2 Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai 519000, China;
3 Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China
Benthic dinoflagellates, e.g., species of Ostreopsis, Gambierdiscus, Fukuyoa, Prorocentrum, Coolia, and Amphidinium, are group of microalgae that attach to substrates such as macroalgae, dead coral, sand, and rocks (Fukuyo, 1981; Boisnoir et al., 2019a). Harmful benthic algal blooms have aroused widespread concern due to benthic-dinoflagellate-produced toxins and bloom-induced anoxia, which endangers human health and marine ecosystem (Yasumoto et al., 1987; Litaker et al., 2010; Mangialajo et al., 2011; Kudela et al., 2017). Palytoxin and its analogs, the metabolites of some Ostreopsis species, are the most toxic non-protein algal toxins (Amzil et al., 2012; Ben Gharbia et al., 2016). The ciguatoxins and maitotoxins generated by species of Gambierdiscus and Fukuyoa, which are responsible for ciguatera fish poisoning (CFP) in humans, are the most serious non-bacterial illness associated with seafood consumption (Litaker et al., 2010). Some species of Prorocentrum produce okadaic acid and its analogs, which are the culprit of diarrheic shellfish poisoning (DSP) in humans (Tripuraneni et al., 1997; Foden et al., 2005). Furthermore, some Coolia and Amphidinium species can threaten marine benthic animals (Pagliara and Caroppo, 2012; Karafas et al., 2017).
In 1979, with the discovery of Gambierdiscus toxicus — a toxic species isolated from a ciguatera-endemic area — the study of harmful benthic dinoflagellates was initiated (Yasumoto et al., 1977; Adachi and Fukuyo, 1979). An increase in number of CFP incidences and Ostreopsis blooms reported from around the world has led to the increasing studies highlighting their ecological impacts (Litaker et al., 2010; Rhodes, 2011). In recent decades, scholars have paid more attention to the taxonomy, toxicity, and ecology of benthic dinoflagellates, as well as the geographical distribution of toxic and harmful species (Kudela et al., 2017). CFP producers, species of Gambierdiscus and Fukuyoa, are mainly reported in tropical and subtropical waters (Bienfang et al., 2008; Chinain et al., 2020). A review summarized that the Caribbean Sea and the Western Pacific Ocean are the areas where these two genera are most requently reported (Chinain et al., 2021). This distribution pattern has also expanded to higher latitude temperate waters, including Korea (Jang et al., 2018) and the northeast Mediterranean Sea in the past decade (Aligizaki and Nikolaidis, 2008). Ostreopsis species are distribution globally. Blooms of Ostreopsis have frequently recorded in waters of Mediterranean Sea (Mangialajo et al., 2011; Rhodes, 2011; Kang et al., 2013; Acaf et al., 2020; Nascimento et al., 2020). Besides Ostreopsis, other genera Coolia, Amphidinium, Prorocentrum, and Sinophysis is with higher biodiversity of benthic dinoflagellates (Rhodes, 2011; Ben Gharbia et al., 2016, 2019; Reñé et al., 2020). Benthic dinoflagellates are abundantly distributed on coral reefs and island ecosystems. Fukuyo (1981) isolated eleven benthic dinoflagellate species belonging to five genera from coral reefs and systematically described the whole benthic dinoflagellate community. Some islands in the South Pacific, such as French Polynesia, the Cook Islands, and the Republic of Kiribati, are considered to be "hot spots" of Gambierdiscus biodiversity (Chinain et al., 2016). So far studies of benthic dinoflagellates have been mostly focused on a certain genus or toxic species. There have been relatively few studies on entire benthic dinoflagellate communities (Nishimura et al., 2013; Boisnoir et al., 2018; Lim and Jeong, 2021). Those studies of community composition and substrate preferences have only been carried out in a few areas (Yong et al., 2018; Mustapa et al., 2019; Accoroni et al., 2020).
To date, three species of Gambierdiscus (Zhang et al., 2016), one species of Fukuyoa (Leung et al., 2018), two species of Ostreopsis (Zhang et al., 2018), four species of Coolia (Leung et al., 2017), eleven species of Amphidinium (Luo et al., 2022), and more than ten Prorocentrum species, in which some of the species were okadaic acid and/or DSP toxins producers (Luo et al., 2017; Zou et al., 2021), have been reported in the South China Sea. Recently, a harmful Prorocentrum concavum bloom was reported at Hainan Island, which is the first benthic dinoflagellate bloom in China at present (Zou et al., 2020).
To date, most studies on benthic dinoflagellates have been concentrated in intertidal areas, bays, lagoons, and islands, especially in the Caribbean Sea and Mediterranean Sea (Mangialajo et al., 2011; Durán-Riveroll et al., 2019; Chinain et al., 2021). A study of how water movement, depth, and diﬀerent habitats influenced on the abundance and distribution of benthic dinoflagellate communities was carried out at Johnston Atoll, Pacific Ocean (Richlen and Lobel, 2011). It was found that moderate hydrodynamic disturbance promoted the distribution of benthic dinoflagellate community. The sampling depth of this study was concentrated below 5 m and the maximum depth was only 13 m. Lee et al. (2020) studied the eﬀects of substratum and depth on benthic harmful dinoflagellate assemblages at Perhentian Islands and Terengganu, Malaysia. Though the deepest sampling depth was 25 m, sampling sites were mostly on the coasts of the islands. In short, there are few studies on reefs of more than 20-m depth, especially the areas far away from the mainland. Compared with the coasts of continents or large islands, reef ecosystems with water depths of more than 20 m are subject to stronger hydrodynamic disturbances. The Zhongsha Islands are the typical tropical coral reef ecosystems composed of reef-building corals and the marine organisms that grow in and around the reefs. The main body of the Zhongsha Islands, Zhongsha Great Atoll, is fully submerged beneath water depth of 13–26 m. The seabed surface of Zhongsha Great Atoll is coral sand and shell debris. In this study, we investigated the biodiversity and distribution of benthic dinoflagellates in the Zhongsha Islands based on morphology, phylogeny, and cell counting. Samples of sediments and macroalgae were collected to analyze the epibenthic dinoflagellate community composition and abundance at diﬀerent shoals.2 MATERIAL AND METHOD 2.1 Study area and sampling strategy
The Zhongsha Islands (13°57′N–19°33′N, 113°02′E–118°45′E), are composed of 26 named shoals on Zhongsha Great Atoll and Huangyan Island, and four scattered shoals: the Yitong, Xianfa, Shenhu, and Zhongnan Shoals. The Zhongsha Islands belong to a tropical monsoon climate zone, with the nature characteristics of strong sunshine exposure, low temperature variation, and high temperature and salinity. In addition, the Zhongsha Islands have distinct dry and wet seasons, and abundant precipitation that increases from north to south.
Sediments and macroalgal samples were collected by scuba diving between June 24 and July 5, 2020, at the Zhongsha Great Atoll (six stations), Yitong Shoal, and Shenhu Shoal, China (Fig. 1 & Table 1). Sediment samples were collected in triplicate using a 10-cm×10-cm×3-cm mud sampler and were transferred with seawater into a 1-L sample bag (Fig. 2). Sediment samples were placed for 10 min for sediment setting, and then seawater was slowly collected and the volume was recorded. Later, the seawater was filtered with a 120-μm sieve to collect the epibenthiccells. Benthic dinoflagellates were retained by a sieve with 20-μm mesh, then immediately fixed in 1.5% Lugo's solution. Water samples were collected using a water sampler. Macroalgae were sampled carefully with the surrounding seawater and transferred into sampling bags. Macroalgal samples were shaken for 30 s to detach attached dinoflagellate cells and then rinsed twice with 0.22-μm filtered seawater. The seawater containing epibenthic algal cells were filtered using a 120-μm sieve and then epibenthic algal cells were retained by a 20-μm mesh, and immediately fixed in 1.5% Lugol's solution. The separated macroalgae was dried using absorbent paper before weighing (Boisnoir et al., 2019b).2.2 Dinoflagellate cell abundance
Cells were counted by 1-mL Sedgewick Rafter Counting Cell using the light microscope (Olympus BX 61, Tokyo, Japan). The cell abundance of the sediment was described as cells per 100 cm2. Abundance on macroalgae was measured as cells per gram of fresh macroalgal weight.2.3 Isolation and culture
Live cells during the exponential period were isolated with micropipette under the light microscope. Isolated benthic dinoflagellates cells were cultured in a 96-well plate with L1 medium (Guillard and Hargraves, 1993). The culture plate was under a dark꞉light cycle of 12 h꞉12 h with 100-μmol/(m2·s) irradiance, 25 ℃, and salinity of 30. Strains that divided a number of cells were ported to a 25-mL culture flask with a same culture conditions.2.4 Morphological observation
Cell during the exponential period was observed under a microscope. Morphological data (cell shape, length, and width) were obtained using Image-Pro Plus 6.0 Image.
The morphology and distribution of chloroplasts and nuclei were observed using a fluorescence microscope, then image acquisition was conducted using the QImaging Retiga 400R digital camera (QImaging, Surrey, BC, Canada) at 400 magnifications.
Sample preparation for the scanning electron microscope (SEM) was performed according to Lim et al. (2019). Cells grown in the exponential phase were fixed in glutaraldehyde (final concentration: 2.5%) for 12 h at 4 ℃. One milliliter of algal cells was pipetted onto a round glass slide and soaked in 10% polylysine for more than 2 h. The slide was observed under a microscope to ensure that a large number of cells were adsorbed on the surface. The cells were soaked using a gradient of 100%, 80%, 60%, 40%, and 20% of the seawater and Milli-Q water for 5 min. Dehydration was carried out through a series of ethanol gradients (10%, 30%, 50%, 70%, and 90% and three times at 100%, for 5 min each). The dehydrated cells were dried by a CO2 critical point dryer (Leica Microsystems, Mannheim, Germany) and putter-coated with gold (Leica, Germany). Finally, the morphology and structure of cell was presented and photographed under a scanning electron microscope (Carl Zeiss Inc., Germany).2.5 DNA isolation and gene amplification
The clonal cells of all benthic dinoflagellate strains were harvested at 12 000×g for 1 min at 4 ℃. Total genomic DNA of exponential cells were extracted by MiniBEST Universal Genomic
DNA Extraction Kit (TaKaRa, Japan) according to the instructions. A 50-μL PCR reaction system includes: 2-μL DNA, 25-μL Accurate TaqMaster Mix (2X) (Zoman Biotechnology, China), each 1 μL of the forward primer and reverse primer, and 21-μL ultrapure water. The D1–D3 regions of large subunit (LSU) was amplified using general primers (D1R: 5ʹ-ACCCGCTGAATTTAAGCATA-3ʹ, D3B: 5ʹ-TCGGAGGGAACCAGCTACTA-3ʹ) described in Scholin et al. (1994). The PCR cycle procedure: pre-denaturation at 94 ℃, 4 min and followed by 36 cycles, each cycle included denaturation at 94 ℃ for 20 s, annealing at 56 ℃ for 30 s, and extension at 72 ℃ for 1.5 min, and a final extension step at 72 ℃ for 10 min (Zou et al., 2020). All sequences were uploaded to GenBank and the accession numbers were listed in Supplementary Table S4.2.6 Phylogenetic analysis
34 sequences of the LSU (D1–D3) regions obtained by sequencing was aligned and edited by ContigExpress software. The obtained sequences from our study and related species sequences from GenBank, which consist of 41 Amphidinium sequences, 43 Coolia sequences, 61 Ostreopsis sequences, and 64 Prorocentrum sequences, were analyzed for multiple alignments at the website (https://www.ebi.ac.uk/Tools/msa/muscle/). Sequence group of diﬀerent genera were aligned separately. Then the aligned results were break oﬀ both ends using BioEdit (Version 7.2.9) and then were analyzed using the maximum likelihood (ML) analysis in an online program (https://www.phylo.org/) with RAxML-HPC2 and XSEDE V. 8.2.10. The best models were determined by the program MrModeltest2.3 under the Akaike Information Criterion. In addition, Bayesian inference (BI) was conducted at Mr. Bayes 3.2.7 with four Markov Chain Monte Carlo (MCMC) chains and each chain was run for 1 000 000 cycles and sampling every 100 cycles using the best fit model (GTR). The uncorrected P-distances among all species and its closely related species were used by the software Mega X and the results are displayed in the Supplementary Table S5.3 RESULT 3.1 Biodiversity of benthic dinoflagellates species
Fifteen species of benthic dinoflagellates belonging to four genera were identified in morphology: Amphidinium carterae, A. magnum, A. massartii, A. operculatum, Coolia canariensis, C. malayensis, C. palmyrensis, C. tropicalis, Ostreopsis cf. ovata, Prorocentrum concavum, P. cf. sculptile, P. emarginatum, P. hocmﬀcmmannianum, P. lima, and P. rhathymum. We focus the morphology of a species first discovered in the waters of China (A. magnum), and potentially widespread toxic species (O. cf. ovata, P. lima, and P. hocmﬀcmmannianum). The morphological micrographs of P. cf. sculptile and A. carterae were not obtained because of cultivation failure.3.1.1 Morphology of benthic dinoflagellates 126.96.36.199 Amphidinium magnum (Fig. 3)
Cells are 26.94–38.10 μm long (mean 34.05±2.31 μm, n=30) and 20.04–29.70 μm wide (mean 24.04±1.93 μm, n=30). The length/width (L/W) ratio is from 1.03 to 1.67 (mean 1.42±0.10, n=30). The cells are generally ovoid, and the epicone extends to the right with an approximate triangular structure. The antapex of the hypocone is rounder, while its right is almost vertical (Fig. 3a). No pyrenoid is observed but numerous chloroplasts fill the whole cell with a block distribution (Fig. 3b). The nucleus is posterior (Fig. 3c). The longitudinal flagellum is located approximately a third of the way up the cell (Fig. 3e). Irregular scales are scattered across the hypocone surface. The ventral ridge is long and narrow, which is about half the length of the cell, connecting the two flagella insertion points (Fig. 3e).188.8.131.52 Amphidinium massartii and Amphidinium operculatum (Fig. 4; Supplementary Fig.S1 and Table S1)
Amphidinium massartii (Fig. 4a–c; Supplementary Fig.S1a–b): Cells are 16.44–21.92 μm long (mean 19.52±1.57 μm, n=30) and 10.31–18.08 μm wide (mean 15.34±1.72 μm, n=30). The ratio of L/W is from 1.21 to 1.59 (mean 1.29±0.20, n=30). Cell shape varies greatly from round to elliptical and the epicone extends to the left with a crescent structure (Fig. 4a). No pyrenoid is observed. The nucleus is oval and locates at posterior (Fig. 4c). The antapex of hypocone is ovoid generally while its right is almost vertical (Fig. 4a; Supplementary Fig.S1a–b).
Amphidinium operculatum (Fig. 4d–f; Supplementary Fig.S1c–d): Cells are 27.57–37.95 μm long (mean 31.89±2.66, n=30) and 20.04–29.70 μm wide (mean 18.73–26.39 μm, n=30). The ratio of L/W is from 1.24 to 1.63 (mean 1.43±0.09, n=30). Cell shape varies greatly from round to elliptical and the epicone extends to the right with an approximate triangular structure and the hypocone is relatively round and broad (Fig. 4d; Supplementary Fig.S1c–d). Numerous chloroplasts are fill the whole cell with a block distribution (Fig. 4e). The nucleus is oval and located at posterior (Fig. 4f).184.108.40.206 Coolia canariensis, Coolia malayensis, Coolia palmyrensis, and Coolia tropicalis (Fig. 5; Supplementary Fig.S2 and Table S2)
Coolia canariensis (Fig. 5a–c; Supplementary Fig. S2a): Cells are round or oval in ventral view (Fig. 5a; Supplementary Fig.S2a). They are 25.28–36.34 μm long (dorsal to ventral, mean 30.93±2.46 μm, n=30), 23.53–33.53 μm wide (transdiameter, mean 25.51±2.93 μm, n=30). The ratio of L/W is from 1.02 to 1.24 (mean 1.11±0.06; n=30). The chloroplast is granular and fills the entire cell (Fig. 5b). The elongated nucleus locates at the posterior (Fig. 5c).
Coolia malayensis (Fig. 5d–f; Supplementary Fig. S2b): Cells are round or oval in ventral view (Fig. 5d; Supplementary Fig.S2b). They are 20.21–33.79 μm long (dorsal to ventral, mean 25.56±2.87 μm, n=30), 17.47–26.90 μm wide (transdiameter, mean 22.38±2.36 μm, n=30). The ratio of L/W is from 1.01 to 1.30 (mean 1.14±0.08; n=30). The chloroplast is granular and fills the entire cell (Fig. 5e). A bean-shaped nucleus locates at the right posterior end of cell. (Fig. 5f).
Coolia palmyrensis (Fig. 5g–i; Supplementary Fig.S2c): Shape of cells is nearly spherical (Fig. 5g; Supplementary Fig.S2c). They are 21.01–25.97 μm long (dorsal to ventral, mean 23.89±1.41 μm, n=30), 17.43–24.04 μm wide (transdiameter, mean 21.46±1.67 μm, n=30). The ratio of L/W is from 1.01 to 1.22 (mean 1.12±0.07; n=30). The granular chloroplast was filled the entire cell (Fig. 5h). A bean-shaped nucleus locates at the right posterior end of cell (Fig. 5i).
Coolia tropicalis (Fig. 5j–l; Supplementary Fig. S2d): Shape of cells is nearly spherical (Fig. 5j; Supplementary Fig.S2d). They are 28.74–40.84 μm long (dorsal to ventral, mean 34.82±3.08 μm, n=30), 25.34–35.71 μm wide (transdiameter, mean 32.07±3.33 μm, n=30). The ratio of L/W is from 1.02 to 1.19 (mean 1.09±0.05; n=30). The granular chloroplast are fill the entire cell (Fig. 5k). A U-shaped nucleus locates at the middle posterior end of cell (Fig. 5l).220.127.116.11 Ostreopsis cf. ovata(Fig. 6)
The cells of this species are compressed and tear-shaped in general (Fig. 6a). In the ventral view, they are 32.55–42.87 μm long (dorsal to ventral, mean 36.04±2.72 μm, n=30), 22.30–32.95 μm wide (transdiameter, mean 25.51±2.93 μm, n=30). The L/W ratio ranges from 1.24 to 1.60 (1.42±0.09; n=30). An oval nucleus located at the posterior end of the cell (Fig. 6c). Thecal plate pattern: Po, 3ʹ, 7ʹʹ, pyrenoid (arrowhead), 5ʹʹʹ, 2ʹʹʹʹ. The plate surface is smooth with scattered pores (Fig. 6d–f). The pore plate (Po) is narrow and long in shape with a few pores and generally looks like a slit (Fig. 6f). In the epitheca, it is hard to observe apical plate 2ʹ, which is very close to Po in the ventral view. Compared with apical plate 2ʹ, the other two apical plates are much larger. Apical plate 1ʹ is a broad hexagon. The first apical plate (1ʹ) is much longer and broader than the other two apical plates (Fig. 6d). Additionally, apical plate 1ʹ occupies the middle in the ventral view. The hypotheca is made up of eight plates. Antapical plates 2ʹʹʹʹ are hexagonal and smaller than postcingular plates 2ʹʹʹ, 3ʹʹʹ, and 4ʹʹʹ (Fig. 6e). All postcingular plates are about the same size, except postcingular plate 1ʹʹʹ. The ventral pore is not observed.18.104.22.168 Prorocentrum hoﬀmannianum (Fig. 7)
Cells of P. hocmﬀcmmannianum are oval and symmetrical (Fig. 7a). They are 46.81–53.91 μm long (mean 50.52±1.60 μm, n=30) and 40.05–51.60 μm wide (mean 47.32±2.66 μm, n=30). The L/W ratio varies from 0.98 to 1.27 (mean 1.13±0.05, n=30). The pyrenoid is locates at the cell center, with abundant chloroplasts scattered all around the cell (Fig. 7b). The nucleus is located at the posterior (Fig. 7c). Kidney-shaped pores are scattered across the thecal surface, with no pore in the central area (Fig. 7d–e). The number of thecal pores varies from 139 to 159 and 0.52–0.96 μm length, 0.23–0.42 μm. Numerous small and shallow depressions are observed in the thecal plate and the intercalary band is surrounded by marginal pores varies from 82 to 93 (Fig. 7d). Additionally, there is a big depression in the plate center. Small round to ovoid pores were found within some depressions. The left thecal margin exhibited a flared and flattened curved apical collar that bordered the periflagellar area (Fig. 7e). An apical collar is just above the flagellar region. The periflagellar area is wide and V-shaped, with a large flagellar pore (fp) and an accessory pore (ap) (Fig. 7f). The plate patten is 1, 2, 3, 4, 5, 6, 7, and 22.214.171.124.6 Prorocentrum lima (Fig. 8)
Cells of P. lima are oval, slightly thin, and symmetrical (Fig. 8a). They are 34.39–41.17 μm long (mean 38.27±1.68 μm, n=50) and 21.36–29.65 μm wide (mean 30.29±1.69 μm, n=50) with a L/W ratio varies from 1.34 to 1.67 (mean 1.45±0.07, n=50). The pyrenoid is observed in cell center with a starch ring (Fig. 8a). Numerous chloroplasts fill the whole cell (Fig. 8b). The thecal plate is smooth, and round or kidney-shaped pores are dispersed across the surface (Fig. 8d–f). Approximately 56–71 pores are observed, but no pore is observed in the center of the plate (Fig. 8d). The nucleus is oval and locates at the posterior (Fig. 8c). The intercalary bands are surrounded by a series of 44–56 neatly arranged pores (Fig. 8d–e). The periflagellar area is wide V-shaped. Eight platelets and two large pores are observed (Fig. 8f).126.96.36.199 Prorocentrum concavum, Prorocentrum emarginatum, and Prorocentrum rhathymum (Fig. 9; Supplementary Fig.S3 and Table S3)
Prorocentrum concavum (Fig. 9a–c; Supplementary Fig.S3a): cells of Prorocentrum concavum are oval and symmetric (Fig. 9a; Supplementary Fig.S3a). They are 43.77–49.29 μm long (mean 46.7±1.67 μm, n=30) and 35.80–44.70 μm wide (mean 41.52±2.17 μm, n=30). The ratio of L/W is from 1.04 to 1.27 (mean 1.13±0.05, n=30). A pyrenoid could be brightly observed in the center of cell, which be rounded by a starch ring (Fig. 9a). Numerous chloroplasts fill the whole cell (Fig. 9b). Nucleus locates at the posterior of the cell (Fig. 9c).
Prorocentrum emarginatum (Fig. 9d–f; Supplementary Fig.S3b): cells of Prorocentrum emarginatum are broad, oval and asymmetric (Fig. 9d; Supplementary Fig.S3b). They are 28.56–40.56 μm long (mean 35.36±2.63 μm, n=30) and 20.67– 33.77 μm wide (mean 27.63±3.22 μm, n=30). The ratio of L/W is from 1.07 to 1.73 (mean 1.29±0.16, n=30). No pyrenoid is observed, while numerous chloroplasts fill the whole cell (Fig. 9e). The nucleus is oval and locates at the posterior of the cell (Fig. 9f).
Prorocentrum rhathymum (Fig. 9g–i; Supplementary Fig.S3c): cells of this species are oval and asymmetric (Fig. 9g; Supplementary Fig.S3c). They are 27.79–36.86 μm long (mean 31.73±2.23 μm, n=30) and 19.22–26.43 μm wide (mean 22.55±1.98 μm, n=30). The ratio of L/W is from 1.25 to1.73 (mean 1.41±0.11, n=30). A pyrenoid could be observed in the center of cell, which be rounded by a starch ring (Fig. 9g). Numerous chloroplasts fill the whole cell (Fig. 9h). The nucleus is oval and located at the posterior of the cell (Fig. 9i).3.1.2 Phylogenetic analysis 188.8.131.52 Amphidinium
In the present study seven strains, comprising four species, were used for phylogenetic analysis of the Amphidinium genus with related species sequences (Fig. 10). Three A. massartii strains isolated from this study were grouped together and they formed a sister clade to the Brazilian strain Am_Cub_1. The A. carterae strain ZS130 diﬀered from the Chinese strain 3WZD8, Australian strain CS740, and New Zealand strain CAWD57. Amphidinium magnum was clustered with three other Bahamian strains with maximal support. Two A. operculatum strains were used for phylogenetic analysis. Strain ZS701 diﬀered from strain WZD225, all of which were isolated from the South China Sea. Strain ZS716 was clustered with the Brazilian strain Ao-Rec-1 and Australian strain K_06184.108.40.206.2 Coolia
In the present study, a total of eight strains, consisting of four species, were used for phylogenetic analysis of the Coolia genus with related species sequences (Fig. 11). The C. malayensis strains ZS719 and ZS733 were grouped with two Hong Kong strains SKLMP_W059 and SKLMP_W072, and the USA strain CCMP1345 with a 53 Bootstrap value. In addition, C. palmyensis strain ZS601 clustered together with the northeast Atlantic strain Cp1412_1 and two Hong Kong strains, SKLMP_S017 and SKLMP_W085. Three C. canariensis strains acquired in the present study clustered together and shared identical sequences with the Hainan Island strain D1C2. Two C. tropical strains, ZS602 and ZS619, grouped together with strain XS554 isolated from the Xisha Islands, South China Sea.220.127.116.11 Ostreopsis
Five Ostreopsis sequences belonging to this study were aligned with related species sequences from GenBank. The phylogenetic relationship is shown in Fig. 12. The O. cf. ovata clade contained three groups, one consisted of some strains isolated from the western Pacific (South China Sea and Gulf of Thailand) and Indonesia. A Brazil and two Portugal (East Atlantic) strains were formed the Atlantic group. The remaining strains isolated from the Mediterranean Sea (Spain, Greece, Italy, Tunisia, and Manoca) and West Atlantic area (Brazil) clustered together as the Mediterranean/West Atlantic group. All O. cf. ovata strains acquired in the present study grouped together with other strains from the western Pacific.18.104.22.168 Prorocentrum
In this study, 14 strains belonging to four Prorocentrum species, were used for phylogenetic analysis of the Prorocentrum genus with related species sequences (Fig. 13). There was one point for which the D1–D3 region sequence of P. concavum was not obtained. Based on the phylogenetic tree, the P. lima complex separated into four subclades, which roughly showed diﬀerent geographical distributions. Nine strains belonging to P. lima were isolated from this study. Prorocentrum lima strain ZS102 was grouped together with other Chinese strains, and P. lima strain ZS516 was grouped together with strain TIO115a and strain S4, isolated from Guangxi, China, and Australia, respectively. Another seven P. lima strains were clustered with strain NMN07, and they were a sister clade of a Martinique strain. Two P. hocmﬀcmmannianum strains isolated from this study were the sister clade of the La Réunion strain PBMA_01. Prorocentrum rhathymum strain ZS405 clustered with the Xisha Islands strain XS501. The P. cf. sculptile strain ZS615 formed a clade and clustered with two Martinique strains. P. cf. emarginatum strain ZS251 was the sister clade of La Réunion strain PERN06. Besides, strain ZS251 grouped with three other strains, two Xisha Islands strains, and one was isolated from the USA.3.2 Distribution of benthic dinoflagellates 3.2.1 Spatial distribution of benthic dinoflagellates
At Oliver Shoal (15 m), Hand Shoal (30 m), and Shenhu Shoal (17 m), no macroalgal sample was collected, so all species at those sites were isolated from coral sand (Table 1). Hand Shoal was located at the middle of Zhongsha Great Atoll, which had a deepest sampled depth of 30 m and only 3 species were isolated from coral sand at this Shoal. Additionally, 8 species were only identified from Padina australis at Magpie Shoal (20 m) (Table 2). At south Zhongsha Great Atoll, 10 benthic dinoflagellates species were identified from all types of samples at Addington Patch, which had a medium sampled depth of 20 m. Also, 7 species were identified from coral sand or Padina australis at Cawston Shoal with a middle sampled depth of 23 m (Table 2). Amphidinium carterae and A. magnum were only isolated from macroalgae Padina australis at Magpie Shoal and coral sand at Oliver Shoal, respectively, and A. massartii appeared at half of the Shoals, whatever sediment or all macroalgae (Table 2). Except Oliver Shoal and Hand Shoal, A. operculatum was found at all shoals from coral sand, Padina australis, and Halimeda sp. In Shenhu Shoal, C. canariensis and C. malayensis were present. C. palmyrensis and C. tropicalis only isolated from coral sand and Padina australis at Addington Patch (Table 2). Additionally, except Oliver Shoal, O. cf. ovata appeared at all Shoal. Prorocentrum cf. sculptile appeared at half of the shoals, including Oliver Shoal, east of Addington Patch, Magpie Shoal, and Yitong Shoal. This species was found in all types of samples (sediment or macroalgae). In addition, P. concavum was only identified from coral sand at Hand Shoal with a 30-m sampled depth. At all shoals, Prorocentrum lima was found in coral sand, Amphiroa fragiroa, Padina australis, and Halimeda sp. (Table 2). However, P. hocmﬀcmmannianum only appeared in coral sand and Padina australis at Oliver Shoal (Table 2). Only identified from coral sand, Padina australis, and Halimeda sp., P. rhathymum were found at Magpie Shoal, Cawston Shoal, and Yitong Shoal (Table 2). Furthermore, although classification data for Gambierdiscus were not obtained in this study, Gambierdiscus spp. appeared at all shoals, no matter coral sand or other macroalgae except Oliver and Cawston Shoal (Table 2). In short, Benthic dinoflagellates are most diversely represented on south of Zhongsha Great Atoll and Yitong Shoal, as well as macroalgae. Additionally, more species were identified from macroalgae samples Padina australis.3.2.2 Quantitative distribution of benthic dinoflagellates
The abundance of benthic dinoflagellates in the sediment was between 88 and 4 345 cells/100 cm2 (Fig. 14). Except for Shenhu Shoal and west of Addington Patch, the genus Prorocentrum was dominant at all sites. The lowest cell number averaged 88 cells/100 cm2 was at Oliver Shoal, which has the lowest sampled depth of 15 m, while the maximum cell abundance averaged 88 cells/100 cm2 was found at Cawston Shoal with a 23-m sampled depth (Fig. 14). The two sites were located at the northernmost and southernmost part of the Zhongsha Great Atoll (Fig. 14a). Only Prorocentrum and Ostreopsis species were found at Oliver Shoal, with an average of 82 cells/100 cm2 and 5 cells/100 cm2, respectively. At Hand Shoal with the deepest sampled depth, a cell abundance of averaged 334 cells/100 cm2 was collected and Coolia species were absent in samples collected (Fig. 14a). At southern Zhongsha Great Atoll, Amphidinium was dominant, with cell abundance averaging 54 cells/100 cm2 at west of Addington Patch. However, at east of Addington Patch, cell abundance was as high as 1 815 cells/100 cm2, which was more than 10 times that west of Addington Patch (142 cells/100 cm2), although their sampling depths are very close (20 m and 19 m, respectively). Yitong Shoal (15 m) and Shenhu Shoal (17 m) had a lower sampled depth and there were similar levels of cell abundance of benthic dinoflagellates were recorded, respectively averaging 642 cells/100 cm2 and 534 cells/100 cm2 (Fig. 14b). Compared with that at other shoals, the maximum cell abundance of Coolia was found at Shenhu Shoal, with an average of 408 cells/100 cm2, representing approximately 76.4% of all benthic dinoflagellates (Fig. 14b). Gambierdiscus species were not observed in samples from Cawston Shoal, or Shenhu Shoal (Fig. 14a–b). As the dominant genus, Prorocentrum occupied more than 50% of the total abundance of benthic dinoflagellates at all shoals, except west of Addington Patch, where they comprised only 6.8%. Diﬀerent distribution patterns in the abundance of benthic dinoflagellates were observed on the east and west sides of Addington Patch. The benthic dinoflagellates abundance near the outside of Zhongsha Great Atoll was higher, while that of the inside was much lower. At the same time, the abundance in the south of Zhongsha Great Atoll was higher than that in the northern (Fig. 14a). In addition, the relationship between depth and abundance was not very close, but the abundance at a medium depth of 20 m was much higher (averaged 1 815 cells/100 cm2 at East of Addington Patch and averaged 4 345 cells/100 cm2 at Cawston Shoal).
Since only three kinds of macroalgae (Padina australis, Halimeda sp., and Amphiroa fragiroa) were collected from all sites, macroalgal preference is not discussed in this study. The abundance of benthic dinoflagellates on macroalgae was between 10 and 91 cells/g and Prorocentrum was dominant on macroalgae at all sites (Fig. 15). The northernmost site of Zhongsha Great Atoll, Magpie Shoal, had a total abundance of benthic dinoflagellates averaged 57 cells/g on Padina australis with a sampled depth of 20 m. However, the abundance of Gambierdiscus and Ostreopsis was as low as 1 cell/g (Fig. 15a). At west of Addington Patch, which had a sampled depth of 19 m, benthic dinoflagellates cell abundance averaged 49 cells/g on Padina australis, the diﬀerence was that Coolia comprised 33.1% of total abundance. At east of Addington Patch, which had the same depth as Magpie Shoal, the cell abundance of benthic dinoflagellates was lowest on Amphiroa fragiroa, averaging 10 cells/g, which was significantly diﬀerent from that at other sampling sites (Fig. 15a). Additionally, no Gambierdiscus spp. or Coolia spp. were observed on Amphiroa fragiroa. At Yitong Shoal, with the lowest sampled depth of 15 m, the highest abundance of benthic dinoflagellates was found on Halimeda sp., with an average of 91 cells/g (Fig. 15a). In addition, all five benthic dinoflagellate genera were recorded from macroalgal samples. The benthic dinoflagellates abundance of Yitong Shoal was higher than that of Zhongsha Great Atoll because of its low water depth (Fig. 15a–b). At north Zhongsha Great Atoll, Addington Patch had the lowest abundance. On macroalgae samples, the highest abundance was observed at Halimeda sp. (91 cells/g at Yitong Shoal), while the lowest abundance appeared at Amphiroa fragiroa (10 cells/g at east of Addington Patch).4 DISCUSSION 4.1 Biodiversity of benthic dinoflagellates in Zhongsha Islands 4.1.1 Amphidinum
Amphidinium magnum identified in Zhongsha Islands is the first record in Chinese waters. It is also the first report in addition to type species locality, Grand Bahama Beach, Bahamas (Karafas et al., 2017). Its morphological characteristics were consistent with those originally described. A long and narrow ventral ridge was clearly observable, but the pusule was absent in our strain. Scales were visible on the hypocone surface, which were absent in the strain from the type locality.
The phylogenetic tree based on the LSU region showed that the species isolated from Zhongsha Islands can be divided into two clades, one clade consisting of A. massartii and A. carterae, both of which are smaller than A. operculatum, which formed a clade on its own, with 100% bootstrap support in the phylogeny. Amphidinium magnum grouped together with the three type locality strains, with maximum support. Additionally, the position of A. magnum was close to that of A. cf. thermaeum and A. theodori, which is consistent with the results of Karafas et al. (2017).4.1.2 Coolia
Morphological characteristics (size, plate arrangement, and shape) of all four Coolia species, including C. canariensis, C. malayensis, C. palmyrensis, and C. tropicalis, identified from Zhongsha Islands, were generally similar to that of the type species descriptions. The four species were reported previously from Hong Kong and Hainan Island in China (Leung et al., 2017; Zhang et al., 2021).
The phylogenetic tree of Coolia showed that the C. malayensis closely related to the Hong Kong strains and New Zealand strain, which was consistent with that conducted by Zhang et al. (2021) in Hainan Island. The results reveal that all C. malayensis strains isolated from Chinese waters have a common ancestor. Strains of C. palmyensis from Zhongsha Islands and from NE Atlantic were a sister clade of Hong Kong strain, which suggested that there were few diﬀerences between Zhongsha Islands strain and Hong Kong strain in phylogeny (Leung et al., 2017). Two strains of C. tropical isolated from Zhongsha Islands grouped together with Xisha Islands, which also showed that there are diﬀerences among strains even they are geographically adjacent.4.1.3 Ostreopsis
Morphologically, it is diﬃcult to distinguish the various species of Ostreopsis, which are similar to the type species O. siamensis. The cell size of O. cf. ovata strain from the Zhongsha Islands was 32.55–42.87 μm long (dorsal to ventral, mean 36.04±2.72 μm), 22.30–32.95 μm wide (transdiameter, mean 25.51±2.93 μm), which is significantly smaller than that of O. siamensis (dorso-ventral 60–100 μm; transdiameter 45–90 μm). In addition, the morphological characteristics of the Zhongsha Islands strain (ZS718) in this article were generally consistent with those of strains isolated by Fukuyo (1981), which were generally tear-shaped with a L/W ratio of 1.5–2.0 (Fig. 6). In original description, a large antapical plate of O. cf. ovata was designated as postcingular intercalary plate (1p) (Fukuyo, 1981), now it is antapical plate (2ʹʹʹʹ). Compared with the original description, our plate pattern (Po, 3ʹ, 7ʹʹ, ?C, ?S, 5ʹʹʹ, 2ʹʹʹʹ) has been reported in recent literatures (Chomérat et al., 2020; Nascimento et al., 2020).
Penna et al. (2010) revealed that O. cf. ovata could phylogenetically divide into three clades, including Atlantic/Mediterranean/Pacific clade, Indian/ Pacific clade, and Atlantic/Indian/Pacific clade. All O. cf. ovata strains from this study were clustered together to form an Indian/Pacific clade. The Indian/ Pacific clade included all strains collected from the South China Sea (including the newly discovered Zhongsha Islands strain, Vietnamese strain, and Malaysian strain) and adjacent waters (Thai strain and Indonesian strain). The consistency in genetic characteristics and geographic distribution may be because of the similarity in ecosystems and the influence of ocean circulation. The islands and land surrounding the South China Sea make it a marginal sea, and the unique genetic characteristics of O. cf. ovata suggest that it is genetically separated from the Mediterranean/Indian Ocean O. cf. ovata strain. Thus, geographical isolation has had a strong influence on phylogenetic divergence.4.1.4 Prorocentrum
A number of studies have confirmed that P. lima shows genetic diversity and phenotypic plasticity (Hoppenrath et al., 2013; Zhang et al., 2015). Similar to O. cf. ovata, P. lima is also a cosmopolitan distributed species. Based on morphology and phylogeny constructed with LSU and internal transcribed spacer (ITS) regions, of strains of P. lima from Hainan Island, China were classified as five morphotypes (Zhang et al., 2015). Then Nascimento et al. (2017) defined morphotype 4 of P. lima as a new species, P. caipirignum. A study also discovered two morphological types (morphotypes 1 and 2) of P. lima in the Caribbean (Chomérat et al., 2019). The results showed that these two types had obvious morphological diﬀerences. Morphotype 2 was oblong oval, the cell length and width were 37.1–38.2 μm and 27.8–32.3 μm, respectively (L/W ratio was 1.18– 1.36). The morphology and cell measurements of a P. lima strain (ZS102) from Zhongsha Islands was in accordance with the morphotype 2 in the Caribbean. The cell of P. hocmﬀmannianum strain from Zhongsha Islands was broader than that of the type species, but it was similar to that of P. belizeanum (Faust, 1990). Herrera-Sepúlveda et al. (2015) summarized that P. hoﬀmannianum and P. belizeanum have some micromorphological similarities (cell size, surface ornamentation, and periflagellar area architecture) and molecular data lack suﬃcient evidence to distinguish these two species. Therefore, they are considered to be conspecifics.
On the base of phylogenetic tree of LSU rDNA (D1–D3 region), we also identified our strains (ZS102 and ZS516) from Zhongsha Islands belonged to P. lima morphotype 2. Furthermore, the P. lima complex could be clearly distinguished from the similar species P. caipirignum and had high genetic diversity, based on phylogenetic analysis of D1–D3 region. This same result is observed in previous studies (Zhang et al., 2015; Chomérat et al., 2019). Normally, the same geographical origin have similar phylogenies. However, the P. lima complex in this study is divided into three groups. The first group has a neighboring geographical origin, the second is distributed in the northern and southern hemispheres, and the third strains are geographically separated from each other. This result suggests that P. lima in the same area also has diﬀerent genetic characteristics.
Hoppenrath et al. (2013) summarized that P. belizeanum, P. hocmﬀcmmannianum, and P. maculosum were highly similar in morphology with small genetic diﬀerences. But their toxin-producing characteristics were diﬀerent, which blurred the boundaries between these species. However, with the application of more precise molecular techniques, these species are all attributed to the P. hocmﬀcmmannianum species complex. In this study, the Zhongsha Islands strains of P. hocmﬀcmmannianum were a sister clade of a La Réunion strain, which was once defined as P. belizeanum. Therefore, we further demonstrate that our P. hocmﬀcmmannianum strains are closer to P. belizeanum, which is consistent with the morphological results. However, these were our subclades of P. hocmﬀcmmannianum and these were directly related to geographic locations, indicating that geographical isolation has had a considerable influence on phylogenetic divergence. Therefore, another marker is needed to identify P. hocmﬀcmmannianum species more accurately.4.2 Distribution of benthic dinoflagellates from Zhongsha Islands 4.2.1 Geographical distribution of benthic dinoflagellates
There are approximately 200 species of Amphidinium, both freshwater and seawater (Guiry and Guiry, 2022). Many species were heterotrophic (Taylor, 1971; Calado et al., 1998). Most of Amphidinium species identified in Zhongsha Islands were widespread and commonly reported in tropical and subtropical waters, as well as in temperate higher latitude regions (Murray and Patterson, 2002; Lee et al., 2013; Jiang, 2019; Luo et al., 2022).
Leaw et al. (2016) provided a new scenario for the phylogeographic history of Coolia and it was speculated that the divergence of the two species, C. malayensis and C. monotis, may be due to geographic segmentation caused by vicariant events. Four Coolia species, C. canariensis, C. malayensis, C. palmyrensis, and C. tropicalis identified from the Zhongsha Islands, were widely distributed in the western Pacific (C. tropicalis and C. canariensis in Vietnam, C. canariensis and C. malayensis in the Korean Sea, C. malayensis, C. palmyrensis, and C. tropicalis in Australia) (Jeong et al., 2012; Ho and Nguyen, 2014; Larsson et al., 2019). Aside from the western Pacific, C. malayensis was also found in Florida, Belize, and the Cook Islands (Leaw et al., 2016). Coolia palmyrensis has been found in the Caribbean Sea (Karafas et al., 2015) and western Atlantic Ocean (De Azevedo Tibiriçá et al., 2020). Coolia tropicalis was first isolated from an Atlantic barrier reef ecosystem (Faust, 1995) and has then been expanded to Brazil (Nascimento et al., 2019) and many areas of the western Pacific (Larsson et al., 2019; Zhang et al., 2021).
The globally distributed species O. cf. ovata has been reported in the Caribbean Sea, Mediterranean Sea, Atlantic, Indian, and Pacific Oceans (Monti et al., 2007; Mangialajo et al., 2008; Rhodes, 2011; Kang et al., 2013; Zhang et al., 2018). Parsons et al. (2012) summarized the global distribution of Ostreopsis and found that Ostreospsis species were also widely present in the western and southern Pacific, as well as western and northern Atlantic, except for the Mediterranean and Caribbean regions where the species was widespread. The genetic analyses indicated that O. cf. ovata were divided into three geographical areas: Atlantic/Mediterranean/ Pacific, Atlantic/Indian/Pacific, and Pacific (Penna et al., 2010; Mangialajo et al., 2011; Sato et al., 2011). Ostreopsis species were commonly observed in Caribbean and Western Atlantic region such as Florida Keys (Accoroni et al., 2020), Brazil (Nascimento et al., 2020), and Bezile (Faust, 1995). Zhang et al. (2018) reported O. cf. ovata and O. lenticularis from Hainan Island, South China Sea. Zheng et al. (2017) found that the O. cf. ovata isolated in Beibu Bay, southern China, were contain higher concentrations of ovatoxins-c (OVTX-c) and OVTX-d/e, as well as small amounts of OVTX-g and OVTX-f. So far there were only two Ostreopsis species have been reported in tropical waters of the southern China.
Prorocentrum as the dominant genus is a consistent feature of tropical waters (Mohammad-Noor et al., 2005; Parsons and Preskitt, 2007). Prorocentrum lima is a widely distributed Prorocentrum species, which is often observed in water column, on macroalgal surfaces and sediments (Fukuyo, 1981; Faust, 1990; Aligizaki et al., 2008). Prorocentrum hocmﬀcmmannianum was first isolated in Belize (Faust, 1990). Subsequently, P. hocmﬀcmmannianum, P. belizeanum, and P. maculosum were revised as a P. hocmﬀcmmannianum species complex (Herrera-Sepúlveda et al., 2015; Rodríguez et al., 2018). P. hocmﬀcmmannianum species complex were mainly tropical and subtropical distribution, such as the Mediterranean (mez, 2003), Australia, New Zealand (Rhodes and Smith, 2019), and North America (Torres-Ariño et al., 2019). Prorocentrum concavum was widely distributed in the water column and reef ecosystems from tropical to high latitude temperate zones (Faust, 1994; Luo et al., 2017).4.2.2 Quantitative distribution of benthic dinoflagellates
Although the Zhongsha Islands are called an archipelago, its main body, Zhongsha Great Atoll, is actually a series of reefs located below sea surface, far from human activities in water depths of approximately 20–40 m. The habitat is mainly coral reef ecosystems, which are greatly aﬀected by ocean water dynamics such as turbulence. In this study, the environment in which benthic dinoflagellates grow is characterized by weak light intensity, low temperature relative to the intertidal zone, and low nutrient concentrations.
Benthic dinoflagellates are generally epiphyte on the seafloor matrix (sand, silt, or debris shell) or on the surface of macrophytes, and are less abundant in water column unless bloom occurs (Fukuyo, 1981; Boisnoir et al., 2019a). Prorocentrum was dominant on coral sand and three macroalgaes (Padinaaustralis, Halimeda sp., and Amphiroa fragiroa) except for those to the west of Addington Patch and Shenhu Shoal. A similar phenomenon that Prorocentrum was the first dominant genus has also observed in previous researches (Richlen and Lobel, 2011; Widiarti and Anggrain, 2012; Accoroni et al., 2020). Interestingly, our results indicate that Ostreopsis was the least abundant group in the Zhongsha Islands. The maximum density of Ostreopsis was appeared at Yitong Shoal with averaged 64 cells/100 cm2 on coral sand. Lee et al. (2020) studied the eﬀect of substratum and depth on benthic dinoflagellates assemblages and found that Ostreopsis were the second most dominant genera of benthic dinoflagellate assemblages, both in inorganic matrix and macrophytes. The maximum abundance of Ostreopsis were observed in hard coral and turf algae. A similar phenomenon was observed at Johnston Atoll in the Pacific, which has a similar geographical characteristics and environmental conditions with Zhongsha Islands (Richlen and Lobel, 2011). Ostreopsis spp. from Johnston Atoll accounted for the largest proportion at several of hard coral reef sites, an opposite result to our study. Boisnoir et al. (2019a) indicated that Ostreopsis preferred to attach to blade-ramified macrophytes, particularly Florideophyceae. However, the minimum abundance was presented on Padina australis with averaged 8 cells/g. We speculate that other factors may have contributed to this phenomenon, such as the sampling depth. The maximum sampling depth at Johnston Atoll was 13 m, which was lower than the least depth in our study. Although Ostreopsis had generally low abundance on the three macroalgae and coral sand, they were significantly more abundant on blades with larger surface areas, e.g. Halimeda sp. (8 cells/g). Whereas the abundance on branched Amphiroa fragiroa and foliated Padina australis were as low as 1 cell/g.
Like Prorocentrum, Amphidinium was widely distributed in various substrates with a high abundance. Amphidinium was the genus with the second highest abundance in the benthic dinoflagellate community of Zhongsha Islands, both on coral sands and macroalgae. This was inconsistent with those of other studies that the abundance of Amphidinium was generally the lowest (Richlen and Lobel, 2011; Lee et al., 2020). Coolia is present in low abundance. The same results are consistent with previous studies in the Gulf of Mexico (Okolodkov et al., 2007; Hachani et al., 2018; Ben Gharbia et al., 2019).
A study on the depth distribution of benthic dinoflagellates in the Caribbean demonstrated that their distribution was significantly correlated with light intensity. The cell density decreased significantly with increasing depth (Boisnoir et al., 2018). The benthic dinoflagellate density was < 100 cells/g in both wet and dry seasons at a depth of 20 m. It was found that no Ostreopsis cells were observed when the water depth exceeds 10 m. Interestingly, in this study, Ostreopsis spp. were observed at all depths on coral sands and macroalgae except that on Padina australis at Cawston Shoal. A diﬀerent trend was observed by Lee et al. (2020) that at depths greater than 12 m, only Prorocentrum and Coolia were presented. In our study, on coral sand samples, Gambierdiscus were only observed at 15 m (Yitong Shoal) and 19 m (West of Addington Patch). Although Gambierdiscus was also present in the samples of the macroalgae Padina australis at Cawston Shoal (20 m) and Magpie Shoal (23 m), Their abundance was as low as 1 cell/g. On macroalgae samples, the maximum abundance of Prorocentrum were observed at the minimum depth of 15 m (Yitong Shoal). At a maximum depth of 30 m, a second minimum Prorocentrum abundance of 334 cells/100 cm2 on coral sand were presented. However, no Coolia spp. was observed. Macroalgae generally grow in abundance in the intertidal zone with water depth less than 3 m. The Zhongsha Islands have a water depth of more than 15 m, and the bottom is a coral reef ecosystem. Increased depth means less macroalgae. Compared to the coasts of continents or large islands, reef ecosystems with water depths of more than 20 m are subject to stronger hydrodynamic disturbances. Benthic dinoflagellates were significantly more abundant in calm lagoons than on reefs heavily aﬀected by waves (Boisnoir et al., 2018). Hydrodynamic conditions may be the most important factor aﬀecting the growth of benthic-epiphytic dinoflagellates. The abundance in sheltered areas is significantly higher than that in unsheltered areas (Shears and Ross, 2009; Mangialajo et al., 2011). It is speculated that the low abundance of benthic dinoflagellates is closely related to the scarcity of macroalgae and stronger water motion at the depth > 15 m in Zhongsha Islands.5 CONCLUSION
The biodiversity and distribution of benthic dinoflagellates in the Zhongsha Islands have been revealed. In general, 15 species of benthic dinoflagellates belonging to four genera were identified: A. carterae, A. magnum, A. massartii, A. operculatum, C. canariensis, C. malayensis, C. palmyrensis, C. tropicalis, O. cf. ovata, P. cf. sculptile, P. concavum, P. emarginatum, P. hocmﬀcmmannianum, P. lima, and P. rhathymum. Benthic dinoflagellates were distributed broadly on coral sands and three species of macroalgae (Padina australis, Halimeda sp., and Amphiroa fragiroa). However, the total abundance of benthic dinoflagellates in the Zhongsha Islands, which was 88–4 345 cells/100 cm2 on sediment and 10– 91 cells/g on macroalgae, is much lower than that in other areas. Prorocentrum and Amphidinium were the dominant and subdominant genera, respectively. It is speculated that the low abundance of benthic dinoflagellates is closely related to the scarcity of macroalgae and stronger water motion at the depth > 15 m in Zhongsha Islands. This study has provided a deeper understanding of benthic dinoflagellate assemblages in the Zhongsha Islands.6 DATA AVAILABILITY STATEMENT
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Electronic supplementary material
Supplementary material (Supplementary Tables S1–S5 and Figs.S1–S6) is available in the online version of this article at https://doi.org/10.1007/s00343-022-1322-z .
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