Jinxiu WANG , Fanzhou KONG , Yunfeng WANG , Nanjing JI , Minjie SONG ,Zhangxi HU , Zhuang NIU , Chao LIU , Xin WANG , Yuanyuan SUN ,Rencheng YU , Tian YAN

1 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,Qingdao 266071, China

2 Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China

3 University of Chinese Academy of Sciences, Beijing 100049, China

4 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China

5 CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071,China

6 Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Ocean University, Lianyungang 222005,China

Abstract With the development of industrialization and aquaculture in Jiangsu and Shandong Provinces along the South Yellow Sea coast, China, eutrophication has greatly intensif ied in the region, resulting in frequent occurrence of diverse harmful algal blooms. An algal bloom formed by a chain-forming dinof lagellate species was recorded in the Haizhou Bay, South Yellow Sea, in September 2020. The causative species was isolated and studied in morphology, molecular phylogeny, pigment prof ile, presence of paralytic shellf ish toxins, and acute toxicity. The loop-shaped apical groove running anticlockwise around the apex,the presence of peridinin as characteristic pigment, as well as a single phylogenic clade of 28S ribosomal DNA (100% posterior probability), def ined this species as Gymnodinium impudicum, a non-toxic species that exhibited no obvious biotoxicity to the rotifer Brachionus plicatilis, the copepod Artemia salina, and the shrimp Neomysis awatschensis. Gymnodinium impudicum is typically distributed in coastal waters with high nitrate concentrations, where it reaches a maximum density of 2.6×10 5 cells/L. This is the f irst report of a G. impudicum bloom in the Yellow Sea; however, G. impudicum blooms may have been misidentif ied or underreported in Haizhou Bay due to the species morphological similarity with G. catenatum. A combination of multiple methods is recommended to accurately identify new algal bloom species.

Keyword: Gymnodinium impudicum; harmful algal bloom; Yellow Sea; Haizhou Bay; species identif ication;eutrophication

1 INTRODUCTION

Harmful algal blooms (HABs) are intense proliferations of algae that can cause severe economic losses, alter ecosystems, or even endanger human health (Glibert et al., 2014). The frequency,intensity, duration, diversity, and impacts of HABs are increasing worldwide under the inf luence of intensif ied human activities and climate changes(Roelke et al., 2012; Glibert, 2017). Haizhou Bay,a typical open bay located in the South Yellow Sea,is a historically important f ishing ground in China.However, with the development of local agriculture and mariculture industry, eutrophication has become a severe ecological and environmental concern in Haizhou Bay, leading to the frequent occurrence of HABs (Han et al., 2019). In the Haizhou Bay, these blooms show a trend of diversif ication, reduction in cell size and more severe impacts in the last two decades. The dominant species of bloom-forming microalga in the Haizhou Bay have changed from the dinof lagellateNoctilucascintillansto small-sized(6–22 μm) diatomSkeletonemacostatumand many harmful/toxic dinof lagellates (Gao et al., 2017), such as some species of gymnodinoids that are responsible for producing phycotoxins. Multiple blooms ofGymnodiniumcatenatum,Amphidiniumcarterae, andKareniamikimotoihave been recorded in Haizhou Bay (Jiangsu Provincial Oceanic and Fishery Bureau,2011–2017), as well as aKarlodiniumvenef icumbloom in 2020 (unpublished data), that pose severe threats to aquatic and human health. On September 9, 2020, a chain-forming gymnodinioid bloom was observed in Haizhou Bay co-occurring with a bloom of a single-celled gymnodinioidTakayamasp. (Zhang et al., 2022). The increasing occurrence of new gymnodinioid blooms pose a challenge to species identif ication and bloom monitoring in Haizhou Bay.

During HABs monitoring, traditional identif ication of unarmoured gymnodinioids was typically based on morphological characteristics, such as the length of the cingulum displacement, but this approach was clearly inadequate and often problematic because of the species’ fragile cells and highly variable morphology(Re?é et al., 2015). Electron microscopy technology has alleviated this problem, and allowed proposing some new features (e.g., the apical groove) for the identif ication of gymnodinioids (Takayama, 1985).With the development of molecular biology, ribosomal DNA (rDNA) and its internal transcribed spacer (ITS)have been widely used in microalgae phylogeny and classif ication, especially for morphologically similar species. Thus, Gymnodiniales have been redef ined using a combination of morphological and ultrastructural features along with biological phylogeny (Daugbjerg et al., 2000; Re?é et al., 2011;Luo et al., 2018). However, the routine monitoring of phytoplankton in the Haizhou Bay is based on typically algal morphological identif ication by light microscopy, which may lead to the lack of detection or misidentif ication of many gymnodinioids species.

To identify the causative species of the chainforming gymnodinioid bloom on September 9, 2020,a strain of the causative species was isolated and phytoplankton water samples were collected during the bloom. Morphological features, phylogenetic sequences, pigment composition, the putative paralytic shellf ish toxins (PSTs) prof ile, and biotoxicity of the isolate, which were consistent with f ield results,conf irmed a newly recorded bloom-forming species,Gymnodiniumimpudicum, in the Yellow Sea.Phytoplankton composition and spatial distribution during the bloom, as well as their relationships with environmental factors were also analyzed. This study aims to: 1) identify bloom microalgae to species level by applying morphological and molecular methods to the study of algal isolates from a bloom in Haizhou Bay,2) determine pigment and toxin prof iles of the isolated strain and its acute toxicity to invertebrates, and 3)elucidate the occurrence and ecological characteristics of theG.impudicumbloom and potential risk.

2 MATERIAL AND METHOD

2.1 Sample collection, and strain isolation and culture

Surface water samples were collected from eight sites in Haizhou Bay on September 9, 2020(Fig.1). Phytoplankton samples were preserved with Lugol iodine solution and stored in darkness until microscopic analysis. For phytoplankton pigment analysis, water samples were f iltered onto precombusted (450 °C, 6 h) 25-mm Whatman GF/F f iberglass f ilters with a 0.68-μm nominal pore size.The f ilters were frozen in liquid nitrogen and stored at -80 °C until pigment extraction. Filtrates were collected in polyethylene bottles and stored at -20 °C for subsequent analysis of dissolved nutrients. Water temperature and salinity were measured with a RBR concerto CTD (RBR Company, Canada).

A single cell of the bloom-forming species was obtained from live algal blooms samples using an aseptic capillary under an inverted microscope.The isolate was initially cultured in a 48-well plate,and gradually transferred to larger culture volumes.Ultimately, the isolate was cultured in 100-mL Erlenmeyer f lasks using L1-Si medium (Guillard and Hargraves, 1993) prepared with autoclaved seawater at a salinity of 32, at 20±1 °C with a light intensity of 100 μE/(m2·s) and a 14-h?10-h light?dark cycle.

2.2 Laboratory analyses

2.2.1 Characterization of the isolated strain

2.2.1.1 Morphological characterization

Fig.1 Study area and sampling sites in Haizhou Bay

The morphology of the isolated strain was determined by light microscopy (LM) and scanning electron microscopy (SEM). Live cells were observed and photographed with an upright microscope(DM2500, Leica, Germany) at 400× magnif ication.Cells in mid-exponential growth stage for SEM observation were f ixed with 2% OsO4for 1 h, and gravity f iltered through a 5-μm Millipore nylon membrane. The membrane sample was dehydrated through a gradient acetone series, treated with liquid CO2for critical point-drying (EM CPD300,Leica, Austria), then sputter-coated with gold before examination using an S-3400 N SEM (Hitachi,Hitachinaka, Japan). Cell size of the strain was calculated from SEM micrographs.

2.2.1.2 Molecular phylogenetic analysis

Genomic DNA was extracted from 10 mL of clonal culture using a modif ied cetyltrimethylammonium bromide (CTAB) method, as described by Winnepenninckx et al. (2002). The DNA was dissolved in 30-μL Tris-EDTA buff er and frozen at -80 °C until PCR reactions; D1–D2 regions of the 28S large-sub-unit(LSU) rDNA were amplif ied using the primers: LSU D12-F (5?-ACCCGCTGAATTTAAGCATA-3?) and LSU D12-R (5?-CCTTGGTCCGTGTTTCAAGA-3?)(Lenaers et al., 1989). Amplif ication was conducted with an initial denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 20 s, 52 °C for 20 s, 72 °C for 20 s, and a f inal elongation step of 5 min at 72 °C.Targeted DNA bands were electrophoresed in 1%agarose gel, purif ied and sequenced from both ends by Sangon Biotech (Shanghai, China). The obtained sequences were assembled with Vector NTI 11.5.3 ContigExpress (ThermoFisher, USA).

Sequences obtained and 21 other closely related species and outgroup taxa downloaded from GenBank were used for phylogenetic analysis (Table 1).Maximum likelihood analysis was performed using MEGA 7.0 with 500 bootstrap replicates and the K2+G was chosen as the bestf itting nucleotide substitution model. The sequence of this strain was submitted to GenBank (http://www.ncbi.nlm.nih.gov/), with the accession number OL321619.

2.2.1.3 Pigment analysis

Clonal cultures in exponential growth (40 mL)were f iltered onto a 25-mm Whatman GF/F f iberglass f ilter (0.68-μm nominal pore size), and then extracted and analyzed following the method of Zapata et al.(2000) with slight modif ication of the extraction volume and elution gradient prof ile. Pigments were extracted in 1.4-mL 95% methanol, with 100-μL 8?-apo-β, ψ-carotaldehyde (750 μg/L) (Sigma, USA)as internal standard (IS), and analyzed on a Waters E2695 high-performance liquid chromatography(HPLC) system using a Waters Symmetry C8 column(3.5 μm, 4.6 mm×150 mm) with binary gradient elution. The mobile phase and modif ied elution gradient prof ile are shown in Table 2. Pigments were detected with a Waters 2998 diode array detector(DAD) at a wavelength of 440 nm, and identif ied by comparison of their retention time and spectra with pigment standards (DHI Water and Environment,H?rsholm, Denmark).

2.2.1.4 Analysis of paralytic shellf ish toxins

Clonal culture in exponential growth (100 mL) was f iltered onto a Whatman GF/C f iberglass f ilter (1.2-μm nominal pore size), and paralytic shellf ish toxins(PSTs) were extracted following the method of Costaet al. (2015), with some modif ications (Lin et al.,2022). The f ilter was extracted in 3 mL of 0.05-mol/L acetic acid and disrupted with a probe sonicator(Scientz Biotechnology Co. Ltd., Ningbo, China)for 5 min on ice. The extract was centrifuged and the supernatant cleaned by an octadecyl bonded silica cartridge (Supelclean LC-18 SPE cartridge, 3 mL,Supelco, Sigma Aldrich, USA). Water (1.5 mL) was added onto the column to elute residual PSTs. The cartridge was subjected to a second elution with 0.5-mL 20% acetonitrile (V?V), followed by 0.5-mL 80% acetonitrile to capture the hydroxybenzoate analogues or GC toxins. The eluates were then reconstituted and f iltered through 0.22-μm syringe membrane f ilters before analysis.

Table 1 Sequences of 28S large-sub-unit rDNA (D1–D2 regions) of the Gymnodinium or Prorocentrum species used for phylogenetic analysis

Toxin analysis was performed using a Thermo Fisher Ultimate 3000 HPLC system coupled to an AB SCIEX Q-Trap 4500 mass spectrometer (Applied Biosystems, Darmstadt, Germany). The HPLCtandem mass spectrometry (MS/MS) analysis of PSTs including the GC toxins employed during this study was established following the protocol of Costa et al.(2015), with little modif ication (Lin et al., 2022). Theelution procedure was modif ied and set as 65% B to 45% B at a f low rate of 0.2 mL/min in 20 min, and then maintained at 65% B for 5 min. Due to the lack of GC toxin standards, extract ofG.catenatumknown to contain GC toxins was used as a reference. The strain ofG.catenatum(MEL11) was isolated from the coastal waters of Fujian Province, and contains four GC toxins (GC2, GC3, GC5, and GC6) (Lin et al., 2022).

Table 2 Mobile phase and elution gradient prof ile for HPLC pigment analysis

Table 3 Source and test conditions of the three test organisms

Table 4 Initial ratio matrix of the diagnostic pigment to chlorophyll a for CHEMTAX calculations

2.2.1.5 Acute toxicity tests

Acute toxicity tests of the isolated strain were performed with the rotiferBrachionusplicatilis, the brine shrimpArtemiasalinaand the eurytopic mysid speciesNeomysisawatschensisas test organisms.The initial cell density of the isolated strain for acute toxicity tests was 1.0×103cells/mL (slightly higher than the bloom cell density), withChlorellasp. of the same cell density as the negative control and sterilized seawater as the blank.Chlorellasp. was provided by the Algal Culture Center of the Institute of Oceanology,Chinese Academy of Sciences. Both the isolated strain andChlorellasp. used for acute toxicity tests were cultured in L1-Si medium at 20±1 °C under a light intensity of 100 μE/(m2·s) and a 14-h?10-h light?dark cycle. Each treatment was conducted in triplicate with 10 test organisms. The tests were conducted for 24 h without renewing solutions. Mortalities of test organisms were measured at 0, 3, 6, 12, and 24 h after the beginning of the tests (see Table 3 for the source and test conditions of the three test organisms).

2.2.2 Analysis of f ield samples

2.2.2.1 Phytoplankton identif ication and enumeration

Phytoplankton identif ication and enumeration of preserved water samples were performed using an inverted optical microscope (Axiovert, Zeiss,Germany) at 200× or 400× magnif ication. Each sample was counted using a Sedgewick Rafter counting chamber (1 mm2), ensuring that the total number of counted cells was not less than 300. The community taxonomic composition was determined down to the genus and species levels.

2.2.2.2 Pigment analysis and CHEMTAX analysis

Field samples for pigment analysis were treated in the same manner as the cultured strain above. Data were analyzed using CHEMTAX software to calculate the contribution of distinct phytoplankton taxa based on the ratio of diagnostic pigments to chlorophylla(Chla). The initial pigment ratios (Table 4) for CHEMTAX calculation were approximated base on Mackey et al. (1996). Dinof lagellates were divided into two sub-groups. Peridinin (Peri) was used as a diagnostic pigment to represent most dinof lagellates(Dino-peri), while the group “Dino-fuco” representedTakayamasp. (Zhang et al., 2022), dominant species with 19’-butanoyloxyfucoxanthin (But-fuco),fucoxanthin (Fuco) and gyroxanthin-diester (Gyro)as diagnostic pigments. The initial ratio of each diagnostic pigment to Chlafor the “Dino-fuco”group was determined based on the pigment ratio ofTakayamasp. at the bloom site and the culturedTakayamasp. strain (data not shown). The selection of other phytoplankton groups (i.e., prasinophytes,cryptophytes, haptophytes (containing the Type 8 pigment (T8) described by Zapata et al. (2004)),chlorophytes, cyanobacteria, and diatoms) was mainly based on the detection of their diagnostic pigments in Haizhou Bay. The optimized output matrix of pigment ratios is provided in Supplementary Table S1.

2.2.2.3 Nutrient analysis

Nutrients in seawater, including the concentration of nitrate (NO3ˉ), nitrite (NO2ˉ), ammonium (NH4+),phosphate (PO43ˉ), silicate (SiO32ˉ), dissolved total nitrogen (DTN), and dissolved total phosphorus(DTP), were measured with a QUAATRO Continuous Flow Analyzer (QuAAtro 39, Seal, Germany).The principle of nutrient analysis is based on the“Specif ication for oceanographic survey” (GB/T 12763.4-2007). The concentration of NO3ˉ, NO2ˉ,NH4+, PO43ˉ, and SiO32ˉ were analyzed by the coppercadmium reduction method, diazo azo method,sodium bromate oxidation method, molybdenum blue colorimetry, and silicon-molybdenum blue method, respectively. DTN and DTP were analyzed following persulfate oxidation (120 °C, 30 min).The sum of NO3ˉ, NO2ˉ, and NH4+was considered as dissolved inorganic nitrogen (DIN), and PO43ˉ was considered as dissolved inorganic phosphorus (DIP).The concentrations of dissolved organic nitrogen(DON) and dissolved organic phosphorus (DOP)were determined by subtracting DIN and DIP from DTN and DTP, respectively.

2.3 Statistical analysis

Statistical signif icance of the diff erence between controls and treatments in acute toxicity tests was determined using a two-tailed pairedt-test and results were considered statistically signif icant atP<0.05.The correlation analysis between microscopic enumeration and CHEMTAX calculation was performed by Pearson correlation tests. Principal component analysis (PCA) performed with CANOCO 5.1 software was used to examine the relationships between phytoplankton groups and environmental factors.

3 RESULT

3.1 Biological characteristics of algal bloom causative species

3.1.1 Morphology

Light microscope (Fig.2a) and SEM (Fig.2b–i)images ofGymnodiniumimpudicumreveal that the unarmoured cells were ovoid, from 10.5- to 20.3-μm long (15.0±2.8 μm standard error,n=27) and from 15.1- to 20.0-μm wide (17.3±1.3 μm standard error,n=27). Chains of 2, 4, and 8 cells were observed,although the most common were chains of 4 cells(Fig.2a–c). The anterior and posterior cells of the chains were longer than the intermediate cells.The anterior cells had a dome-shaped epicone and f lattened hypocone, while the posterior cells showed the opposite shape (Fig.2d–e). The epicone and hypocone of intermediate cells in the chain were f lattened (Fig.2f). There were round pores in the middle apex and antapex (Fig.2g), and the pores seemed to be connected by f ilaments in some cells(Fig.2c), through which the cells form chains. The cingulum was deeply incised with a displacement of ca. 1/3–1/4 of the cell length. The sulcus was narrow, penetrated into the epicone directly to the apex forming the apical groove. The latter was loopshaped, running anticlockwise viewed from the apex(Fig.2d & h). It contained three elongated vesicles,and possessed a string of knobs on the outer row of vesicles (Fig.2i).

3.1.2 Phylogeny

The 28S rDNA sequence of the strain isolated from the Haizhou Bay was aligned with other related microalgae belonging to theGymnodiniumgenus selected from GenBank and usingProrocentrumclipeusas an outgroup (Fig.3). The well resolvedGymnodiniumcladesensustrictocomprisedG.impudicumas well as other species, such asG.dorsalisulcum,G.litoralis,G.aureolum,G.plasticum,G.microreticulatum,G.catenatum, andG.nolleri. The strain in this study grouped well with f ive strains ofG.impudicumisolated from other sea areas (the Bohai Sea, coastal waters of France, Australia, and South Korea) to form a single phylogenic clade with a posterior probability of 100%. All otherGymnodiniumsequences formed distinct clades.Gymnodiniumimpudicumspecies were closely related to a clade ofG.dorsalisulcumwith moderate ML bootstrap support (92). Molecular phylogeny was also analyzed using 18S rDNA and ITS rDNA sequences with a subset of species, and these results were not signif icantly diff erent from those of 28S rDNA phylogenetic analysis (data not shown).

3.1.3 Pigment prof ile

Pigments detected in the culturedG.impudicumisolate included peridinin (Peri), diadinoxanthin(Diad), dinoxathin (Dino), Chla, and a small amount of peridininol, diatoxanthin, zeaxanthin,β,β-carotene and some unknown carotenoids (Fig.4). Results showed thatG.impudicumhas a peridinin-type chloroplast, with Peri (21.2 pg/cell) as the diagnostic pigment, which was the most abundant and slightly higher than Chla(18.6 pg/cell). The molar ratio of Peri to Chlawas about 1.6?1. Diadinoxanthin(Diad) and Dino were major accessory carotenoids,contributing almost equally to the carotenoid pool.

Fig.2 Light microscope (a) and scanning electron microscope (b–i) micrographs of Gymnodinium impudicum

3.1.4 Toxin prof ile and acute toxicity

Compared with HPLC-MS/MS product ion spectra of the PST standards (including the gonyautoxins GTX1-6, neosaxitoxin (NEO), saxitoxin (STX), and decarbamoyl toxins dcGTX2, dcGTX3, dcSTX) and GC toxins fromG.catenatum(including the GC2,GC3, GC5, and GC6), no toxin was detected inG.impudicum, which conf irmed thatG.impudicumdid not contain PSTs (Supplementary Fig.S1).

Acute toxicity tests showed thatG.impudicumhad no obvious biological toxicity towardsB.plicatilis,A.salinaandN.awatschensis(Fig.5).BrachionusplicatilisandA.salinawere insensitive toG.impudicum, and experienced negligible mortalities when exposed to this alga during 24 h. The mortality ofN.awatschensiswas higher than that ofB.plicatilisandA.salina.Neomysisawatschensisbegan to die after 3 h, but there was no signif icant diff erence between the treatments and controls.

3.2 Algal bloom f ield studies

3.2.1 Phytoplankton community composition

Fig.3 Maximum-likelihood phylogenetic tree of Gymnodinium impudicum and selected species inferred from the D1–D2 region of 28S LSU rDNA

Fig.4 Chromatogram of Gymnodinium impudicum pigments

A total of 30 phytoplankton taxa were detected and identif ied from this Haizhou Bay algal bloom. The phytoplankton community was mainly dominated by dinof lagellate and diatom species.GymnodiniumimpudicumandTakayamasp. were the two dominant dinof lagellate species, with a peak abundance of 2.6×105cells/L (site A1) and 1.7×107cells/L (site B1),respectively, which jointly caused the heterogeneous bloom.Ceratiumfurca,C.triposandC.fususwere sub dominant groups, with highest cell abundances of 7.8×104, 8.8×103, and 5.0×103cells/L, respectively.Protoperidiniumspp. was another relatively abundant dinof lagellate group. The abundance of diatoms was lower than that of dinof lagellates, and the major diatom species belonged to the generaChaetoceros,Coscinodiscus, andNitzschia. Distribution patterns of these species are shown in Fig.6. The distribution ofG.impudicum,C.furca, andChaetocerosspp. were similar, with highest abundance in the area closest to Liandao Island.CeratiumfususandProtoperidiniumspp. were also distributed in nearshore waters, mainly at sites A1 and A3. BothTakayamasp. andC.triposwere concentrated at site B1 in the northwest of the study area. The distributions ofCoscinodiscusspp.andNitzschiaspp. were opposite to that ofTakayamasp. in mainly the southeast site B3.

Fig.6 Distribution of major phytoplankton groups in the Haizhou Bay

The contribution of diff erent phytoplankton groups to total Chl-abiomass estimated using CHEMTAX is shown in Fig.7. Consistent with the results of microscopic examination, Dino-peri (mainlyG.impudicum) and Dino-fuco (mainlyTakayamasp.)were the dominant phytoplankters, exceeding diatoms at almost all sites. There was a high and signif icant correlation between the results of microscopic cell counts and CHEMTAX analysis for Dino-peri(Pearson correlation coeffi cient,R=0.89,P< 0.01) and Dino-fuco (Pearson correlation coeffi cient,R=0.88,P< 0.01). Site A2 with the greatest abundance of Dino-peri had the lowest proportion of the Dino-fuco group, while the inverse was observed at site B1. The phytoplankton communities were more complex at sites relatively far from the coast and the algal bloom area, such as B3, C1, and C3. Site C1 in the north had a relatively high abundance of chlorophytes,cyanobacteria, and prasinophytes.

3.2.2 Relationship between phytoplankton groups and environmental parameters

The spatial distributions of temperature, salinity,and major nutrients are given in Fig.8. Water temperatures ranged between 26.45 and 28.81 °C. It is noteworthy that water temperatures were measured at diff erent times during the same day, so they may not eff ectively ref lect the spatial temperature pattern.Salinity was lower in nearshore waters and highest in the north of the study area. The distribution of SiO32ˉ,NO3ˉ, and NO2ˉ was consistent, showing the highest concentrations at sites A1 and A2 near the west coast.Concentrations of NO3ˉ and NO2ˉ were extremely low at site B1, which was the area of high densities ofTakayamasp. The ratio of DIN to DIP ranged from 12?1 (at the B1 site) to 258?1, with an average of 144?1, thus far exceeding 16?1 Redf ield ratio (Redf ield et al., 1963). In contrast to NO3ˉ and NO2ˉ, PO43ˉ, DON,and DOP concentrations were the highest at site B1.Ammonium concentration was maximal at site C1 in the north, but was lower in nearshore waters.

Principal component analysis (PCA) was performed to discern the relationship between major phytoplankton groups and environmental parameters(Fig.9).Gymnodiniumimpudicum,C.furca,C.fusus,Chaetoceros, andProtoperidiniumspp. were positively correlated with NO3ˉ, NO2ˉ, and SiO32ˉ, but negatively correlated with temperature and salinity,which were mainly attributable to their nearshore distribution. In contrast,Takayamasp.,C.tripos,NitzschiaandCoscinodiscusspp. were positively correlated with DON, DOP, and PO43ˉ, negatively correlated with inorganic nitrogen and were not signif icantly aff ected by temperature and salinity.Ammonium was negatively correlated with most phytoplankton groups, but had little eff ect.

Fig.7 Chlorophyll- a biomass contributed by diff erent phytoplankton groups during the Haizhou Bay bloom on September 9, 2020

Fig.8 Distribution of temperature, salinity, and major nutrients in the Haizhou Bay

4 DISCUSSION

4.1 Identif ication of Gymnodinium impudicum and possible misidentif ication of Gymnodinium catenatum in Haizhou Bay

Naked dinof lagellates are diffi cult to diff erentiate under the light microscope, especially from f ixed samples.Gymnodiniumimpudicumwas initially namedGyrodiniumimpudicumbased on morphological features (Fraga et al., 1995).Gymnodiniumspecies were originally characterized by a cingulum displacement ≤20% of the cell length, whileGyrodiniumspecies have a cingulum displacement ≥20% of the cell length. However,variation of the cingulum displacement within the same species often hindered generic identif ication.Therefore, additional morphological features, such as the apical structure complex (ASC), have been proposed as new characteristics for the taxonomy of gymnodiniods. Thus,Gyrodiniumimpudicumwas renamedGymnodiniumimpudicumbased on an anticlockwise loop-shaped apical groove and molecular analyses (Daugbjerg et al., 2000). Other gymnodinioid species were also transferred to either known or newly described genera based on the shape of the apical groove and other important morphological criteria. For example,Gymnodiniumfususwas successively transferred toCeratoperidinium(Re?é et al., 2013) andPseliodinium(Gómez, 2018). The new generaLevanderina(Moestrup et al., 2014),Pellucidodinium(Onuma et al., 2015),Wangodinium(Luo et al., 2018) etc., were established to incorporateGymnodinium-like species.

Fig.9 Principal component analysis (PCA) of the association of major phytoplankton groups with environmental parameters

Gymnodiniumimpudicumhas been confused with toxigenicG.catenatumdue to their similar chainlike morphology based on which the diff erentiation in light microscopy is diffi cult (Carrada et al., 1991;Gómez, 2003). Compared withG.catenatum(isolated from coastal waters of Fujian Province) (Fig.10a &d),G.impudicumcan be distinguished by smaller cell size and shorter chains of eight cells at the most, whileG.catenatumcan form chains of up to 64 cells. Chain cells ofG.impudicumare closely attached, while inG.catenatumthere are elongated connections (Fraga et al., 1995). In terms of cell shape, the hypocone ofG.impudicumis “plump” whereas that ofG.catenatumis trapezoidal. However, all these distinguishing features are vague and often inaccurate, especially for inexperienced phytoplankton identif ication personnel.Additionally, f ixation with Lugol solution (Fig.10b& e) and formaldehyde solution (Fig.10c & f) cause deformation and degradation ofG.impudicumandG.catenatum. Fixed cells are nearly spherical, and the taxonomic characteristics are almost lost, making species identif ication more diffi cult.Gymnodiniumcatenatumhas become the second dominant algal bloom species in Haizhou Bay in addition toS.costatumin the past two decades (Gao et al.,2017), but detailed species identif ication has not been undertaken, and no associated poisoning event has been reported. Water samples from the routine monitoring of algal blooms in Haizhou Bay are typically f ixed with Lugol solution or formaldehyde solution and identif ied by light microscopy, which is known to be insuffi cient for species identif ication.Additionally,G.impudicumandG.catenatumoverlap ecologically (Band-Schmidt et al., 2020),and sometimes co-occur in the same bloom (Lee etal., 2001; Glibert et al., 2002). It is thus probable thatG.catenatumblooms have been misidentif ied and thatG.impudicumhas been underrepresented in the Haizhou Bay in the past.

Fig.10 Light microscope micrographs of live cells (a, d) and cells f ixed with Lugol solution (b, e) and formaldehyde solution(c, f) of Gymnodinium impudicum (a, b, c) and Gymnodinium catenatum (d, e, f)

To overcome these confounding factors, pigment prof iling, DNA sequencing and more sophisticated microscopy techniques, such as scanning or transmission electron microscopy, are essential to distinguish unarmoured dinof lagellates. Results of the analyses conducted in this study support the conclusion that the bloom occurred on September 9,2020 in Haizhou Bay was formed byG.impudicumrather thanG.catenatum. Most of the morphological features and the pigment prof ile ofG.impudicumin this study are consistent with those reported in other sea areas (Fraga et al., 1995; Yim and Lee, 2004; Luo et al., 2018). Phylogenetic analysis of 28S rDNA showed thatG.impudicumdiff ered signif icantly fromG.catenatumin terms of molecular phylogeny with a nucleotide sequence similarity of 86%, although they have similar morphological characteristics.Gymnodiniumimpudicumisolated in this study was non-toxic, and did not cause signif icant mortality of marine test organisms both in acute toxicity tests and in the bloom area, which was consistent with the description of Fraga et al. (1995). Given thatG.catenatumis a PSTs producer, to distinguishG.catenatumandG.impudicumin the future algal bloom monitoring in Haizhou Bay, it is f irstly recommended by microscopic observation combined with rapid toxin kits or HPLC-MS/MS toxin analysis,secondly by accurate molecular analysis at the same time for species verif ication if conditions permit.

4.2 Trophic modes of Gymnodinium impudicum and concurrent Takayama sp.

Dinof lagellates are characterized by diff erent trophic modes, including exclusively autotrophic,mixotrophic, and heterotrophic, which contribute to the proliferation of diff erent types of algae under varying nutrient proportions and forms (Heisler et al.,2008). Haizhou Bay is aff ected by multiple nutrient sources, such as industrial effl uents, agricultural runoff , municipal sewage, and intensive mariculture,creating a complex nutritional environment via the introduction of organic and inorganic nutrients that can support a variety of algal species (Zhang et al.,2022).

Gymnodiniumimpudicumhas been described as an autotrophic species (Yahia-Kéf i et al., 2005). It showed a signif icant positive correlation with nitrate concentration in the Gulf of Tunis (Aissaoui et al.,2012), and its growth rate increased signif icantly with increasing nitrate concentration below 40 μmol/L in a laboratory experiment (Lee et al., 2001). In the present study, the abundance ofG.impudicumin the Haizhou Bay was highest in coastal waters near Liandao Island with high nitrate levels (33 μmol/L)and a high N/P ratio (128?1), which may be related to its autotrophic characteristics. Based on literature descriptions and f ield observations, the red seaweedPorphyrayezoensisis intensively cultured in this area (Su et al., 2020).Porphyrayezoensisshowed high absorption of nitrate and ammonium during its growth period (from October to April of the following year), resulting in the decrease of inorganic nitrogen concentration in seawater (Wang and Cao,2020). In September, four months after the harvest ofP.yezoensis, nutrient accumulation coupled with increased input from runoff may provide suffi cient nitrogen forG.impudicumbloom.

Species of all three genera of the family Kareniaceae have been found to adopt a mixotrophic strategy, with the ability to assimilate dissolved organic nutrients or consume prey. In contrast toG.impudicum, the highest abundance ofTakayamasp. appeared in the area with the lowest nitrate but the highest organic nitrogen,which is close to an oyster and mussel culture area. It has long been known thatKareniacan utilize a wide variety of organic N and P substrates (Yamaguchi et al., 2004; Killberg-Thoreson et al., 2014), but little is known about the nutrient requirements ofTakayama.In the Johor Strait, Singapore, the occurrence of a variety of algal blooms includingTakayamasp. is considered to be supported by the high concentration of dissolved organic material (Leong et al., 2015). The close relationship betweenTakayamasp. and DON and DOP concentrations in Haizhou Bay indicates thatTakayamasp. may be able to remain competitive by utilizing organic compounds.

Phagotrophy has been noted forT.helixandT.tasmanica, and both of them can feed on algae much larger than themselves (Jeong et al., 2016; Lim et al., 2018).Ceratiumtripos, which was closely related toTakayamasp., is considered a better food forFragilidiumsubglobosumthanC.furcaandC.fusus,supporting higher growth rates ofF.subglobosum(Hansen and Nielsen, 1997).Takayamasp. in Haizhou Bay may prey on largeC.triposcells by sucking intracellular contents, or perhaps mixotrophicC.triposhave similar ecological characteristics to those ofTakayamasp. Since these suggestions are based only on one-day studies, the specif ic relationship between algae and nutrients needs further investigation via laboratory and f ield results.

4.3 Occurrence of novel algal blooms associated with eutrophication in the Haizhou Bay

Reports ofG.impudicumhave usually been localized, the species has been reported to form blooms in Korea (Park and Park, 1999; Lee et al.,2001), the Arabian Gulf (Glibert et al., 2002), and diff erent areas of the Mediterranean Sea (Spain, Italy,Tunisia, and Egypt coastal waters) (Fraga et al., 1995;Yahia-Kéf i et al., 2005; Mikhail et al., 2020). Most of these blooms develop in eutrophic coastal waters with high levels of nitrate or phosphate and usually occur in summer and autumn at high temperatures (22–31 °C). In China,G.impudicumwithAlexandriumsp. and other algal species caused large-scale blooms(630 km2) along the coast of Tianjin in the Bohai Sea from August to October 2016 (Ocean Administration of Tianjin, 2017). Cysts ofG.impudicumhave been detected in the North Yellow Sea and in Zhejiang and Fujian coastal waters (Sun et al., 2006; Lin et al., 2016;Weng, 2017), indicating the potential of this species to form blooms throughout a wide range of China’s coastal waters, but detailed studies ofG.impudicumin China have rarely been carried out.

The present study provides the f irst record of aG.impudicumbloom in the Haizhou Bay,and in the South Yellow Sea. Like most otherG.impudicumblooms in warm and eutrophic waters, that occurring in Haizhou Bay shared similar environment characteristics. With socioeconomic development and accelerated industrialization,various anthropogenically produced nutrients are continuously discharged into Haizhou Bay, leading to eutrophication and frequent occurrence of HABs.Intensive shellf ish mariculture activity in the Haizhou Bay is another potential cause of eutrophication by altering the natural cycling of nutrients as point sources of regenerated nutrients. Due to the low assimilation effi ciency, shellf ish can act as pumps along the coast to transform the nutrients in algal biomass into dissolved and particulate detrital nutrients (Bouwman et al., 2013). Since the 1980s, nitrate concentrations in Haizhou Bay increased markedly, from 1.30 to about 20–30 μmol/L in 2015, and the N/P ratio also increased from 8.3 to 91.9 (Zhu et al., 2017). Nitrate concentrations inf luenced by river f lood discharge,are usually highest in August, September, and October(Tian et al., 2006). The weak water exchange capacity of this area aggravates the accumulation of nutrients,creating a favorable environment for the occurrence of various algal blooms, which extend from hundreds to thousands of square kiliometers in area.

In recent decades, toxic and harmful dinof lagellates,including presumedG.catenatum,Gonyaulaxpolygramma,A.carteraeandK.mikimotoi,have gradually replaced diatoms as the dominant taxonomic group blooming in Haizhou Bay under high N/P conditions. From June to November 2020 alone, successive blooms caused byK.venef icum,Takayamasp.,G.impudicumand the raphidophyteHeterosigmaakashiwowere observed in the Haizhou Bay (unpublished data), i.e., without including other bloom events that may have been missed. Given the high bloom species variety and frequency in Haizhou Bay, we need to be vigilant of new algal blooms, which present new challenges for algal bloom monitoring and prevention, as well as unknown ecological and socioeconomic risks.

5 CONCLUSION

A newly-recorded algal bloom caused byG.impudicumin the Haizhou Bay was scrutinized based on morphological features and 28S rDNA gene analyses. Typical dinof lagellate pigments were found and no paralytic shellf ish toxins were detected. The non-toxicG.impudicumtended to be distributed in coastal waters rich in nitrate, while the concurrentTakayamasp. preferred to organic nutrients, which might indicate diff erential trophic modes of these dinof lagellates. Complex eutrophication of Haizhou Bay has led to the occurrence of a variety of novel algal blooms, which needs to be considered to better manage the unexpected risks and impacts.

6 DATA AVAILABILITY STATEMENT

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGMENT

The authors greatly appreciate the constructive advice of anonymous reviewers. They also thank Wei LIU, Institute of Oceanology, Chinese Academy of Sciences (IOCAS), for his help in the use of SEM,Xiuqi YU, IOCAS, for her assistance in performing acute toxicity tests, and Zhuoru LIN, IOCAS, for her assistance in the analysis of paralytic shellf ish toxins.