Journal of Oceanology and Limnology   2022, Vol. 40 issue(6): 2146-2163     PDF       
http://dx.doi.org/10.1007/s00343-022-1321-0
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

LÜ Songhui, CHAO Aimin, LIANG Qianyan, CEN Jingyi, WANG Jianyan, JIANG Tao, LI Si
Is the dinoflagellate Takayama xiamenensis a synonym of Takayama acrotrocha (Kareniaceae, Dinophyceae)?
Journal of Oceanology and Limnology, 40(6): 2146-2163
http://dx.doi.org/10.1007/s00343-022-1321-0

Article History

Received Oct. 1, 2021
accepted in principle Nov. 14, 2021
accepted for publication Jun. 3, 2022
Is the dinoflagellate Takayama xiamenensis a synonym of Takayama acrotrocha (Kareniaceae, Dinophyceae)?
Songhui LÜ1,2,3#, Aimin CHAO4#, Qianyan LIANG1,2, Jingyi CEN1,2, Jianyan WANG5, Tao JIANG6, Si LI1,2     
1 Research Center of Harmful Algae and Marine Biology, Jinan University, Guangzhou 510632, China;
2 Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China;
3 Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai 519000, China;
4 Zhejiang Ecological and Environmental Monitoring Center, Hangzhou 310012, China;
5 Department of Science Research, Beijing Museum of Natural History, Beijing 100050, China;
6 School of Ocean, Yantai University, Yantai 264005, China
Abstract: The naked dinoflagellate Takayama acrotrocha was identified as responsible for a bloom in Shenzhen Bay, Guangdong, China, in early spring 2021. The identification was confirmed by light, scanning, and transmission electron microscopy and molecular data based on the LSU ribosomal DNA (rDNA) and ITS rDNA sequences. This is the first record of T. acrotrocha bloom in the South China Sea. The sulcus of T. acrotrocha was wide and extended onto the epicone as a short intrusion in general, sometime the intrusion was not apparent and some were finger-like. The apical groove was deeply sigmoid. The nucleus was large, ovoid to cup-shaped and occupied most of the epicone. A large, rounded pyrenoid surrounded by a starch sheath was located at the left side to the centre of the hypocone. Under epifluorescence illumination, a row of large vesicular knobs was observed on the upper border of the cingulum. The intraspecific morphological variabilities in the clonal cultures of T. acrotrocha were investigated carefully. Cells that share the same diagnostic characters used for the description of Takayama xiamenensis such as the finger-like sulcus, a large nucleus located in the epicone and the similar pyrenoid type were observed. The LSU rDNA sequences of T. acrotrocha and T. xiamenensis only differed in 3 base pairs (bp) for a sequence length of 673 bp (with a similarity of 99.55%). For these reasons, we propose T. xiamenensis as a junior synonym of T. acrotrocha.
Keywords: Takayama spp.    harmful algal bloom    morphology    phylogeny    South China Sea    
1 INTRODUCTION

The genus Takayama was proposed by de Salas et al. (2003) and was later classified into the family Kareniaceae with two other genera, Karenia and Karlodinium (Bergholtz et al., 2006). Typicalcharacteristics of this genus are a sigmoid apical groove on the epicone, and fucoxanthin and/or its derivatives as the main accessory pigments. Species in the genus Takayama were formerly classified under the genera Gymnodinium and Gyrodinium, for example Takayama acrotrocha (syn. Gyrodinium acrotrochum) (Larsen, 1996), Takayama cladochroma (syn. Gyrodinium cladochroma) (Larsen, 1994) and Takayama pulchella (syn. Gymnodinium pulchellum) (Larsen, 1994). Seven species have now been reported in the genus Takayama: T. acrotrocha, T. cladochroma, Takayama helix, T. pulchella, Takayama tasmanica, Takayama tuberculata, and Takayama xiamenensis (Larsen, 1994, 1996; de Salas et al., 2003, 2008; Gu et al., 2013); Takayama tasmanica is the type species.

Takayama species have been detected in Australia (Larsen, 1996), China (Gu et al., 2013), Italy (Siano et al., 2009), Japan (Omura et al., 2012), Korea (Cho et al., 2021), Singapore (Tang et al., 2012; Leong et al., 2015), and Florida, USA (Steidinger, 1998). Blooms dominated by Takayama species have been reported in China (Gu et al., 2013), Singapore (Tang et al., 2012; Leong et al., 2015), Japan (Omura et al., 2012), Italy, and the USA (Steidinger, 1998). Takayama species have been suspected to be toxic to marine organisms. For example, T. pulchella (reported as Gymnodinium pulchellum) blooms in the Indian River (Florida, USA) in 1990 and 1996 caused the deaths of many species of fish (Centropomus undecimalis, Mugil cephalus, Arius felis, Sciaenops ocellatus, Archosargus probatocephalus, and Pogonias cromis) and invertebrates (Callinectes sapidus and Penaeus spp.) (Steidinger, 1998).

In China, 4 species in the genus Takayama have been identified: T. xiamenensis, T. pulchella, T. tuberculata, and T. tasmanica (Gu et al., 2013; Law and Lee, 2013; Cheng et al., 2020). Takayama xiamenensis (identified as T. pulchella when it was first reported by Gu et al. (2006)) was isolated from Xiamen Bay during a bloom in 2003 (Gu et al., 2013). Takayama pulchella was commonly detected with a low density in Hong Kong coastal waters (Law and Lee, 2013). In 2011, a bloom of T. pulchella was reported in Tolo Harbor, Hong Kong coastal waters, although no fish kill was reported during the bloom (Law and Lee, 2013). Takayama tuberculata bloomed discontinuouslyfrom March to June in 2021 in Hong Kong coastal waters (https://www.afcd.gov.hk/english/publications/publications_press/pr2223.html) (Cheng et al., 2020). Takayama tasmanica was reported in the East China Sea (Gu et al., 2013) and Hong Kong waters (Law and Lee, 2013), and its toxicity is uncertain.

Takayama species are characterized by a sigmoid groove on the epicone, which is distinctly different from the linear apical groove of the genera Karenia and Karlodinium (Daugbjerg et al., 2000). Species in the genus Takayama are distinguished mainly by the details of cell nucleus, chloroplasts, pyrenoids, and sulcal intrusion onto the epicone (de Salas et al., 2003, 2008). However, the morphological interspecies differences between Takayama spp. are minimal, and accurate identification to species-level is extremely difficult, even for a taxonomy specialist. It has been considered that large subunit (LSU) rDNA, the commonly used molecular marker for identifying marine dinoflagellates, is even insufficient to distinguish Takayama species (de Salas et al., 2008; Gu et al., 2013), and thus they may be misidentified or omitted in traditional surveys based on morphological identification.

In this study, 5 isolates of T. acrotrocha were established from the South China Sea (SCS), and two of these were isolated from seawater during Takayama blooms. Morphological features of the isolates were carefully checked based on light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and analysis of their phylogenetic relationship with the related dinoflagellates was based on the LSU rDNA and the internal transcribed spacer (ITS) region. Examination of the lethality of T. acrotrocha on brine shrimp (Artemia salina) and marine medaka (Oryzias melastigma) was also conducted.

2 MATERIAL AND METHOD 2.1 Algae isolation and cultivation

Seawater was sampled from the coastal waters of the SCS for kareniaceans collection. Specific isolation for the bloom-causing species was conducted during an unarmoured dinoflagellate bloom in Shenzhen Bay (22°28′30″N, 113°57′46″E), Guangdong Province, from March 19th to April 15th, 2021. The peak cell density of the bloom was 7.1×107 cells/L. The bloom area was about 6.5 km2. The sea surface temperature during the bloom was 22.97–26.57 ℃. The sea surface salinity was 23.62–29.02. Microalgae in the seawater were concentrated using a phytoplankton net with a pore size of 10 μm, and the samples were taken to the laboratory for cell isolation. The algal clone was established by isolating a single cell from the net sample using a capillary pipette under the microscope. The single cell was first cultured in a well of a 96-well cell plate, and then transferred to a test tube for mass cultivation. The isolates were cultivated in L1 medium (Guillard, 1975) with a salinity of 28±1 and incubated at 20±1 ℃ with a photoperiod of 12-h light꞉12-h dark under 60±10 μmol photons/ (m2·s). The isolates were deposited at the Research Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou, China.

2.2 Light microscopy

Live cells were observed using an Olympus BX61 microscope (Olympus, Tokyo, Japan) with magnifications of 400× and 1 000×. Micrographs of more than 45 randomly selected cells were taken using a QImaging Retiga 4000R digital camera (QImaging, Surrey, BC, Canada). Cell length and width were measured by Image-Pro Plus 6.0 image acquisition software (QImaging, Surrey, BC, Canada). For epifluorescence microscopy, 2 mL of algal culture at the exponential growth stage was stained with SYBR Green stain (Thermo Fisher, Waltham, MA, USA). The shape, size, and location of the cell nucleus and chloroplasts were identified under an Olympus BX61 epifluorescence microscope.

2.3 Scanning electron microscopy

Cells collected from the Takayama bloom and cells of the isolates were fixed by OsO4 with a final concentration of 2%. Fixed samples were filtered, dehydrated, critical point dried, and sputter coated as described in Wang et al. (2018). The mounted samples were examined using a Zeiss Ultra 55 field emission scanning electron microscope (Zeiss, Jena, Germany).

2.4 Transmission electron microscopy

Vegetative cells in the exponential phase were double fixed by OsO4 (with a final concentration of 0.5%) and glutaraldehyde (with a final concentration of 2%) at room temperature for 30 min. The fixed samples were dehydrated in a series of ethanol solutions (10%, 30%, 50%, 70%, 90%, and 95%), followed by three changes in 100% ethanol. The dehydrated cells were embedded in Spurr's resin via propylene oxide. The embedded blocks were sectioned on an Ultracut E Ultramicrotome (Leica Microsystems, Wetzlar, Germany) using a diamond knife. The sections were transferred to a formvar film and double-stained with uranyl acetate and lead citrate. The stained sections were examined in a JEOL JEM-1010 electron microscope (Jeol Ltd., Tokyo, Japan) operated at 30–50 kV.

2.5 Genomic DNA extraction and PCR amplification

A 2-mL aliquot of the isolates in the exponential growth phase was harvested by centrifugation. The genomic DNA was extracted using a Takara MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to the protocol provided by the manufacturer. Partial LSU rDNA and the ITS sequences were amplified by PCR using primers D1R-F (5′-ACCCGCTGAATTTAAGCATA-3′) (Scholin et al., 1994), D3B-R (5′-TCGGAGGGAAC-CAGCTACTA-3′) (Nunn et al., 1996), ITS1F (5′-TCGTAACAAGGTTTCCGTAGGTG-3′) and ITS1R (5′-ATATGCTTAAGTTCAGCGGG-3′) (Pin et al., 2001). The PCR amplification protocols for LSU rRNA and ITS rDNA were followed as described in Cen et al. (2020). The LSU rDNA sequences of the causative species in the Takayama blooms on March 19th 2021 were determined by single-cell PCR using the same PCR protocol mentioned above.

2.6 DNA sequence alignment and phylogenetic analysis

The partial LSU rDNA and ITS rDNA of the isolates were deposited in GenBank with accession numbers MZ948839–MZ948843 for the LSU rDNA sequences and MZ948833–MZ948837 for the ITS rDNA sequences. LSU rDNA and ITS rDNA sequences of the kareniacean species closely related to the Takayama spp. were searched from GenBank using the online software BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were aligned using Clustal X software in MEGA 7.0 (Kumar et al., 2016). BioEdit 7.0.5 was used to edit and excise the redundant bases at the two ends of the sequences for the phylogenetic analysis (Hall, 1999). The aligned lengths used for phylogenetic tree construction were 673 base pairs (bp) for the LSU rDNA and 588 bp for the ITS rDNA. The genetic divergence (p-distance) between Takayama species was calculated by MEGA 7.0. The phylogenetic trees were constructed by MEGA7.0 software using the maximum likelihood (ML) (model T3+G+I) in 1 000 bootstrap running. The optimal model of Bayesian inference (BI) was selected by MrModeltest 2.2 and phylogenetic analysis was conducted using MrBayes 3.2 (Ronquist and Huelsenbeck, 2003).

2.7 Pigment extraction and analysis

A 10-mL algal culture in the mid-exponential phase was filtered onto a fiber membrane with a 0.45-μm pore size (Whatman, Maidstone, UK), and was stored in a refrigerator at -80 ℃ for later treatment. Pigment extraction and analysis were performed according to the methods described in Zapata et al. (2000) and Wang et al. (2018).

2.8 Cell acute toxicity analysis

Artemia salina bioassay: A. salina was incubated at 25 ℃ in a 500-mL glass beaker for 24 h. Seawater collected from the Takayama bloom with a concentration of 2.5×107 cells/L was used for the lethality test. A Prorocentrum obtusidens (3×107cells/L) culture was used as the blank control and non-bloom seawater was used as the negative control. Twenty specimens of A. salina were used in each test, and tests were performed in triplicate. Survival of A. salina were recorded every 6 h for 48 h.

Marine medaka (Oryzias melastigma) bioassay: Brood stock of O. melastigma were kindly provided by the State Key Laboratory in Marine Pollution of the City University of Hong Kong. Ten healthy, adult O. melastigma were transferred into a 5-L bucket containing 3-L bloom seawater at 25 ℃. A photoperiod of 12-h light꞉12-h dark was used, and the fish were fed twice daily. Acute toxicity was assessed by measuring the lethal effect of the seawater collected from the Takayama bloom (with a concentration of 2.5×107 cells/L) on the marine medaka over 48 h. The non-bloom seawater and P. obtusidens culture (1×107 cells/L) were used as the negative control and the blank control, respectively. The survival ratio of O. melastigma was observed at 0, 4, 8, 12, 24, and48 h. Three replications were set for each test.

3 RESULT

Five strains of Takayama spp. were established in this study, and detailed information for the isolates is listed in Table 1. All strains have an apparent sigmoid groove around the cell apex, which is consistent with the typical apical groove of the genus Takayama. All the 5 strains and single cells from the Takayama bloom shared the same LSU rDNA and ITS rDNA sequences. After a careful and comprehensive analysis of the taxonomy and phylogeny, the 5 isolates were identified as T. acrotrocha. The causative species of the dinoflagellate bloom in Shenzhen Bay, Guangdong Province from March 19th to April 15th 2021 was determined to be T. acrotrocha.

Table 1 Information for the Takayama acrotrocha isolates from the South China Sea
3.1 Morphological characteristics of Takayama acrotrocha 3.1.1 Morphology of Takayama acrotrocha under LM

Cells of T. acrotrocha were unarmoured, small and oval, with a length of 14–20 μm and a width of 11–17 μm (Supplementary Table S1). Cells were slightly flattened dorso-ventrally, with a length to width ratio of 1.02–1.64 (Supplementary Table S1). Some small cells less than 14 μm in length and no more than 10 μm in width were also observed. The epicone was hemispherical (Fig. 1ab). The hypocone was irregular in shape, and slightly longer than the epicone (Fig. 1cd). In the ventral view, the hypocone was bilobed, truncated, and incised antapically (Fig. 1ab). The cingulum was shallow, with a displacement of 1–1.5 times of the cingulum width (approximately 1/4 of the cell length) (Fig. 1ab). The sulcus was wide in the hypocone, becoming narrow in the inter-cingular region, and extended onto the epicone as a short intrusion (sometimes the intrusion was not apparent) (Fig. 1ab). The apical groove was sigmoid and curved around the cell apex, starting above the anterior of the sulcal intrusion and extending to the dorsal part of the epicone (Fig. 1a & c). A large, rounded pyrenoid was located at the left side or in the middle of the hypocone from the ventral view (Fig. 1d). The chloroplasts were numerous, irregularly shaped, and distributed peripherally in the cell, arranged in a helicoidal pattern on the epicone and more abundant in the hypocone (Fig. 1ac & e-f). The nucleus was large, ovoid to cup-shaped, and occupied the most part of the epicone (Fig. 1di). Under epifluorescence illumination, a row of large vesicular knobs was clearly observed on the upper border of the cingulum (Fig. 1ef).

Fig.1 Light microscopy images of vegetative cells of Takayama acrotrocha (strain No. TAG9) a. ventral view of a cell focus on the cell surface, showing the general shape of the cell, apical groove (arrowhead), sulcal intrusion (black arrow), and the tube-like structure (white arrow); b. ventral view of a cell focus on the sulcus intrusion (black arrow) and the tube-like structure (white arrows); c. dorsal view of a cell showing the posterior end of the apical groove (black arrow) and amphiesmal knobs (white arrow); d. dorsal view of a cell showing the big ovoid nucleus (n) and a large rounded pyrenoid (py), the cell surface was covered by amphiesmal vesicles (black arrow); e–f. epifluorescence micrograph for cells stained by SYBR Green dye showing the distribution of chloroplasts (in red), and the position of nucleus (in green), and a row of vesicular knobs on the upper rim of the cingulum (white arrows); g. epifluorescence micrograph showing a cell with a cup-shaped nucleus; h–i. sister cells in division. Scale bars=10 μm.

Generally, there was no significant morphological variance among the different strains, except for the minimal variability in the length and width of the sulcus intrusion onto the epicone. For cells sampled from the bloom seawater, the sulcal intrusion was short but could be observed under LM (Fig. 2ab). The sulcal intrusion in strain TAG9 was of a similar length and shape as in the field cells (Fig. 2cd). Most cells of strain TAH8 had a relatively long and finger-like sulcal intrusion (Fig. 2ef), and some cells with a short intrusion were also observed (Fig. 2gh). The sulcal intrusion was not obvious in strain TAF10 (Fig. 2ij), while a short intrusion in some cells was visible (Fig. 2kl).

Fig.2 Light microscopy images of vegetative cells of Takayama acrotrocha in ventral view, focused on the sulcal intrusion (arrows) Strain Nos: a–b. field samples; c–d. TAG9; e–h. TAH8; i–l. TAF10. Scale bars=10 μm.
3.1.2 Morphology of Takayama acrotrocha under SEM

Cells were oval (Fig. 3ad). The epicone was hemispherical and the hypocone was slightly irregular in shape (Fig. 3ae). The posterior of the hypocone was truncated and incised by the sulcus (Fig. 3cd). The cingulum displaced approximately 1/4–1/3 of the cell length (Fig. 3b). The sulcus extended briefly onto the epicone (Fig. 3ad), and the sulcus intrusion was not obvious in some cells (Fig. 3ef). A tubular structure was observed in the sulcus (Fig. 3ab & ef). The apical groove was coiled along the apex of the cell in a 'S' shape (Fig. 3ab, e, & gh). The apical groove started from the right of the sulcal intrusion, ran around the cell apex and extended approximately 1/3 of the dorsal epicone (Fig. 3bc). Both rims of the apical groove were slightly ridged (Fig. 3bc). The ventral pore was small, sometimes slit-like, situated on the left side of the epicone and well above the anterior of the cingulum (Fig. 3ab & ef). A tube-like structure was positioned along the sulcus in the inter-cingular region (Fig. 3ab & ef). The epicone surface on the right lobe, which lies between the ventral end of the apical groove and the sulcus was swollen. A slightly slit-like ventral pore was observed on the swollen structure (Fig. 3ef). In some well-preserved cells, a row of amphiesmal knobs was neatly arranged along the upper rim of the cingulum (a structure that has not been reported in the genus Takayama before) (Fig. 3bc & g). A dorsal pore was observed in the posterior end of the apical groove of some (but not all) cells (Fig. 3hi).

Fig.3 Scanning electron microscopy of the cells of Takayama acrotrocha collected from Shenzhen Bay, Guangdong during a bloom a. ventral (the left) and apical view (the right) of cells showing the cell general morphology. The sigmoid apical groove, the ventral pore (arrowhead), a short sulcus intrusion (black arrow), and the tube-like structure (white arrow) were clearly observed under SEM; b. ventral view of a cell showing the cell surface was cover by a layer of vesicles. A line of amphiesmal vesicles arranged neatly along the upper border of the cingulum (white arrows with dash line). A ventral pore was positioned on the left side of the epicone (arrowhead). A short sulcus intrusion (black arrow) and the tube-like structure (white arrow) were observed; c. dorsal view of a cell showed the apical groove extending to approximately 1/3 of the dorsal epicone. A line of amphiesmal vesicles located along the upper border of the cingulum from the dorsal view (white arrows with dash line); d. posterior view of a cell, showing the hypocone was incised by the sulcus; e–f. ventral view of a cell, focused on the common ventral pore (black arrowhead) and a pore on the lower right part of epicone (white arrowhead), the anterior of the sulcus on the epicone (black arrow) and the tube-like structure (white arrow). A shallow furrow was found above and parallel to the entire anterior edge of the cingulum (red arrow); g. lateral view (from the left of cell) showing the cell was slightly dorso-ventrally flattened and a line of amphiesmal knobs on the upper border of the cingulum; h–i. lateral view (from the right of cell) of a cell showing the sigmoid apical groove, the ventral pore (black arrowhead), and a dorsal pore on the posterior of the apical groove (white arrowhead). Scale bars=2 μm.

The cell surface was covered by a thick layer of large and wart-like amphiesmal vesicles, especially on the hypocone (Fig. 4ah). The amphiesmal vesicles on the epicone were arranged in approximately 6 rows, running in a direction that followed the sigmoid apical groove (Fig. 4c). The amphiesmal vesicles on the hypocone were much bigger, pentagonal to hexagonal in shape, and were also in approximately 6 rows (Fig. 4h). The SEM photos showed that the sulcus intrusion onto the epicone of T. acrotrocha was short in general (Figs. 35); however, some finger-like sulcus intrusions were also observed (Figs. 4de, 5h, & 5k).

Fig.4 Scanning electron microscopy of the vegetative cells of Takayama acrotrocha (Strain Nos: a–f, TAG9; g–i, TAF10) a. ventral view of a cell showing the general morphology of T. acrotrocha; b. dorsal view of a cell showing the amphiesmal vesicles on the cell surface, and the extension of the apical groove on the dorsal epicone (arrow); c. lateral view of a cell showing slight dorso-ventral compression, and the 6 rows of amphiesmal vesicles on the epicone (e1–e6); d–e. ventral view of cells, showing the short finger-like sulcus intrusion onto the epicone. Note that sometimes the ventral pore on the epicone was unapparent; f–g. ventral view of cells showing the inconspicuous sulcus intrusion (arrows); h. dorsal view of a cell showing the cell surface was covered by a layer of amphiesmal vesicles, and the 6 rows of vesicles on the hypocone (h1–h6); i. ventral view of cells showing the inconspicuous sulcus intrusion (arrow). Scale bars=2 μm.
Fig.5 Scanning electron microscopy of vegetative cells of Takayama acrotrocha (strain Nos: a–f, TAH8; g–l, TAD11) a. ventral view of a cell showing the open sulcus intrusion (black arrow) onto the epicone; b. apical view of a cell showing the apical groove and the anterior of the sulcus intrusion (black arrow); c. dorsal view of a cell showing the apical groove on the dorsal epicone; d–f. cells in the ventral view focused on the sulcus intrusion (arrows); g. lethal view of a cell, showing a row of relatively large amphiesmal knobs lay on the upper border of the cingulum (asterisks); h. ventral view of a cell showing the short finger-like sulcus intrusion (arrow); i. dorsal antapical view of a cell showing the irregular shaped amphiesmal vesicles on the hypocone; j. dorsal view of a cell showing the posterior of the apical groove (black arrow); k. magnification of the short finger-like sulcus intrusion (black arrow); l. magnification of the apical groove, showing the amphiesmal vesicles (asterisks) on the epicone running along the apical groove. Scale bars=2 μm.
3.1.3 Ultrastructure of Takayama acrotrocha under TEM

The longitudinal section of the cell from the ventral side revealed the general ultrastructure of T. acrotrocha (TAH8): a dinokaryotic nucleus withcondensed chromosomes, chloroplasts, a pyrenoid and trichocysts (Fig. 6ac). The nucleus was oval, situated in the epicone and extending to the cell centre (Fig. 6a). The trichocysts were scattered sparsely in the cell (Fig. 6a & d). Numerous chloroplasts were distributed peripherally in the cell, and were more abundant in the hypocone (Fig. 6ac). Stacks of three thylakoids were arranged parallel to each other along the longitudinal axis of the chloroplasts (Fig. 6ae). A large, rounded pyrenoid surrounded by a starch sheath was observed in the hypocone, situated at the anterior of the chloroplast and sometimes separated from it (Figs. 6ef & 7ab). The pusular system consisted of a central collecting chamber and a number of pusular vesicles which opened directly into the collecting chamber and connected with the canal of the flagellum (Fig. 7c). The amphiesmal vesicles were regularly distributed on the surface, and were larger on the hypocone than on the epicone (Figs. 6ae & 7de). Sections through the apical groove showed that the apical groove was composed of 4 rows of amphiesmal vesicles (Fig. 7de). Several small vacuoles of electron-opaque material were gathered beneath the apical groove, and some of these were fused with the cytoplasm membrane (Fig. 7de). Rows of cortical microtubules were arranged beneath the inner membrane, sometimes in groups of five (Fig. 7e).

Fig.6 Transmission electron microscopy images of Takayama acrotrocha (TAH8) a–b. longitudinal section through the cell in ventral, showing nucleus (N), numerous condensed chromosomes, chloroplasts (Ch), and pyrenoid (py); c. laterally longitudinal section through the cell; d. cross section of cell on the hypocone; e. oblique cross section through the cingulum; f. pyrenoid surrounded by a starch sheath (ss). N: nucleus; Ch: chloroplast; av: amphiesmal vesicles; ag: apical groove; c: cingulum; lc: longitudinal canal; g: Golgi body; py: pyrenoid; pv: pulsular vesicles; s: sulcus; ss: starch sheath; tf: transverse flagellum; tb: transverse basal body; tr: trichocyst. Scale bars=2 μm.
Fig.7 Ultrastructure of Takayama acrotrocha (TAH8) a. longitudinal section of a pyrenoid (py) in a chloroplast (Ch); b. longitudinal section of a pyrenoid surrounding by a starch sheath (ss); c. transverse section of the pusular system which consisted of a central collecting chamber (cc) and a number of pusular vesicles (pv); d–e. longitudinal section of the apical groove (ag) showing details of amphiesmal vesicles (av), which is consisted of the outermost membrane (om), the membrane beneath the outermost membrane (opm), the thin thecal plate (p), pellicular layer (pl), and cytoplasmic membrane (cm). Vacuoles with electron-opaque vesicles (Vov) were observed protruding the amphiesmal membrane; ov: opaque vesicles.. Some microtubes (mt) lay beneath the amphiesmal membrane. Scale bars=0.5 μm.
3.2 Phylogenetic analysis

The 5 isolates of T. acrotrocha from the SCS shared the identical LSU rDNA and ITS rDNA sequences. The divergence based on the partial LSU rDNA sequence (673 bp) between T. acrotrocha from the SCS and the other 17 strainsof Takayama species (Supplementary Table S2) ranged from 0.52% to 2.88%; for example, 0.36% from T. xiamenensis, 2.14% from T. helix, and 2.88% from T. tuberculata (Table 2). The 5 strains of T. acrotrocha from the SCS differentiated from T. xiamenensis (which was isolated from the East China Sea, GenBank ID: AY764178) in only 3 bp. Based on the ITS rDNA (total 589 bp), the difference between the T. acrotrocha from the SCS and T. acrotrocha from Italy (strain No. MC728-D5 and GenBank ID: HM067011) was 0.50%, and from T. xiamenensis (strain No. TPXM and GenBank ID: AY764179) was 0.30%.

Table 2 The genetic divergence (p-distance) between Takayama species based on the partial large subunit ribosomal DNA (LSU rDNA) (673 bp)

Phylogenetic tree inferred from the LSU rDNA showed that the 5 isolates of T. acrotrocha from the SCS clustered into the genus Takayama with the other Takayama species (Fig. 8), and formed a single branchwith T. acrotrocha from Italy (strain No. MC728-D5 and GenBank ID: FJ024703), Korea (strain No. TaLomme01 and GenBank ID: MZ358881), Singapore (strain No. GT15 and GenBank ID: DQ656116) and T. xiamenensis from the East China Sea (strain No. TPXM and GenBank ID: AY764178) with a support value of 80/0.98 (Bootstrap values/ posterior probabilities). Based on the LSU rDNA phylogeny, species in the genus Takayama clustered into two sister clades: clade 1 comprised T. acrotrocha, T. xiamenensis, T. cf. pulchellum, and T. helix (99/1.00), and clade 2 comprised T. tasmanica and T. tuberculata (99/1.00). In the genus Takayama, the ITS rDNA sequences were only available for T. acrotrocha and T. xiamenensis, and the ITS rDNAsequences of the two species formed a single clade with a support value of 67/0.91 (Fig. 9).

Fig.8 Molecular phylogeny of Takayama species and closely related species based on partial LSU rDNA Gymnodinium catenatum and G. fuscum were used as outgroup. The bootstrap analysis ran with 1 000 replication, and the numbers on the branches are the statistical support values for the cluster on the right. Only Bootstrap values > 50% and posterior probabilities > 0.5 were shown. Takayama acrotrocha from the South China Sea are bold.
Fig.9 Molecular phylogeny of Takayama species and closely related species inferred from ITS rDNA Gymnodinium catenatum was selected as outgroup. The bootstrap analysis ran with 1 000 replication, and the numbers on the branches are the statistical support values for the cluster on the right. Only Bootstrap values > 50% and posterior probabilities > 0.5 were shown. Takayama acrotrocha from the South China Sea are bold.
3.3 Pigment composition

The pigment composition of T. acrotrocha (strain No. TAH8) is similar to that of the fucoxanthin-containing dinoflagellates within the genus Takayama (Table 3). A total of 11 pigments: chlorophyll-c2 (chl c2), chlorophyll-c3 (chl c3), chlorophyll a (chl a), Mg-2, 4-divinylpheoporphyrin (MgDVP), 19′-butanoyloxy-fucoxanthin (but-fuco), fucoxanthin (fuco), prasinoxanthin (pras), 19′-hexanoyloxy-fucoxanthin (hex-fuco), diadinoxanthin (diad), canthaxanthin (cantha), and gyroxanthin-diester (gyro) were detected using high performance liquid chromatography (HPLC) based on the absorption spectra and retention time according to the commercial standard pigments (Fig. 10). The main pigment is chl a, and fucoxanthin is the main accessory pigment.The carotenoid pigments were quantified as ratios relative to chl a and were hex-fuco (0.29%), but-fuco (4.92%), fuco (338.30%), diad (16.88%), cantha (5.66%), and gyro (1.83%). Seven unidentified pigments were detected at retention times of 10.20, 22.70, 24.00, 26.30, 28.60, 29.00, and 32.60 min.

Table 3 Mass pigment ratios (w꞉w, carotenoids꞉Chl a) of Takayama kareniacean species
Fig.10 Pigment chromatogram of Takayama acrotrocha (strain No. TAH8)
3.4 Cell toxicology on brine shrimp and marine medaka

Acute toxicity of T. acrotrocha on A. salina and adult O. melastigma was checked by exposing the two organisms to seawater samples containing the blooming T. acrotrocha with a concentration of 3.05×107 cells/Lfor 48 h. No lethality of T. acrotrocha on A. salina and adult O. melastigma was observed over 48 h.

4 DISCUSSION 4.1 Morphology

Takayama acrotrocha was first described by Larsen (1996) as Gyrodinium acrotrochum, an unarmoured dinoflagellate with a sigmoid apical groove, discshaped chloroplasts with individual pyrenoids and no sulcal intrusion onto the epicone. The original description of T. acrotrocha was based on eight cells from field samples from Hobsons Bay, Australia (referred to in Larsen (1996): 'At the laboratory the water samples were kept in culture cabinets at 15 ℃ until concentrated for microscopy by continuous centrifugation'). The description in Larsen (1996) was based on light microscopy, and no SEM details or DNA sequences were given. Siano et al. (2009) redescribed T. acrotrocha (based on clonal cultures from the Gulf of Naples, Italy) with more morphological features such as the arrangement of the apical groove, the presence of a pore on the ventral epicone, the slit in the sulcal zone, the small sulcal intrusion into the epicone and the tube-shaped structure along the sulcus in the inter-cingular region, as well as LSU rDNA sequences. Four cultures of T. acrotrocha from tropical waters in Singapore were established for a bloom monitoring project by Tang et al. (2012). The Singapore isolates of T. acrotrocha had morphological characteristics which were consistent with the descriptions by Larsen (1996) and Siano et al. (2009). Numerous multilateral plate-like surface vesicles, and possibly a peduncle between the two emerging points of flagellates, were additionally observed in the Singapore isolates of T. acrotrocha under SEM.

Takayama acrotrocha can be distinguished under LM by the presence of a large, oval nucleus in the epicone, a sigmoid apical groove and chloroplasts which are located mostly in the hypocone (Larsen, 1996; Siano et al., 2009). Under SEM, T. acrotrocha was characterized by a sigmoid apical groove starting from the ventral epicone and extending to less than halfway along the dorsal epicone, passing mostly on the left side instead of the middle of the cell apex. A small and deep sulcal intrusion onto the epicone of T. acrotrocha was clearly observed by Siano et al. (2009, their figure 27) and Tang et al. (2012, their figure 2C).

The isolates of T. acrotrocha from the SCS shared the typical features described by Larsen (1996), Siano et al. (2009) and Tang et al. (2012) as the presence of a large oval nucleus in the epicone, the sigmoid apical groove that encircles the cell epicone and the chloroplasts located mostly in the hypocone, allowing the identification of our isolates as T. acrotrocha. According to the original description, the sulcus of T. acrotrocha does not intrude onto the cell epicone, and this is a key feature differentiating T. acrotrocha from the other Takayama species. However, with the high resolution of SEM, sulcus intrusion was found in T. acrotrocha by Siano et al. (2009) and Tang et al. (2012). The sulcus intrusion was apparent in our study both under LM and SEM, though cells with a less obvious sulcus intrusion were observed in strain TAF10 (see Fig. 2il). The original micrographs in Larsen (1996, their figures 24) were focused on the anterior and posterior of the apical groove and nucleus, not on the sulcus. An extension of the sulcus onto the epicone in T. acrotrocha was clearly observed in our strains (Fig. 2). Our finding was confirmed in T. acrotrocha from Korean coastal waters that the sulcus was not apparent in figure 2A but was visible in figure 2B in Cho et al. (2021), despite it was not marked by the author.

The shape and position of the nucleus is another feature for the identification of Takayama species. As illustrated in de Salas et al. (2008), the nucleus in Takayama species can be classified into two shapes, oval or cup-shaped, located mostly in the epicone or on the left side of the cell from epicone to hypocone, see figure 10 in de Salas et al. (2008). T. acrotrocha differed from T. tasmanica and T. tuberculata in having a nucleus that is oval, not cup-shaped, and differed from T. pulchella and T. cladochroma in having a nucleus located in the epicone rather than on the left side of the cell. Takayama acrotrocha was similar to T. helix in having elongated chloroplasts arranged in spiraling bands in the epicone, and with spiraling surface impressions that parallel the chloroplasts on the epicone. However, the very shallow sigmoid groove of T. helix distinguished it from the other members in the genus Takayama.

Chloroplasts of different shapes and with/without individual pyrenoids were the most apparent difference among the Takayama species. Chloroplasts in the genus Takayama can be grouped into 2 types: chloroplasts radiating from a central pyrenoid (in T. tasmanica and T. tuberculata) and chloroplastscontaining individual pyrenoids (in T. cladochroma, T. helix, and T. pulchella). As described by Larsen (1996), chloroplasts of T. acrotrocha had individual pyrenoids: 'Several irregular, disc-shaped chloroplasts are present, mostly located in the hyposome, each with a pyrenoid'. However, the individual pyrenoids in chloroplasts of T. acrotrocha were not confirmed in the later re-examinations by Siano et al. (2009) and Tang et al. (2012). In the present study, an oval pyrenoid surrounded by a starch sheath under the nucleus was observed using both LM (Fig. 1d) and TEM (Fig. 6a), and such an oval pyrenoid was also observed in the T. acrotrocha from Korea, see figure 2C in Cho et al. (2021).

The amphiesma vesicles of T. acrotrocha were clearly present on the surface of some well-preserved cells in Tang et al. (2012) and also in our isolates. The vesicles on the epicone were distributed in approximately 6 rows, extending along the 'S'-shaped apical groove. The amphiesma vesicles on the hypocone were large and wart-like, and were also arranged in 6 layers. A line of amphiesma vesicles lying on the upper layer of the cingulum and forming a pearl necklace-like structure in the well-fixed cells were observed in our isolates, this feature has not been reported in Takayama spp. before. Wart-like amphiesma vesicles on the cell surface were also recorded in T. tuberculata, after which the species was named. We suggest that the wart-like amphiesmal vesicle might not be a species-specific characteristic only for T. tuberculata. Irregularly shaped vesicles could exist on the surface of other Takayama species, and may have been lost in the preparations for SEM, and this has led to a lack of useful features to differentiate T. tasmanica from T. tuberculata.

In the first report of T. acrotrocha by Larsen (1996), the common ventral pore on the left side of the epicone was not found, and this pore was also not mentioned by Tang (2012). A very small ventral pore was present in the Italian strain of T. acrotrocha under SEM (Siano et al., 2009). In this paper, a ventral pore above the sulcal extension on the left side of the epicone was observed both in field cells and clonal cultures, although sometimes it was not obvious in cells covered by the amphiesmal vesicles or cell external mucus. In addition to the ventral pore, a slit-like ventral pore well below the anterior of the apical groove was observed both in the Italian strains and in our cells. Another narrow, shallow slit, above and parallel to the entire anterior edge of the cingulum observed in the Singaporean strains (see figure 2C in Tang et al. (2012)), was not detected in all our strains; however, an unapparent narrow and shallow furrow (rather than a slit) was found in our cultures (see Fig. 3ef), at the same position mentioned by Tang et al. (2012).

4.2 Phylogeny

Gu et al. (2013) considered that the LSU rDNA was too conservative to set boundaries for Takayama species. No DNA sequence was available for the holotypes of T. acrotrocha, T. cladochroma, and T. pulchella, and DNA sequences are only available for T. acrotrocha, T. helix, T. xiamenensis, T. tasmanica, and T. tuberculata in GenBank. Both ML and the BI phylogenetic tree allow us to infer that species in the genus Takayama cluster into 3 subclades: the five T. acrotrocha strains established in this study formeda branch with the Singapore strain of T. acrotrocha (DQ656117) and T. xiamenensis (Tang et al., 2012), T. tasmanica and T. tuberculata formed the secondbranch and T. helix was the third branch. The 3 branches of Takayama in the tree were consistent with the morphological differences among the Takayama species: the first clade, which included T. acrotrocha and T. xiamenensis, has a deep 'S'-shaped apical groove, an oval nucleus occupies most of the epicone and some chloroplasts have bulging pyrenoids. The second branch, which consists of T. tasmanica and T. tuberculata, has a deep S-shaped apical groove, acup-shaped nucleus on the epicone and chloroplasts radiating from a central pyrenoid agglomeration. The third branch, T. helix, has a very shallow, sigmoid, apical groove. Additionally, the wart-like amphiesma vesicle in our strains of T. acrotrocha meant that there were no useful features left to distinguish T. tasmanica from T. tuberculata, and the two speciesmight be conspecific; thus, we concluded that the LSU rDNA is still a valid marker for separating Takayama morphotypes.

The genera Takayama and Karlodinium formed a monophyletic group in the phylogenetic trees, while Brachidinium capitatum, Asterodinium gracile, and Karenia species clustered in a single clade (Fig. 8). The main character of Brachidinium and Asterodinium is an unarmored pelliculate cell with four or five radiating elongated extensions. The extensions of brachidiniaceans are quite variable, with some intermediate specimens resembling cells of Karenia papilionacea and Karenia bidigitata (Gómez et al., 2005), which was further validated by DNA evidence demonstrating that Brachidinium is phylogenetically related to Karenia species (Henrichs et al., 2011; Benico et al., 2019). In 2012, Gómez (2012) proposed that the genera Takayama, Karlodinium, and Karenia should be included in the family Brachidiniaceae, alongside the genera Asterodinium, Brachidinium, Gynogonadinium, Microceratium, Pseliodinium, and Torodinium. However, no cell with long extensionshas been found in kareniaceans, implying that more life cycle and morphology studies are needed for members of these two families. Recently, new taxa Shimiella and Gertia have been placed to the familyKareniaceae (Takahashi et al., 2019; Ok et al., 2021). As a result, we will revisit the definitions for members of the two families Brachidiniaceae and Kareniaceae in the future, and we will continue to include the genus Takayama in the Kareniaceae family in this publication.

4.3 Pigment composition

The pigment profile of T.acrotrocha was categorized as Type-3 following the pigment classification of dinoflagellates by Zapata et al. (2012). The Type-3 profile was characterized with fucoxanthin/19′-acyloxyfucoxanthins/gyroxanthin diesters/chl c2 and chl c3. Gyroxanthin diester, which is usually a marker pigment for Karenia spp. and Karlodinium spp. (Bergholtz et al., 2006; Johnsen et al., 2011; Chang and Gall, 2013), was also detected in T. acrotrocha, but not in T. helix. Takayama acrotrocha from the SCS contained fucoxanthin as the most abundant carotenoid among the 5 Takayama spp., with a ratio to chl a as high as 3.383 0. Usually, species in the family Kareniaceae are known for containing fucoxanthin and/or its derivatives as major carotenoids, and lack peridinin; however, Gertia stigmatica, a species with the peridinin-type chloroplast has been reported in the family Kareniaceae recently (Takahashi et al., 2019), suggesting a more-diverse plastid origin in this family.

4.4 Toxicity

The genus Takayama is affiliated to the toxigenic family Kareniaceae, and some species have been associated with toxic bloom events. However, no ichthyotoxicity for T. pulchellum was determined in vivo (Steidinger, 1998). No toxicity or stress was observed in the bioassay by all SCS T. acrotrocha cultures and the bloom seawater in the laboratory; and no fish kill was associated with the T. acrotrocha bloom in the Zhujiang (Pearl) River estuary, nor with the Singaporean isolates of T. acrotrocha, suggesting the toxicity protocol of Takayama species need more investigation. Multi-dinoflagellate blooms dominated by T. xiamenensis, Karlodinium veneficum, and K. australe have been suspected of being associatedwith massive fish kills in Singapore (Leong, 2015). Future research into accurate species-specific identification of these naked and harmful algal bloom species should be carried out.

4.5 Comparison between T. acrotrocha and T. xiamenensis

In 2013, Gu et al. (2013) identified a new species T. xiamenensis from Xiamen Harbour in the East ChinaSea. Partial LSU rDNA sequences of the two strains of T. xiamenensis were identical to the T. acrotrocha strains from Italy, and had a high similarity (99.72%) to the Singapore strain GT15. Takayama xiamenensis was characterized by a distinctive finger-like intrusion onto the epicone; a large, cup-shaped nucleus on the epicone; and some chloroplasts with large, bulging pyrenoids. As described by Gu et al. (2013), the main difference between T. xiamenensis and T. acrotrocha was the finger-like sulcus in the former, while no such intrusion was found in the latter. However, in our study, cells with or without a sulcus intrusion were both observed in the different strains of T. acrotrocha, and some sulcus intrusions were finger-like, suggesting that T. acrotrocha also had a finger-like sulcus intrusion. Although the author described the nucleus of T. xiamenensis as cup-shaped, an ovoid nucleus was also recognized in T. xiamenensis (Gu et al., 2013, figure 4). Moreover, different from the cup-shaped nucleus in T. tasmanica and T. tuberculata (which surrounds the central pyrenoid agglomeration dorsally, laterally, and apically to form a cup shape), the cup-shaped nucleus of T. xiamenensis was seemed as a shading by the large, round pyrenoid in the hypocone (see figure 4 in Gu et al. (2013)). Figure 13 in Gu et al. (2013) also suggests that the nucleus did not surround the pyrenoid. A bulging pyrenoids surrounded by a sheath of starch or starch granules in T. xiamenensis were also clearly recognized in our T. acrotrocha strains (Fig. 6ab & ef). Additionally, the partial LSU rDNA sequences of T. xiamenensis were identical to the T. acrotrocha strains from Italy, and had a high similarity (99.7%) to the Singapore strain GT15 and our strains. All the data suggested that T. xiamenensis and T. acrotrocha are conspecific species.

5 CONCLUSION

In this study, the morphology and phylogeny of a bloom species from the SCS, T. acrotrocha is studied. The inter- and intra-species morphological and phylogenetic differences for T. acrotrocha were analyzed and compared. Takayama acrotrocha is characterized by a deeply sigmoid apical groove, a sulcus extending onto the epicone as a short intrusion, an ovoid to cup-shaped nucleus occupying most of the epicone, and a rounded pyrenoid located between the left side and the center of the hypocone. The main accessory pigment of T. acrotrocha is fucoxanthin. In the phylogenetic trees inferred from the LSU rDNA and ITS rDNA, the T. acrotrocha from the SCS formed a subclade with the T. acrotrocha from Singapore and T. xiamenensis. Takayama acrotrocha from the SCS has the typical morphological characteristics of T. xiamenensis, and the two species shared the identical LSU rDNA and ITS rDNA sequences, suggesting that T. acrotrocha and T. xiamenensis are the same species, with the priority for T. acrotrocha.

6 DATA AVAILABILITY STATEMENT

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

7 ACKNOWLEDGMENT

Special appreciation is expressed to Dr. Zhaohe LUO from the Third Institute of Oceanography, the State Oceanic Administration for helpful discussions regarding the TEM photos. We would also like to thank Dr. Li LI from the Shenzhen Marine Monitoring and Forecasting Center for her help in the seawater sampling.

Electronic supplementary material

Supplementary material (Supplementary Tables S1–S2) is available in the online version of this article at https://doi.org/10.1007/s00343-022-1321-0 .

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