Home Journals Articles Updates About
CN
image
收藏切换
Morphology and phylogeny of the rediscovered Prorocentrum formosum from the Zhongsha Islands, South China Sea
收藏切换
PDF
Jingyi CEN1, Hang XIE1, Yuan HUANG1, Linjian OU1, Songhui LÜ1, **, Jianyan WANG2, **
Journal of Oceanology and Limnology | 2026, 44(3) : 1224 - 1238
Less
收藏切换
Journal of Oceanology and Limnology | 2026, 44(3): 1224-1238
• Biology •
Morphology and phylogeny of the rediscovered Prorocentrum formosum from the Zhongsha Islands, South China Sea
Full
Jingyi CEN1, Hang XIE1, Yuan HUANG1, Linjian OU1, Songhui LÜ1, **, Jianyan WANG2, **
Affiliations
  • 1Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, College of Life Science and Technology, and Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Jinan University, Guangzhou510632, China
  • 2Department of Life Sciences, Natural History Museum of China, Beijing100050, China
Published: 2026-05-01 doi: 10.1007/s00343-025-5123-z
Outline
收藏切换

A strain of benthic dinoflagellate Prorocentrum formosum was isolated from the Anding Lianjiao (Contiguous Reef) (also known as Addington Shoal), Zhongsha Islands. Morphological observation using light and scanning electron microscopy revealed vegetative cells measuring 24–27 μm in length and 17–22 μm in width, which displayed an asymmetrical oval shape with smooth surfaces. The left and right valves contained two distinct types of radially arranged pores with differing diameters, characterized by sparsely distributed marginal pores and a seamless connection to the parallel horizontal intercalary band. The periflagellar area comprised 9 platelets arranged in a wide V-shape, containing both larger flagellar pores and smaller accessory pores. Phylogenetic analysis based on ITS and LSU rRNA gene sequences confirmed its classification within Prorocentrum, with strong bootstrap support (94% for ITS; 98% for LSU) and posterior probabilities (0.97 for ITS; 1.0 for LSU). Genetic distance analysis showed its closest relationship to Prorocentrum elegans (P=0.359 for ITS; P=0.118 for LSU). HPLC analysis detected chlorophyll a, chlorophyll c2, peridinin, diadinoxanthin, and diatoxanthin. Although trace levels of okadaic acid (OA) were detected in the initial cultures, its production was no longer observed after prolonged cultivation of the strain. Toxicity tests demonstrated 72- and 96-h mortality rates of 18.75%±1.07% and 34.54%±2.71% in Brachionus plicatilis at 2 660 cells/mL, whereas Artemia salina exhibited no mortality after 96-h exposure. Hemolytic activity was not detected in algal extracts. This study provides the first molecular sequences of P. formosum.

Prorocentrum formosum  /  okadaic acid  /  morphology  /  phylogeny  /  Zhongsha Islands
Jingyi CEN, Hang XIE, Yuan HUANG, Linjian OU, Songhui LÜ, Jianyan WANG. Morphology and phylogeny of the rediscovered Prorocentrum formosum from the Zhongsha Islands, South China Sea[J]. Journal of Oceanology and Limnology, 2026 , 44 (3) : 1224 -1238 . DOI: 10.1007/s00343-025-5123-z
Full Text
Less
1 　INTRODUCTION
Less
The genus Prorocentrum was established by Ehrenberg in 1834, with Prorocentrum micans as the type species (Fukuyo, 1981; Tillmann et al., 2019). Currently, a total of 138 species have been described within the genus, of which 85 are taxonomically accepted species according to AlgaeBase (Guiry and Guiry, 2025). Most Prorocentrum species are typically planktonic in nature, whereas approximately 35 are benthic or epibenthic (Hoppenrath et al., 2013; Zhang et al., 2015; Nascimento et al., 2017; Verma et al., 2019; Nishimura et al., 2020; Zou et al., 2022). These benthic species inhabit diverse substrates, including macroalgae, seagrass, corals, marine sediments, and floating detrital aggregates (Faust, 1990, 1993a, b; Grzebyk et al., 1998). Prorocentrum species are distributed globally, primarily in tropical and subtropical coastal regions (Faust, 1991; Murray et al., 2007; Faust et al., 2008; Chomérat et al., 2010, 2011).
The morphology of Prorocentrum is defined by two large valves, the absence of a sulcus, and an apical flagellar insertion (Dodge, 1975). A taxonomically critical feature is the periflagellar region, a complex structure containing two pores at the anterior end of the right valve, surrounded by 5–14 smaller platelets (Hoppenrath et al., 2013). Key morphological traits used for identifying Prorocentrum species include cell size, cell symmetry, thecal plate ornamentation (e.g., presence or absence of apical spines, plate depressions, shape and distribution of pores), and periflagellar region morphology (Tomas, 1996; Hoppenrath et al., 2013). Despite its apparently simple cellular organization, Prorocentrum taxonomy faces challenges due to intraspecific morphological variation and the occurrence of cryptic species (Leander and Hoppenrath, 2008; Han et al., 2016; Nascimento et al., 2017; Chomérat et al., 2019; Lim et al., 2019).
The detailed structural features of the periflagellar region serve as critical diagnostic characters for species identification. While morphological descriptions are increasingly supported by ultrastructural observations of the periflagellar region, many original species descriptions, particularly those lacking cultured strains, have not accurately characterized this region (Hoppenrath et al., 2013; Gómez and Gast, 2022).
Thirteen Prorocentrum species are recognized as harmful algal bloom formers and included in the IOC-UNESCO Taxonomic Reference List of Harmful Microalgae (Lundholm et al., 2025). These species produce diverse toxins, including OA, its analogues, and other bioactive compounds such as dinophysistoxins (DTX-1, DTX-2), borbotoxins, fast-acting toxins, and hemolytic toxins, which induce diarrhetic shellfish poisoning (DSP) to humans via trophic transfer (Hoppenrath et al., 2013). OA, along with DTXs like DTX-1 and DTX-2, are primary inducers of DSP (Alexander et al., 2019). Their lipophilic properties facilitate bioaccumulation in marine organism lipid tissues, impeding metabolic detoxification (Bauder et al., 2001). OA and DTXs function by inhibiting the effects of serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) (Takai et al., 1992), which in turn disrupts various cellular functions (Bialojan et al., 1988; Vale and Botana, 2008; Louzao et al., 2015). To date, at least eight Prorocentrum species (P. faustiae, P. caipirignum, P. lima, P. concavum, P. steidingerae, P. cf. fukuyoi, P. hoffmannianum, P. leve) in the genus Prorocentrum have been verified to produce OA (Hoppenrath et al., 2013; Zou et al., 2022; www.marinespecies.org/). Simultaneously, OA, DTX-1, and a DTX analogue were identified in P. lima from the Xisha (or known as Paracel) Islands (Zou et al., 2022). Recently, the first benthic dinoflagellate bloom caused by P. concavum in Hainan Island was reported (Zou et al., 2020), highlighting the imperative to study the potentially toxin-producing capabilities of these Prorocentrum strains.
Prorocentrum formosum M. A. Faust, 1993 was first identified from the Lair and Lair Channel, Twin Cays, Belize. This photosynthetic species has small, oval cells measuring 25–28 µm in length and 15–16 µm in width. The valves are smooth, with 42–55; the apical area of the left valve is either flat or inclined. The right valve features a V-shaped periflagellar region composed of 5–6 platelets. It lacks an apical pore but has a large flagellar pore and a prominent apical plate. The intercalary band is transversely striated (Faust, 1993a). Since its original description, P. formosum has been recorded in Malaysian waters, though no molecular data have been reported for this species (Faust, 1993a; Mohammad-Noor et al., 2007).
A P. formosum strain isolated from the Zhongsha Islands, South China Sea, was identified through integrated morphological and molecular analyses (ITS/LSU rRNA gene sequencing). LC-MS analysis confirmed OA production, while toxicity assessments evaluated hemolytic activity and acute toxicity using standardized bioassays.
2 　MATERIAL AND METHOD
Less
2.1 　Sampling location and establishing cultures
Benthic dinoflagellates were collected from macroalgal substrates on 22 May 2019 at Anding Lianjiao (Contiguous Reef) (also known as Addington Shoal), Zhongsha Islands, China (114°27.727′E, 15°36.155′N) at a depth of 19 m via diving (Fig.1). Single cell of Prorocentrum was isolated under an Olympus BX61 (Olympus, Japan) light microscope using micropipette aspiration. Isolates were aseptically transferred to 96-well culture plates containing L1 medium (salinity 30). The monoclonal P. formosum strain was maintained in the Algal Culture Collection of Jinan University’s Red Tide and Marine Biology Research Center, cultured under 12-h꞉12-h LD cycle with 100-μmol photons/(m2·s) illumination at 25 °C.
2.2 　Morphological and structural observation using light and scanning electron microscopy
Cells of P. formosum were examined under a light microscope (BX61, Olympus, Japan) equipped with a digital camera (QImaging Retiga 4000R, British Columbia, Canada). Live cell images were captured at 400× magnification using Image-Pro Plus 6.0 software, with cell length and width measured from 30 randomly selected individuals. To investigate the morphology and distribution of chloroplasts and the nucleus, cells were stained with Fluorescent Brightener 28 (Sigma, USA) and SYBR Safe DNA gel stain (Invitrogen, Thermo Fisher Scientific, USA) and then imaged at 1 000× magnification.
Scanning Electron Microscopy (SEM) was employed for a more detailed morphological exploration of algal cells. The sample preparation for SEM was followed the protocol described in Lim et al. (2019). Cells in the exponential growth phase were fixed in glutaraldehyde in a final concentration of 2.5% at 4 °C overnight. The fixed sample was dehydrated using a series of ethanol gradients (10%, 30%, 50%, 70%, and 90%), and this step was repeated three times with 100% ethanol for 15 min each. After dehydration, samples were dried in a CO2 critical point dryer (CPD 300, Leica, Wetzlar, Germany), and then coated with a gold layer using the Leica EM SCD 500 (Leica, Wetzlar, Germany). Final observations were conducted using a Zeiss Ultra 55 field-emission SEM (Carl Zeiss, Germany).
2.3 　Molecular characterization: DNA extraction, PCR amplification and sequencing, phylogenetic analysis
One milliliter of P. formosum algal culture in exponential growth phase was collected by centrifugation (12 000×g, 4 °C, 2 min) for DNA extraction. The genomic DNA was extracted using the MiniBEST Universal DNA Extraction Kit (TaKaRa, Tokyo, Japan) following the manufacturer’s instructions. The partial ITS rDNA and LSU rDNA were amplified by the primers ITS1F/ITS2R to target the ITS domain, and primers D1R/D3B for the D1/D3 region of the 28S rRNA gene (Scholin et al., 1994; Pin et al., 2001). The PCR mixture comprised of 2-μL DNA extract, 22-μL sterile Milli-Q water, 1 μL of each primer at 1-μmol/L concentration, and 25 μL of the 2X Accurate Taq Master Mix (Accurate Biotechnology Co., Ltd.), totaling 50 μL. The PCR protocol was as follows: denaturation was started at 94 °C for 4 min, followed by 36 cycles where denaturation was performed at 94 °C for 20 s, annealing was conducted at 56 °C for 30 s, and extension was carried out at 72 °C for 1.5 min. The process was concluded with a final extension at 72 °C for 10 min (Zou et al., 2020). The PCR product was sequenced at Sangon Bioengineering Co., Ltd.
For phylogenetic analysis, GenBank sequences of Prorocentrum species showing >97% similarity to P. formosum were retrieved. The target sequences were then subjected to multiple alignments using the online tool, MUSCLE (Edgar, 2004). Pairwise genetic distances were computed with MUSCLE and MEGA-X (Kimura 2 parameter model) (Kumar et al., 2018). For the phylogenies of P. formosum, construct Maximum Likelihood (ML) trees were constructed using RAxML-HPC2 on XSEDE v. 8.2.12 with 1 000 bootstrap replicates (Stamatakis, 2014). The most suitable model is chosen based on the Akaike Information Criterion using MrModeltest 2.3. With this model, a Bayesian Inference (BI) tree was generated via MrBayes 3.2.7, running for 10 000 000 iterations at 100-generation intervals (Ronquist and Huelsenbeck, 2003). Trees and associated confidence values are visualized using MEGA X and Tree View software (Page, 1996).
2.4 　Toxins extraction and analysis
Prorocenturum formosum was cultured in a 2-L Erlenmeyer flasks for toxin extraction. Cells in the late exponential growth phase were harvested by centrifuging at 12 000×g for 10 min and then transferred to 50-mL centrifuge tubes maintained at 4 ℃. The cell precipitate was dissolved in 3 mL of 95% methanol, and the mixture was ultra-sonicated for 40 min in an ice bath. Supernatant was then collected by centrifuging at 12 000×g for 10 min at 4 ℃. This step was repeated once more. The supernatant (0.5 mL) was subsequently filtered using a 0.22-μm spin-filters (Pall Corporation, USA), transferred to a 2-mL vial, and stored at ­80 ℃ until further HPLC analysis.
Toxin analysis was conducted using an HPLC system (Shimadzu prominence LC-20ADXR) equipped with a 5500 QTRAP LC-MS system (AB Sciex Instruments, Foster City, CA). Toxins were separated using a Phenomenex Kinetex XB-C18 (150 mm×2.1 mm, 2.6 μm) chromatographic column. The analytical setup for toxin determination included a mobile phase comprising acetonitrile (solvent A) and 0.05% formic acid in water (solvent B). The gradient began with 20% solvent A, ramping up to 90% over 7 min, holding at 90% solvent A for 3 min, and then returning to 20% solvent A within 0.1 min. The sample injection volume was set at 5 μL, with separation taking place at a consistent temperature of 25 ℃ and a flow rate of 0.35 mL/min.
2.5 　Pigment composition
The pigment composition of P. formosum was analyzed using a Waters E2695 HPLC system (Waters, Milford, USA). The protocol followed Zapata et al. (2000) with modifications: 50-mL algal culture was concentrated and filtered through a 47-mm Whatman GF/F membrane. The membrane was sectioned, transferred to a 15-mL centrifuge tube with 2-mL 95% methanol, and stored at 4 °C for 12 h prior to 30-min sonication. The supernatants were filtered using 0.2-μm spin filters (Pall Corporation, USA), and then transferred to a tube for pigment analysis. A C8 column (150 mm×4.6 mm, 3.5 μm, by Waters) was used for the chromatographic separation of the pigments. The mobile phase solution A consisted of methanol, acetonitrile, and pyridine water in a 50꞉25꞉25 (V꞉V꞉V) ratio, while solution B was a mixture of methanol, acetonitrile, and acetone in a 20꞉60꞉20 (V꞉V꞉V) ratio. The injection volume was set at 100 μL with a flow rate of 1 mL/min at 25 ℃. The mobile phase gradient proceeded as follows: (1) 100% solution A, 0% solution B (0 min), (2) 60% solution A, 40% solution B (22 min), (3) solution A 5%, solution B 95% (28 min), (4) solution A 5%, solution B 95% (38 min), and (5) solution A 100%, solution B 0% (40 min). Pigments were identified based on their retention time and spectral characteristics compared to known pigment standards.
2.6 　Haemolytic assay and the bioassay toxicity
Hemolytic activity of P. formosum was analyzed using exponentially growing cultures (2 264 cells/mL). Cells were centrifuged (5 000×g, 10 min) and extracted following Eschbach et al. (2001) with modifications: the pellet was resuspended in 2-mL hemolysis buffer, sonicated at 4 °C for 30 min, and centrifuged. The supernatant was discarded, and the pellet was assayed for hemolytic activity using rabbit erythrocytes (Zhou et al., 2007). The hemolytic toxin in PBS buffer and erythrocytes suspension in PBS buffer were used as blank control, and the erythrocytes mixed with equal volume of 1% Triton X-100 were used as positive control. Cultures of Prorocentrum obtusidens (with a concentration of 2 540 cells/mL) were extracted and used as negative control. Each test was conducted in triplicate (Zhou et al., 2007).
Toxicity bioassays of P. formosum were performed using Brachionus plicatilis and Artemia salina, with the culture medium as a blank control. All assays were conducted in triplicate using exponentially growing P. formosum cultures. Before testing, both species were starved for 24 h under controlled conditions (25±1 °C, salinity 30). For B. plicatilis, 10 per well were exposed in 24-well plates containing 2 mL of test solution supplemented with algal liquid at two densities: 2 660 cells/mL (high-density group) and 555 cells/mL (low-density group). For A. salina, 10 per well were exposed in 12-well plates with 5 mL of test solution plus algal suspensions at 5 090 cells/mL (high-group) or 1 200 cells/mL (low-group). Survival was recorded at 3-, 6-, 12-, 24-, 48-, 72-, and 96-h post-exposure (Wang et al., 2023).
The data obtained were analyzed using Origin 2022 for graphical representation and SPSS Statistics 27 for statistical analysis. Differences between groups were determined by one-way ANOVA, and post-hoc comparisons were conducted using Fisher’s least significant difference (LSD) test. Significance levels were set at P<0.05 and P<0.01.
3 　RESULT
Less
3.1 　Morphological analysis
The cell of Prorocentrum formosum is oval and composed of two valves (Fig.2a–b, h). Their valve surfaces are covered with pores (Fig.2h). The cell is slightly asymmetric, with the right apical (or anterior) region being slightly higher than the left in ventral view (Fig.2a–b, d). The cells measure 23.81–27.06 μm in length (mean 25.52±1.01 μm, n=30) and 17.49–21.89 μm in width (mean 19.72±1.35 μm, n=30), with a length-to-width ratio ranging from 1.23 to 1.39 (mean 1.29±0.06, n=30) (Table 1). Under light microscopy, the cells contain numerous irregularly shaped, yellow-brown chloroplasts distributed throughout the cytoplasm (Fig.2a–e), and a posterior nucleus (Fig.2f). No pyrenoid was observed.
Under SEM (Fig.3a–g), the apical region of the cell features a large V-shaped depression on the anterior right valve (Fig.3a). The thecal surface is smooth, lacking pronounced thick flanges on the upper right region or collars on the left valve (Fig.3a–b). Two distinct pore types, varying in diameter, are present on the valve surfaces (Fig.3a–f), primarily distributed along the margins (Fig.3a–d), with no pores in the central area (Fig.3a–b). The periflagellar region is characterized by radial rows of pores (Fig.3a–c). The periflagellar area is located at the apical end of the right valve, characterized by a shallow triangular depression (Fig.3e–g). This region contains both an accessory pore and a flagellar pore, with the accessory pore being significantly smaller than the flagellar pore (Fig.3e). Based on the nomenclature proposed by Hoppenrath et al. (2013), nine periflagellar platelets (1a, 1b, 2, 3, 4, 5, 6, 7, and 8) are identified in this species (Fig.4a, b). The surface of the platelets is predominantly smooth, and platelet 1a exhibits a wing-like structure (Fig.3e & f), adjacent to the accessory pore (Fig.3e–g). The accessory pore is small and often partially obscured by protrusions from platelets 1, 6, and 8 (Fig.3e–g). The flagellar pore is surrounded by protrusions, with an additional protrusion located between platelets 4 and 5 (Fig.3g). The intercalary band displays a smooth surface with broad transverse striations (Fig.3c, d).
3.2 　Genetic distance and phylogenetic analysis
LSU and ITS rDNA sequences of strain ZSG2 (Zhongsha Islands, China) were obtained and deposited in GenBank under accession numbers PV524993 and PV524994. These sequences were selected for phylogenetic tree construction. In the LSU rDNA phylogeny (Fig.5), P. formosum (strain ZSG2) was resolved as a strong-supported clade (98%/1.0), grouping most closely with P. rhathymum, P. koreanum, P. micans, P. elegans, P. obtusidens, and P. cordatum. Within the ITS phylogenetic tree (Fig.6), P. formosum formed a distinct clade with strong bootstrap support (--/1.0), clustering closest to P. koreanum, P. rhathymum, P. micans, P. elegans, P. triestinum, P. cordatum, P. balticum, and P. obtusidens.
Pairwise genetic distances of the D1–D3 LSU and ITS rDNA sequences were compared among Prorocentrum sequences and other related species (Tables 2–3). The interspecies genetic distances within the genus Prorocentrum, based on the LSU rDNA sequence, ranged 0.025%–0.158%. The genetic distance of P. formosum to its closest relative, P. elegans, was 0.134% (11 bp out of 819 bp of LSU rDNA), which is notably higher than the minimum interspecific variability observed within the genus, such as the distance between P. elegans KF835600 and P. elegans MH381771, which was only 0.002%. In comparison, the genetic distances between P. formosum and other Prorocentrum species were generally higher, with values ranging from 0.118% to 0.155%. The shortest genetic distance was 0.118%, observed between P. formosum and P. elegans, which is still higher than the genetic distances fluctuating between 0.053% and 0.080% among recognized distinct species of Prorocentrum, such as P. elegans, P. micans, and P. koreanum. These findings indicate a significant genetic divergence between P. formosum and the other species within the genus, supporting its classification as a distinct species.
Similar trends were observed for genetic distances based on ITS rDNA sequences, which ranged from 0.016% to 0.449% among Prorocentrum species. The closest genetic relationship observed was found between P. formosum and P. elegans MH356284, with a divergence of 0.364% (31 base pair differences out of 85 bp of ITS rDNA). This genetic divergence is again higher than the minimal interspecific variability within the genus, as seen in distances among other Prorocentrum species, which ranged 0.002%–0.304%. The genetic divergence between P. formosum and other Prorocentrum species, such as P. cordatum and P. obtusidens, was even more pronounced, with genetic distances reaching up to 0.449% based on ITS sequences. In comparison, genetic distances among other Prorocentrum species, such as P. elegans and P. micans, ranged 0.002%–0.291%, further reinforcing the genetic distinctness of P. formosum within the genus Prorocentrum.
3.3 　Toxin and toxicity analysis
Trace amounts of the lipophilic toxin OA were detected in the initial cell extracts of P. formosum during the strain isolation phase via LC-MS. However, after prolonged laboratory cultivation, OA and its derivatives DTX-1 and DTX-2 were not detected in the extracts, indicating a loss of toxin production in this strain over time.
The acute toxicity of P. formosum on Brachionus plicatilis and Artemia salina was listed in Table 4. A 96-h exposure test exhibited pronounced detrimental effects of P. formosum (harmful algal blooms algae liquid and 10% harmful algal blooms algae liquid) on Brachionus plicatilis. At 72 h, the mortality rates were 18.75%±1.07% and 14.79%±1.23% respectively, while at 96 h, the rates increased to 34.54%±2.71% and 25.49%±0.45% respectively, both significantly higher than the blank control group (P<0.05). Regarding Artemia salina, no deaths were observed after exposure to P. formosum pure cultured algae in 24 h. The mortality rate of Artemia salina showed an upward trend in 48 and 96 h without significant differences among the three experimental groups, implying that P. formosum did not induce acute lethality in Artemia salina. Hemolysis experiments on rabbit blood cells confirmed that P. formosum did not pose hemolytic activity (Table 5).
3.4 　Pigment analysis
Pigment composition of P. formosum (ZSG2) was analyzed through HPLC analysis. Six pigments, chlorophyll c2 (chl c2), peridinin, diadinoxanthin, chlorophyll a (chl a), peridinol, and diatoxanthin, were identified (Fig.7). Among them, chl a, peridinin, and diadinoxanthin were the primary pigment constituents. The specific pigment concentrations for the three main pigments were as follows: peridinin (peri) at 0.133±0.010 mg/L, diadinoxanthin (diato) at 0.095±0.008 mg/L, and chl a at 0.098±0.019 mg/L. Some unidentified pigments in trace amounts were also detected.
4 　DISCUSSION
Less
4.1 　Identification of P. formosum and its closely related species
Many species of the genus Prorocentrum were initially described solely based on morphological characteristics, frequently resulting in taxonomic synonymy among morphologically convergent taxa. For instance, P. lima was described by Ehrenberg in 1834 and later revised by Dodge (1975), who documented its morphological plasticity under light microscopy. Subsequent studies demonstrated both high morphological variability and genetic divergence among its morphotypes, suggesting that these morphotypes constitute a P. lima species complex despite their genetic distinctness (Zhang et al., 2015). Similarly, P. mexicanum and P. rhathymum were distinguished primarily by differences in cellular outline and periflagellar area ultrastructure, despite their pronounced morphological overlap (Loeblich et al., 1979; Fukuyo, 1981). This variability led to confusion and multiple descriptions of the same species under different names. The present study integrates morphological observations with molecular phylogenetic evidence, providing conclusive support for the taxonomic validity of P. formosum as a distinct species.
Strain ZSG2 was identified as P. formosum based on morphological congruence with Faust (1993a)’s original description. Ventral view reveals slight asymmetry with the right apical plate elevated relative to the left (Figs.2a–b, 3a–b), matching Faust’s observation of a flat/inclined left apical margin (figs.14–15 in Faust, 1993a). The valves bear two morphologically distinct pore classes: small pores (0.06–0.15 μm) and large pores (0.20–0.43 μm), showing marginal distribution and a central pore-free zone—consistent with Faust’s description of large trichocyst pores and smaller peripheral pores (Faust 1993a). Strain ZSG2 lacks a pyrenoid, a feature confirmed in the original taxon (Faust, 1993a; Mohammad-Noor et al., 2007). The periflagellar area of P. formosum is described as V-shaped region of 5–6 platelets lacking an accessory pore, featuring a ventral wing bordering Platelet 1 and a dorsal list on Platelet 4 (Faust, 1993a), along with an annular ridge surrounding the flagellar pore (Faust, 1993a; Mohammad-Noor et al., 2007). Strain ZSG2 recapitulates this platelet configuration, including a ventral flange on Platelet 1 and an angled plate (Figs.3e–g). The accessory pore in Strain ZSG2 did not report by Faust (1993a) and Mohammad-Noor et al. (2007). Due to its inconspicuous positioning, this pore remains indistinct in ventral view (Fig.3g) but becomes discernible at specific angles, appearing as a small aperture obscured by Platelet 1 and protrusions adjacent to the flagellar pore (Fig.3c, e–f). Although P. formosum was originally defined by the absence of an accessory pore—a key diagnostic trait distinguishing it from congeners (e.g., P. elegans), the periflagellar area morphology of Strain ZSG2 (Fig.3g) and other features align with the species’ taxonomic criteria. Consequently, Strain ZSG2 is classified herein as P. formosum.
In this study, light microscopy confirmed the absence of a pyrenoid in P. formosum, a trait first noted by Faust (1993a) and critical for distinguishing it from P. mexicanum and most Prorocentrum species, which typically possess this structure (Hoppenrath et al., 2013). Morphologically, P. formosum shares a broadly V-shaped periflagellar region with P. elegans (Faust, 1993a, b). The P. formosum (23.81–27.06 μm in length, 1.23–1.39 length-to-width ratio) displays three protrusions in the periflagellar area (Fig.3) and nine platelets (Fig.3f–g), compared to P. elegans (15–20 μm length), which lacks protrusions and possesses eight platelets (Faust, 1993b; Hoppenrath et al., 2013). Additionally, P. formosum exhibits a radial pore pattern on its smooth thecal surface (Fig.3a–d), contrasting with the apical row of pores in P. elegans (Faust, 1993b). Contrasting with P. emarginatum and P. fukuyoi, which have narrow V-shaped periflagellar regions with thick flanges and central pyrenoids, P. formosum lacks pyrenoids and displays a smooth thecal surface without flanges (Murray et al., 2007; Aligizaki et al., 2009). P. fukuyoi displays randomly scattered pores with central platelet aggregation, while P. formosum demonstrates radially aligned pore rows and a distinct central pore-free zone (Fig.3a–c) (Murray et al., 2007). The P. koreanum differs significantly in cell shape, periflagellar structure, and pore distribution, with large pores concentrated centrally and lacking small pores (Han et al., 2016). Earlier reports suggested P. formosum lacked an accessory pore (Faust, 1993b; Murray et al., 2007), but our SEM observations confirm its presence, although partially obscured by platelet protrusions (Fig.3e–g). In conclusion, P. formosum is unequivocally distinct from related species based on its unique platelet architecture (nine platelets with a wing-like structure on Platelet 1a), radial pore pattern, posterior nucleus, and molecular divergence. Morphologically similar P. formosum and P. elegans exhibited 0.134% (LSU rDNA) and 0.364% (ITS rDNA) genetic divergence, surpassing both the interspecific threshold (0.1%) and maximum intraspecific variability (0.3%) established for Prorocentrum (Zhang et al., 2007), confirming their distinct species status (Zhang et al., 2007). Phylogenetically, P. formosum forms a distinct clade, positioned as sister to taxa such as P. cf. emarginatum, P. elegans, and P. rhathymum (Fig.5). This distinct clustering supports its classification as an independent species.
4.2 　The pigment of P. formosum
HPLC analysis of laboratory-cultured P. formosum (strain ZSG2) identified six pigments: chlorophyll c (chl c), peridinin, diadinoxanthin, chl a, peridinol, and diatoxanthin (Fig.7). Quantitative analysis revealed peridinin as the dominant carotenoid (0.133±0.010 mg/L), followed by chl a (0.098±0.019 mg/L) and diadinoxanthin (0.095±0.008 mg/L). Many research results show that the major pigments in dinoflagellates are composed of peridinin, chl a and chl c (Hu et al., 1992; Zhang et al., 2007), but peridinin was the major carotenoid present in Prorocentrum cells (Faust et al., 1982; Hu et al., 1992; Krakhmalnyi et al., 2004; Hou et al., 2007; Berden-Zrimec et al., 2008). Different from the previous research results (Hu et al., 1992), the chl c detected in P. formosum was not the main pigment component compared with other pigments in the cell. The pigment component diatoxanthin was detected in P. formosum, P. micans, P. cordatum, P. hoffmannianum, and P. lima in the genus of Prorocentrum (Faust et al., 1982; Krakhmalnyi et al., 2004; Hou et al., 2007; Berden-Zrimec et al., 2008), but not in P. obtusidens and P. rhathymum (Hu et al., 1992; Krakhmalnyi et al., 2004). The co-occurrence of diadinoxanthin and diatoxanthin indicates active operation of the photoprotective xanthophyll cycle, a mechanism triggered by high irradiance. This is corroborated by experimental evidence: diadinoxanthin-to-diatoxanthin conversion escalates under light stress in Alexandrium (Demers et al., 1991) and during late exponential growth phases in Prorocentrum (Faust et al., 1982; Hou et al., 2007; Berden-Zrimec et al., 2008). The presence of both diadinoxanthin and diatoxanthin in P. formosum may reflect its adaptation to benthic light environments influenced by tidal fluctuations. However, pigment composition alone cannot definitively resolve ecotype classification in Prorocentrum. Although the absence of diatoxanthin in some species and its presence in P. formosum imply divergent light acclimation strategies, this discrepancy may reflect microenvironmental light heterogeneity rather than obligate lifestyle differences (Faust et al., 1982; Krakhmalnyi et al., 2004; Hou et al., 2007; Berden-Zrimec et al., 2008).
5 　CONCLUSION
Less
In this study, we isolated Prorocentrum formosum from Anding Lianjiao (Contiguous Reef) (or Addington Shoal), Zhongsha Islands, confirmed its identity through morphology and molecular phylogeny. Genetic distances indicated closest affinity to P. elegans. Pigment analysis detected chlorophylls and xanthophylls but no okadaic acid. While non-toxic to Artemia salina and non-hemolytic, it caused moderate mortality in Brachionus plicatilis. This work provided the first molecular sequences for P. formosum.
6 　DATA AVAILABILITY STATEMENT
Less
The data that support the findings of this study are available from the corresponding author upon request.
7 　ACKNOWLEDGMENT
Less
We thank Dr. Heng CHEN from Wenzhou Medical University for his help in pigment composition analysis.
References
Less
Alexander J, Benford D, A Boobiset al. 2019. Marine biotoxins in shellfish—Summary on regulated marine biotoxins. The EFSA Journal, 1306: 1-23, https://doi.org/10.2903/j.efsa.2009.1306.
Aligizaki K, Nikolaidis G, P Katikouet al. 2009. Potentially toxic epiphytic Prorocentrum (Dinophyceae) species in Greek coastal waters. Harmful Algae, 8(2): 299-311, https://doi.org/10.1016/J.HAL.2008.07.002.
Bauder A G, Cembella A D, V M Briceljet al. 2001. Uptake and fate of diarrhetic shellfish poisoning toxins from the dinoflagellate Prorocentrum lima in the bay scallop Argopecten irradians. Marine Ecology Progress Series, 213: 39-52, https://doi.org/10.3354/meps213039.
Berden-Zrimec M, Flander-Putrle V, L Drinovecet al. 2008. Growth, delayed fluorescence and pigment composition of four Prorocentrum minimum strains growing at two salinities. Biological Research, 41(1): 11-23, https://doi.org/10.4067/S0716-97602008000100003.
Bialojan C, Rüegg J C, Takai A. 1988. Effects of okadaic acid on isometric tension and myosin phosphorylation of chemically skinned guinea-pig taenia coli. The Journal of Physiology, 398(1): 81-95, https://doi.org/10.1113/jphysiol.1988.sp017030.
Chomérat N, Bilien G, A Derrienet al. 2019. Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): a revisit of its morphology, molecular phylogeny and toxicity. Harmful Algae, 84: 95-111, https://doi.org/10.1016/j.hal.2019.02.004.
Chomérat N, Sellos D Y, F Zentzet al. 2010. Morphology and molecular phylogeny of Prorocentrum consutum sp. nov. (Dinophyceae), a new benthic dinoflagellate from South Brittany (northwestern France). Journal of Phycology, 46(1): 183-194, https://doi.org/10.1111/j.1529-8817.2009.00774.x.
Chomérat N, Zentz F, S Boulbenet al. 2011. Prorocentrum glenanicum sp. nov. and Prorocentrum pseudopanamense sp. nov. (Prorocentrales, Dinophyceae), two new benthic dinoflagellate species from South Brittany (northwestern France). Phycologia, 50(2): 202-214, https://doi.org/10.2216/10-12.1.
David H, Laza-Martínez A, K García-Etxebarriaet al. 2014. Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny. Journal of Phycology, 50(4): 718-726, https://doi.org/10.1111/jpy.12200.
Demers S, Roy S, R Gagnonet al. 1991. Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism. Marine Ecology Progress Series, 76(2): 185-193, https://doi.org/10.3354/meps076185.
Dodge J D. 1975. The Prorocentrales (Dinophyceae). II. Revision of the taxonomy within the genus Prorocentrum. Botanical Journal of the Linnean Society, 71(2): 103-125, https://doi.org/10.1111/j.1095-8339.1975.tb02449.x.
Edgar R C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5): 1792-1797, https://doi.org/10.1093/nar/gkh340.
Faust M A, Sager J C, Meeson B W. 1982. Response of Prorocentrum mariae‐lebouriae (Dinophyceae) to light of different spectral qualities and irradiances: growth and pigmentation. Journal of Phycology, 18(3): 349-356, https://doi.org/10.1111/j.1529-8817.1982.tb03195.x.
Faust M A. 1990. Morphologic details of six benthic species of Prorocentrum (Pyrrophyta) from a mangrove island, Twin Cays, Belize, including two new species. Journal of Phycology, 26(3): 548-558, https://doi.org/10.1111/j.0022-3646.1990.00548.x.
Faust M A. 1991. Morphology of ciguatera-causing Prorocentrum lima (Pyrrophyta) from widely differing sites. Journal of Phycology, 27(5): 642-648, https://doi.org/10.1111/j.0022-3646.1991.00642.x.
Faust M A. 1993a. Three new benthic species of Prorocentrum (Dinophyceae) from Twin Cays, Belize: P. maculosum sp. nov., P. foraminosum sp. nov. and P. formosum sp. nov. Phycologia, 32(6): 410-418, https://doi.org/10.2216/i0031-8884-32-6-410.1.
Faust M A. 1993b. Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, three new benthic species (Dinophyceae) from a mangrove island, Twin Cays, Belize. Journal of Phycology, 29(1): 100-107, https://doi.org/10.1111/j.1529-8817.1993.tb00287.x.
Faust M A, Vandersea M W, S R Kibleret al. 2008. Prorocentrum levis, a new benthic species (Dinophyceae) from a mangrove island, Twin Cays, Belize. Journal of Phycology, 44(1): 232-240, https://doi.org/10.1111/j.1529-8817.2007.00450.x.
Fukuyo Y. 1981. Taxonomical study on benthic dinoflagellates collected in coral reefs. Bulletin of the Japanese Society of Scientific Fisheries, 47(8): 967-978, https://doi.org/10.2331/suisan.47.967.
Gómez F, Gast R J. 2022. Rediscovery of the gonyaulacoid dinoflagellate Schuettiella. The Journal of Eukaryotic Microbiology, 69(4): e12920, https://doi.org/10.1111/jeu.12920.
Grzebyk D, Sako Y, Berland B. 1998. Phylogenetic analysis of nine species of Prorocentrum (Dinophyceae) inferred from 18S ribosomal DNA sequences, morphological comparisons, and description of Prorocentrum panamensis, sp. nov. Journal of Phycology, 34(6): 1055-1068, https://doi.org/10.1046/j.1529-8817.1998.341055.x.
Guiry M D, Guiry G M. 2025. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway, https://www.algaebase.org.
Han M S, Wang P B, Kim J Het al. 2016. Morphological and molecular phylogenetic position of Prorocentrum micans sensu stricto and description of Prorocentrum koreanum sp. nov. from southern coastal waters in Korea and Japan. Protist, 167(1): 32-50, https://doi.org/10.1016/j.protis.2015.12.001.
Hoppenrath M, Chomerat N, T Horiguchiet al. 2013. Taxonomy and phylogeny of the benthic Prorocentrum species (Dinophyceae)—A proposal and review. Harmful Algae, 27: 1-28, https://doi.org/10.1016/j.hal.2013.03.006.
Hou J J, Huang B Q, Cao Z Ret al. 2007. Effects of nutrient limitation on pigments in Thalassiosira weissflogii and Prorocentrum donghaiense. Journal of Integrative Plant Biology, 49(5): 686-697, https://doi.org/10.1111/j.1744-7909.2007.00449.x.
Hu T M, Marr J, de Freitas A S Wet al. 1992. New diol esters isolated from cultures of the dinoflagellates Prorocentrum lima and Prorocentrum concavum. Journal of Natural Products, 55(11): 1631-1637, https://doi.org/10.1021/np50089a011.
Krakhmalnyi A F, Tishaeva M V, Z A Paninaet al. 2004. A problem on identity Prorocentrum cordatum (Ostf.) Dodge and P. minimum (Pavill.) Schiller (Dinophyta). International Journal on Algae, 6(4): 331-340, https://doi.org/10.1615/InterJAlgae.v6.i4.30.
Kumar S, Stecher G, Li Met al. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6): 1547-1549, https://doi.org/10.1093/molbev/msy096.
Leander B S, Hoppenrath M. 2008. Ultrastructure of a novel tube-forming, intracellular parasite of dinoflagellates: Parvilucifera prorocentri sp. nov. (Alveolata, Myzozoa). European Journal of Protistology, 44(1): 55-70, https://doi.org/10.1016/j.ejop.2007.08.004.
Lim Z F, Luo Z H, Lee L Ket al. 2019. Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae). Harmful Algae, 83: 95-108, https://doi.org/10.1016/j.hal.2019.01.007.
Loeblich A R III, Sherley J L, Schmidt R J. 1979. The correct position of flagellar insertion in Prorocentrum and description of Prorocentrum rhathymum sp. nov. (Pyrrhophyta). Journal of Plankton Research, 1(2): 113-120, https://doi.org/10.1093/plankt/1.2.113.
Louzao M C, Fernández D A, P Abalet al. 2015. Diarrhetic effect of okadaic acid could be related with its neuronal action: changes in neuropeptide Y. Toxicology Letters, 237(2): 151-160, https://doi.org/10.1016/j.toxlet.2015.06.004.
Lundholm N, Bernard C, C Churroet al. 2025. IOC-UNESCO Taxonomic Reference List of Harmful Micro Algae. https://www.marinespecies.org/hab, https://doi.org/10.14284/362.
Luo Z H, Zhang H, B Krocket al. 2017. Morphology, molecular phylogeny and okadaic acid production of epibenthic Prorocentrum (Dinophyceae) species from the northern South China Sea. Algal Research, 22: 14-30, https://doi.org/10.1016/j.algal.2016.11.020.
Mohammad-Noor N, Moestrup Ø, Daugbjerg N. 2007. Light, electron microscopy and DNA sequences of the dinoflagellate Prorocentrum concavum (syn. P. arabianum) with special emphasis on the periflagellar area. Phycologia, 46(5): 549-564, https://doi.org/10.2216/06-94.1.
Murray S, Nagahama Y, Fukuyo Y. 2007. Phylogenetic study of benthic, spine-bearing prorocentroids, including Prorocentrum fukuyoi sp. nov.. Phycological Research, 55(2): 91-102, https://doi.org/10.1111/j.1440-1835.2007.00452.x.
Nascimento S M, Mendes M C Q, M Menezeset al. 2017. Morphology and phylogeny of Prorocentrum caipirignum sp. nov. (Dinophyceae), a new tropical toxic benthic dinoflagellate. Harmful Algae, 70: 73-89, https://doi.org/10.1016/j.hal.2017.11.001.
Nishimura T, Uchida H, R Noguchiet al. 2020. Abundance of the benthic dinoflagellate Prorocentrum and the diversity, distribution, and diarrhetic shellfish toxin production of Prorocentrum lima complex and P. caipirignum in Japan. Harmful Algae, 96: 101687, https://doi.org/10.1016/j.hal.2019.101687.
Page R D M. 1996. Tree View: an application to display phylogenetic trees on personal computers. Bioinformatics, 12(4): 357-358, https://doi.org/10.1093/bioinformatics/12.4.357.
Pin L C, Teen L P, A Ahmadet al. 2001. Genetic diversity of Ostreopsis ovata (Dinophyceae) from Malaysia. Marine Biotechnology, 3(3): 246-255, https://doi.org/10.1007/s101260000073.
Ronquist F, Huelsenbeck J P. 2003. Mrbayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19(12): 1572-1574, https://doi.org/10.1093/bioinformatics/btg180.
Scholin C A, Herzog M, M Soginet al. 1994. Identification of group- and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. Journal of Phycology, 30(6): 999-1011, https://doi.org/10.1111/j.0022-3646.1994.00999.x.
Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9): 1312-1313, https://doi.org/10.1093/bioinformatics/btu033.
Takai A, Murata M, K Torigoeet al. 1992. Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship. The Biochemical Journal, 284(Pt 2): 539-544, https://doi.org/10.1042/bj2840539.
Tillmann U, Hoppenrath M, Gottschling M. 2019. Reliable determination of Prorocentrum micans Ehrenb. (Prorocentrales, Dinophyceae) based on newly collected material from the type locality. European Journal of Phycology, 54(3): 417-431, https://doi.org/10.1080/09670262.2019.1579925.
Tomas C R. 1996. Identifying Marine Diatoms and Dinoflagellates. Academic Press, San Diego. https://do10.1016/b978-012693015-3/50000-0
Vale C, Botana L M. 2008. Marine toxins and the cytoskeleton: okadaic acid and dinophysistoxins. The FEBS Journal, 275(24): 6060-6066, https://doi.org/10.1111/j.1742-4658.2008.06711.x.
Verma A, Kazandjian A, C Sarowaret al. 2019. Morphology and phylogenetics of benthic Prorocentrum species (Dinophyceae) from tropical northwestern Australia. Toxins, 11(10): 571, https://doi.org/10.3390/toxins11100571.
Wang X, Liu C, Zhang Q Cet al. 2023. A dinoflagellate bloom caused by multiple species of Kareniaceae in the coastal waters of Fujian in June 2022 and its adverse impacts on Brachionus plicatilis and Artemia salina. Marine Pollution Bulletin, 196: 115685, https://doi.org/10.1016/j.marpolbul.2023.115685.
Zapata M, Rodríguez F, Garrido J L. 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Marine Ecology Progress Series, 195: 29-45, https://doi.org/10.3354/meps195029.
Zhang H, Bhattacharya D, Lin S J. 2007. A three-gene dinoflagellate phylogeny suggests monophyly of Prorocentrales and a basal position for Amphidinium and Heterocapsa. Journal of Molecular Evolution, 65(4): 463-474, https://doi.org/10.1007/s00239-007-9038-4.
Zhang H, Li Y, Cen J Yet al. 2015. Morphotypes of Prorocentrum lima (Dinophyceae) from Hainan Island, South China Sea: morphological and molecular characterization. Phycologia, 54(5): 503-516, https://doi.org/10.2216/15-8.1.
Zhou C X, Fu Y J, Yan X J. 2007. Hemolytic activity studies of several harmful alga strains. Asian Journal of Ecotoxicology, 2(1): 78-82, https://doi.org/10.3969/j.issn.1673-5897.2007.01.011.(in Chinese with English abstract)
Zou J, Li Q, Liu Het al. 2022. Taxonomy and toxin profile of harmful benthic Prorocentrum (Dinophyceae) species from the Xisha Islands, South China Sea. Journal of Oceanology and Limnology, 40(3): 1171-1190, https://doi.org/10.1007/s00343-021-1045-6.
Zou J, Li Q, Lu S Het al. 2020. The first benthic harmful dinoflagellate bloom in China: morphology and toxicology of Prorocentrum concavum. Marine Pollution Bulletin, 158: 111313, https://doi.org/10.1016/j.marpolbul.2020.111313.
Appendix
Less
Year 2026 volume 44 Issue 3
PDF
13
7
Cite this Article
BibTeX
Article Info
doi: 10.1007/s00343-025-5123-z
  • Receive Date：2025-04-18
  • Online Date：2026-07-29
  • Published：2026-05-01
Article Data
Affiliations
History
  • Received：2025-04-18
Affiliations
    1Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, College of Life Science and Technology, and Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Jinan University, Guangzhou510632, China
    2Department of Life Sciences, Natural History Museum of China, Beijing100050, China

Corresponding:

References
Share
https://castjournals.cast.org.cn/joweb/jol/EN/10.1007/s00343-025-5123-z
Share to
QR

Scan QR to access full text

Cite this article
BibTeX
Citations
表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
关闭全屏
  • BibTeX
  • EndNote
  • RefWorks
  • TxT
Links
Articles
Latest Articles
Most Read
Collections
Updates
Events
News
Multimedia
About
About Us
Contact
No. 86 Xueyuan South Road, Haidian District, Beijing
100081
010-62199257
qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House