell but not in the vegetable cell in the original description (Karafas et al., 2017). In Chinese strains no longitudinal cleave was observed in either the division cells or vegetable cells. A similar structure has been reported in the vegetable cells of A. thermaeum and A. klebsii (Klebs, 1884; Dolapsakis and Economou-Amilli, 2009). The furrows (longitudinal cleave) may only appear at a specific point in the life cycle and their taxonomic significance needs to be validated (Karafas et al., 2017; this study). Amphidinium tomasii was previously only reported in the northwest Atlantic (Dominican Republic, Puerto Rico, and Florida) and New Zealand (Karafas et al., 2017), but here we extend its distribution to the South China Sea.

4.2 Pigment profile

The typical chloroplasts of dinoflagellates possess peridinin, chlorophylls c2 and chlorophyll a as major pigments (Zapata et al., 2012). This type of chloroplast is considered to have originated from red alga via a secondary endosymbiosis (red alga get their chloroplast from cyanobacterium at the primary endosymbiosis) (Schnepf and ElbräChter, 1999; Zhang et al., 1999; Gould, 2012). About half of dinoflagellates have lost their chloroplasts and have become colorless during their evolution (Cavalier-Smith, 1992; Saldarriaga et al., 2004; Moestrup and Daugbjerg, 2007). In addition, a small number of dinoflagellates have replaced the peridinin-containing chloroplast with fucoxanthin-containing chloroplast, which originated from haptophyte alga, via the tertiary endosymbiosis (Morden and Sherwood, 2002; Yoon et al., 2002; Hackett et al., 2004; Curtis et al., 2012; Gould, 2012). According to Zapata et al. (2012), six pigment composition groups are present in photosynthetic dinoflagellate species. In general, most of the dinoflagellates can be classed as peridinin-and fucoxanthin-containing, while the others are alloxanthin-containing or violaxanthin-containing. All of the Amphidinium strains are peridinin-containing and were classed as "chloroplast Type 1", which contain chlorophylls a and c2 and peridinin as the major photosynthetic pigments (Table 3).

Sand-dwelling benthic dinoflagellates produce a far greater diversity of pigments than planktonic forms (Yamada et al., 2015). The finding of pheophorbide and pheophorbide a pigments in all tested Amphidinium strains is unexpected. Pheophorbides are well-known photosensitizers that are used in photodynamic therapy (Dougherty et al., 1998; Dolmans et al., 2003). Pheophorbides are a cancer treatment modality that selectively destroys malignant lesions through a photochemical reaction activated by light of the appropriate wavelength (Dougherty et al., 1998; Dolmans et al., 2003). In particular, pheophorbide a, the dephytylation and demetallation product of chlorophyll a, is formed in both algae and higher plants (Takamiya et al., 2000). This catabolic transformation is mediated by chlorophyllase and Mg-dechelatase enzymes (Matile and Schellenberg, 1996; Rodoni et al., 1997). Pheophorbide a based photodynamic therapy has been shown to completely inhibit the growth of a viral-induced hepatoma cell line Hep 3B (Chan et al., 2006). The cell-free extract of some Amphidinium strains has been reported to exhibit antifungal and antimicrobial properties (Echigoya et al., 2005; Kong et al., 2013; Nuzzo et al., 2014), whereas others have shown strong cytotoxic activity against a human tumor cell line (Bauer et al., 1995; Takahashi et al., 2007; Kumagai et al., 2017). Many polyketide bioactive compounds, such as caribenolide and amphidinolides, have been isolated from the cell-free extract (Bauer et al., 1995; Echigoya et al., 2005), but the pheophorbide / pheophorbide a ratio has not been well studied. Some Amphidinium strains, especially species identified as A. carterae and A. operculatum, have higher growth rates and are easily grown in culture (Murray et al., 2004). The high concentration of pheophorbide found in Amphidinium implies that it may be a good candidate as a natural source of photosensitizers.

5 CONCLUSION

Field surveys were undertaken to investigate the diversity of Amphidinium in shallow waters of the China Sea from 2012 to 2020. Eleven species were identified: A. carterae, A. gibbosum, A. operculatum, A. massartii, A. cf. massartii, A. fijiensis, A. pseudomassartii, A. steinii, A. thermaeum, A. tomasii, and an unidentified species. The last seven species have not been previously reported in Chinese waters. In addition, two strains were attributed to A. trulla based on LSU rRNA sequences. LSU rRNA sequences-based phylogeny revealed two groups (Groups Ⅰ and Ⅱ) within Amphidinium, which were consistent with their sulcus in touch with cingulum or not. High pheophorbide / pheophorbide a ratio was detected in Amphidinium implies that it may be a good candidate as a natural source of photosensitizers, which is a well-known anticancer drug.

6 DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon request.

Electronic supplementary material

Supplementary material (Supplementary Tables S1–S3 and Figs.S1–S5) is available in the online version of this article at https://doi.org/10.1007/s00343-021-1049-2.

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