2 Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering, Ministry of Education, Guangzhou 510632, China;
3 School of Ocean, Yantai University, Yantai 264005, China
Eukaryotic microalgae are important primary producers of marine ecosystems and regarded as the basis of food web structures (Worden et al., 2015). They have diverse nutritional types, including phototrophic, heterotrophic, mixtrophic, and parasitic (reviewed by Brodie et al., 2017). The diversity and community structure of marine microalgae play an important role in global biogeochemical cycles, the stability of marine ecosystems, and the mitigation of global climate change (Martinez et al., 2009; Vogt, 2015). However, some microalgae produce toxins and/or form harmful algal blooms (HABs), which could have negative effects, causing severe economic losses to fisheries, aquaculture, and tourism activities, and exerting major environmental and human health effects (Anderson et al., 2002). Of all phytoplankton, approximately 150 species are categorized as harmful microalgae in the online version of the IOC Taxonomic Reference List of Toxic Plankton Algae (Lundholm et al., 2009 onwards). Those algal species, especially toxin-producing algae, could harm other marine organisms and human health through the food chain, such as fish and shellfish.
Many planktonic microalgal species have been reported to produce dormant stages as a part of their life cycles (Head, 1996; Von Dassow and Montresor, 2011) in the forms of resting spores and resting cysts (hereafter referred to as resting stages). The formation of resting stages is prompted by adverse environmental conditions, nutrient limitation (Kremp et al., 2009), or pressure from grazing (Anderson and Rengefors, 2006). Once the resting stages arrived, they sink to the sea floor and become a part of the benthic assemblages (Head, 1996). Resting stages have resistant cell wall, and they could survive in bottom sediments for long periods (decades to 100 years; reviewed by Ellegaard and Ribeiro, 2018). Germination of resting stages occurs if they are resuspended in the water column and exposed to appropriate light intensity, temperature, nutrients, and most importantly, oxygen availability (McQuoid, 2002). Resting stages are regarded as the "seed bank" of algal blooms, and they play an important role in initiating HABs (Smayda, 2002; Genovesi et al., 2009).
The identification and quantification of the resting stages of eukaryotic microalgae have traditionally been based on microscopic observations and cell counting after separating cells from sediments. These methodologies for separation and observation have been developed over decades; however, some limitations and uncertainties exist, including losses during separation and concentration, biases in the recovered groups, and diﬃculties associated with morphological identification (Piredda et al., 2017). Microscopic observations require professional taxonomic expertise for distinguishing the tiny morphological diﬀerences among species (Shang et al., 2019). Furthermore, only a small proportion of phytoplankton resting stages is known for their corresponding vegetative cells and vice versa. Misidentification is thus common and almost unavoidable due to these diﬃculties (Gómez et al., 2017; Shang et al., 2019).
Advances in the sequencing of DNA extracted from natural water and sediment samples oﬀer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical (reviewed by Lear et al., 2018). DNA sequence analysis using metabarcoding has been widely applied in characterizing the biodiversity and community structure of microeukaryotes in marine sediments (reviewed by Lear et al., 2018; Gran-Stadnicze ko et al., 2019). This method provides information on many organisms aﬃliated with several trophic levels in a biological system (Lanzén et al., 2016), some of which are diﬃcult to identify with classic methods, hard to culture, fragile, and rarely analyzed (Egge et al., 2015). Metabarcoding oﬀers the prospect for determining their distribution in the world's oceans and enables a more complete assessment of anthropogenic eﬀects on ecosystems (Lekang et al., 2020), particularly for unicellular microalgae that often lack distinctive morphological characteristics (Vaulot et al., 1989) and have complex life cycles (Von Dassow and Montresor, 2011; Decelle et al., 2012).
In the past decade, numerous studies have analyzed benthic microeukaryotic community structure and its response to environmental changes. Metabarcoding has been successfully applied to track temporal changes in eukaryotic communities in coastal sediments (Salonen et al., 2019), assess the distribution and diversity of marine eukaryotes (Fonseca et al., 2014; Wu and Huang, 2019), monitor the eﬀects of fish farming and human activities on benthic communities (Pochon et al., 2015; Stoeck et al., 2018), reveal modern and past eukaryotic communities (Garcés-Pastor et al., 2019), and predict the ecological conditions of estuaries and coastal sea areas (Chariton et al., 2015). In addition, DNA metabarcoding has a great potential for the assessment of phytoplankton resting stages in environmental sediments (Dzhembekova et al., 2018; Liu et al., 2020).
The Bohai Sea, China's only inland sea, is a semi-closed, shallow marginal sea in Northeastern China. With the increase in industrialization and urbanization in the Bohai Sea region in recent decades, large quantities of industrial and domestic wastewater have been discharged into the sea and pose a serious environmental risk to the ecosystem (Zhu et al., 2020). As the Bohai Sea exchanges only limited quantities of water with the open sea, it has become one of the most polluted sea areas in China (Duan and Li, 2017). Furthermore, algal blooms, especially those of HABs, have recently become more frequent in the Bohai Sea (Xu et al., 2017). Over the last two decades, a number of studies analyzing environmental conditions and the pollution status in the Bohai Sea have been published (Wei et al., 2019; Yang et al., 2019; Zhu et al., 2020). However, the resting stages of eukaryotic communities in sediments from the Bohai Sea have been rarely revealed. Only a few studies described the distribution of benthic diatoms (Wang et al., 2020) and cysts of toxic dinoflagellates (Dai et al., 2020) in the Bohai Bay. In addition, most of these studies have been based on microscopic observations, which may have not allowed species identification for many resting stages.
In this study, high-throughput sequencing (HTS) was used to analyze the biodiversity and distribution of eukaryotic microalgae in surface sediments from the central Bohai Sea. This study aimed to assess the diversity of benthic microalgae by metabarcoding, with a focus on the phytoplankton resting stages; to understand the distribution of HAB species; and to discuss the potential for HABs in the central Bohai Sea.2 MATERIAL AND METHOD 2.1 Study area and sediment collection
The Bohai Sea is a shallow semi-enclosed epicontinental sea. It includes four parts, i.e., Bohai Bay, Laizhou Bay, Liaodong Bay, and central Bohai Basin (Fig. 1). The area of the Bohai Sea is 77 000 km2, and the average water depth is 18 m. Many rivers, including the Huanghe (Yellow) River, Haihe River, Luanhe River, and Liaohe River, flow into the Bohai Sea (Fig. 1). The study area is located in the central Bohai Sea.
Surface sediment samples were collected by a Van-Veen Grab with a 10-cm×10-cm frame at 17 stations in the central Bohai Sea in May 2015 (Fig. 1). Samples of the upper surface sediments (ca. 2 cm in depth) were collected in triplicate at each station with a polyethylene scraper. Before DNA extraction, the sediment samples were stored under dark conditions at low temperatures (4–8 ℃) for 6 months to inactivate most of the microalgal vegetative cells and to ensure that the algal DNA extracted from the sediment samples mostly belonged to the resting stages (Piredda et al., 2017).2.2 DNA extraction, PCR amplification, and sequencing
Environmental DNA was extracted and purified from 0.5-g sediment samples by using the DNeasy PowerSoil Kit (Qiagen GmbH, Germany) following the manufacturer's protocols (Liu et al., 2020). PCR amplification of the V4 region of 18S rDNA was performed using universal eukaryotic primers 3NDf 5′-GGCAAGTCTGGTGCCAG-3′ (Cavalier-Smith et al., 2009) and V4_euk_R2 5′-ACGGTATCTATCTCTTCG-3′ (Brte et al., 2010) with lengths of ca. 450 bp. PCR products were sequenced using the Illumina MiSeq PE300 (San Diego, CA, USA) platform by Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.2.3 Operational taxonomic unit (OTU) analysis and taxonomic assignment
Raw sequence reads were denoised, trimmed, and filtered using the UPARSE (v8.0.1623) algorithm of USEARCH (Edgar, 2013) and the Ribosomal Database Project (RDP, version 2.2, http://sourceforge.net/projects/rdp-classifer, Caporaso et al., 2010). OTUs were generated based on 97% similarity after removal of singletons and doubletons. Identification of each OTU was inferred using the RDP classifier against the SILVA 18S rRNA database (release138, www.arb-silva.de). The eukaryotic algal OTUs were confirmed based on taxonomic information with AlgaeBase, a global algal database (Guiry and Guiry, 2020, https://www.algaebase.org/). The raw data were deposited into the National Center for Biotechnology Information Sequence Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra) with the accession number PRJNA722689.2.4 Statistical analysis
MAFFT version 7.475 (Katoh and Standley, 2013) was used to align the eukaryotic algal DNA sequences in this study. A maximum-likelihood tree was constructed with FastTree version 2.1.11 software (Price et al., 2009) with default parameters by using the GTR + CAT substitution model for 1 000 bootstrap replicates. The phylogenetic tree was annotated using the packages ggtree (Yu et al., 2017) and Adobe Illustrator 2020. The spatial distribution of eukaryotic algal reads was illustrated by inverse distance weighting (IDW) interpolation (weighting power of 2.0), and a sampling map and IDW interpolation map were produced by ArcMap 10.2.3 RESULT 3.1 Sequencing statistics and diversity estimates
A total of 813 046 reads of eukaryotic rDNA were generated from 17 surface sediment samples from the central Bohai Sea after removal of sequence errors and chimeras. The number of eukaryotic sequences per sample ranged from 30 821 reads to 72 429 reads, with an average of 48 809 reads (Fig. 2a). The dominant length ranged from 441 bp to 462 bp, with an average of 446 bp. At 97% sequence identity, 930 OTUs were detected, and 144–418 OTUs were recorded per sample, with an average of 302 OTUs (Fig. 2b). Among all eukaryotic sequences, 126 679 reads (15.58%) and 98 OTUs (10.54%) were assigned to the eukaryotic microalgae. The numbers and relative abundances of algal reads ranged between 383 and 33 174 reads and between 0.89% and 81.49% (Fig. 2a), with averages of 7 452 reads and 17.01%, respectively. The algal DNA reads were numerous at stations B4 and B12, exceeding 30 000 reads and contributing > 70% of the total reads. On the contrary, the algal OTU reads were low at stations B9, B13, B16, and B17, at which fewer than 1 000 algal reads were recorded. Algal OTU richness was detected in 17–58 OTUs (Fig. 2b), and it was high at stations B2, B3, B5, and B11 (> 50 OTUs). The proportions of algal OTU richness to eukaryotic richness were comparable among samples (10.4%–16.0%).3.2 Community structure of eukaryotic microalgae
The algal communities were composed of 42 genera belonging to 19 classes of six phyla (i.e., Chlorodendrophyceae, Chloropicophycea, Mamiellophyceae, Prasinophyceae, Pyramimonadophyceae, and Trebouxiophyceae in Chlorophyta; Bacillariophyceae, Coscinodiscophyceae, Mediophyceae, Chrysophyceae, Dictyochophyceae, Pelagophyceae, and Raphidophyceae in Ochrophyta; Dinophyceae, Syndiniophyceae, and Noctilucophyceae in Dinophyta; Katablepharidophyceae in Cryptophyta; Kinetoplastea in Euglenozoa; and Pavlovophyceae in Haptophyta). Sixty algal OTUs (61.22%) were identified at the genus level, and only 42 OTUs (42.86%) were fully identified at the species level. Altogether, 39 species were detected in this study (Supplementary Table S1). Some algal OTUs were rare taxa, with 29 OTUs represented by fewer than 20 reads and 23 OTUs represented by fewer than 10 reads. Among these 29 OTUs (< 20 reads), 12 OTUs were identified at the species level (Supplementary Table S1).
Figure 3 shows the percentage composition of DNA reads and OTU richness of microalgae at the phylum or class level. Chrysophyceae dominated the eukaryotic algal communities, contributing 64.49% of the algal sequences (Fig. 3a). However, only six chrysophyte OTUs were detected in this study, four of which were only identified at the class level. Two chrysophyte OTUs (OTU757 and OTU10) contributed 52.64% and 11.63% to the algal sequences, respectively. Dinophyta (dinoflagellates, including the three classes in Dinophyta) was the second most abundant algal group (28.61%). The common dominant dinoflagellates included Biecheleria halophile (Biecheler) Moestrup, Lindberg & Daugbjerg (OTU511), Azadinium trinitatum Tillmann & Nézan (OTU624), Scrippsiella acuminata (Ehrenberg) Kretschmann, Elbr chter, Zinssmeister, Soehner, Kirsch, Kusber & Gottschling (OTU354), and Polykrikos hartmannii Zimmermann (OTU62), which contributed 28.58%, 23.66%, 3.91%, and 1.76% of the dinoflagellate sequences, respectively. In addition, one Syndiniales OTU (OTU234) and two unclassified dinoflagellate OTUs (OTU366 and OTU498) occurred abundantly, contributing 12.05%, 12.24%, and 9.51% to dinoflagellate sequences, respectively. Diatoms (Bacillariophyceae, Coscinodiscophyceae, and Mediophyceae) contributed to 4.28% of the total algal sequences. Thalassiosira Cleve (OTU927) predominated in diatoms, contributing 86.56% to diatom reads. The sequences of raphidophytes contributed 1.18%, and the others contributed less than 1%. Dinoflagellates were the most diverse microalgal group (Fig. 4b), and they represented 45.92% of OTU richness (45 OTUs), followed by chlorophytes (20 OTUs, 20.41%), diatoms (15 OTUs, 15.31%), and chrysophytes (6 OTUs, 6.12%). The other groups included only 1–3 OTUs.
Dinoflagellates dominated at 12 stations (Fig. 4a), with sample relative abundance ranging between 1.96% and 98.13% and averaging 61.60%. Chrysophytes predominated in five samples, with percentages of 0.09%–97.88% per sample and 23.50% on average. The relative abundances of diatoms ranged between 0.03% and 38.87%, with an average of 10.16%. Raphidophytes occurred abundantly at station B11 (13.66%), and they were less abundant in other samples. Dinoflagellates were the most diverse group in almost all samples (Fig. 4b), with 7–28 OTUs (27.45%–53.19%) per sample. OTU richness was high for chlorophytes and diatoms, with 2–18 (8.70%–35.29%) and 2–11 (3.70%–23.53%) OTUs in each sample, respectively.
A maximum-likelihood tree was constructed to compare the relationships among the 98 algal OTUs (Fig. 5). The OTUs in Haptophyta, Cryptophyta, Chlorophyta, and Ochrophyta were clustered into a large clade. The OTUs in Ochrophyta and Chlorophyta were separately clustered together into a large subclade. One OTU in Cryptophyta (OTU144) and one OTU in Haptophyta (OTU250) were grouped together with low bootstrap support. One OTU in Euglenozoa was ungrouped. The phylogenetic relationships among OTUs in Dinophyta were more diverse, and they included five subclades and one ungrouped OTU. In addition, not all OTUs in the same genus were clustered together, and some OTUs in the same genus or order were distributed in diﬀerent subclades. For example, two OTUs of Fragilidium Balech ex Loeblich III were distinct from each other, three OTUs of Gymnodinium Stein were separately grouped into two subclades, and the OTUs in Syndiniales were grouped into two close subclades. Non-monophyly of genera may be caused by misidentification due to incorrect sequence names deposited in the SILVA database. They could also suggest the high genetic diversity and polymorphism in Dinophyta and reflect the hypervariability of the V4 region of 18S rDNA.3.3 Distribution of dominant and harmful algal bloom species
Sixteen of the detected OTUs were assigned to harmful and/or bloom-forming microalgae, including eight taxa in Dinophyceae (Alexandrium affne (Inoue & Fukuyo) Balech, Azadinium trinitatum, Gonyaulax spinifera (Claparède & Lachmann) Diesing, Noctiluca scintillans (Macartney) Kofoid & Swezy, Polykrikos hartmannii, Polykrikos geminatus (Schütt) Qiu & Lin, Protoceratium reticulatum (Claparède & Lachmann) Bütschli, and Scrippsiella acuminate), four diatom taxa (Chaetoceros debilis Cleve, Chaetoceros decipiens Cleve, Chaetoceros socialis Lauder, and Skeletonema marinoi Sarno & Zingone), one taxon in Pelagophyceae (Aureococcus anophagefferens Hargraves & Sieburth), and three in Raphidophyceae (Chattonella marina (Subrahmanyan) Hara & Chihara, Fibrocapsa japonica Toriumi & Takano, and Heterosigma akashiwo (Hada) Hada ex Hara & Chihara). Some of these harmful species belonged to rare taxa, whose sequences were less than 20 reads, including Alexandrium affne, Noctiluca scintillans, and Aureococcus anophagefferens (Supplementary Table S1). However, some occurred widely and abundantly, such as Azadinium trinitatum, Scrippsiella acuminata, Chattonella marina, and Polykrikos hartmannii.
Many species in Gonyaulax Diesing could produce yessotoxin (YTX), including Gonyaulax spinifera (Howard et al., 2009) and Gonyaulax taylorii Carbonell-Moore ( lvarez et al., 2016). Five OTUs of Gonyaulax (OTU119, OTU488, OTU349, OTU764, and OTU868) were obtained (Supplementary Table S1), two of which were assigned to Gonyaulax spinifera, and one of which was identified as Gonyaulax fragilis (Schütt) Kofoid. Gonyaulax sequences ranged from 0 to 146 reads, with the highest reads at station B15 (Fig. 6a). Another YTX producer, Protoceratium reticulatum (Howard et al., 2009), only occurred at station B5 (Supplementary Table S1). Polykrikos hartmannii is an ichthyotoxin producer (Tang et al., 2013) that was detected at 15 stations and prevalent at station B5 and the surrounding stations (Fig. 6b). Alexandrium Halim is an important source of paralytic shellfish poisoning (PSP, Hallegraeff, 2003). Only one OTU of Alexandrium was detected in this study (Alexandrium affne, OTU 257), and it occurred at three stations (B4, B14, and B15) with low sequence numbers (Supplementary Table S1).
Some species of the planktonic dinoflagellate genus Azadinium Elbr chter & Tillmann, such as Azadinium spinosum Elbr chter & Tillmann and Azadinium poporum Elbr chter & Tillmann, produce azaspiracids (AZAs, Tillmann et al., 2014). One Azadinium OTU (OTU624), which was the nontoxic Azadinium trinitatum, was detected in this study (Supplementary Table S1). The abundance of Azadinium trinitatum was higher in the northwestern area, particularly at stations B1 and B2 (Fig. 6c). Scrippsiella acuminata, Polykrikos geminatus, and Noctiluca scintillans are common HAB species in coastal waters (Hallegraeff, 2003). Scrippsiella acuminata was found at almost all stations (Supplementary Table S1). Sequences of Scrippsiella acuminata peaked at station B5 in the southwestern corner, with high levels in western waters (Fig. 6d). Polykrikos geminatus was found at only three stations (Supplementary Table S1). Noctiluca scintillans appeared at only a few stations, with very low numbers of reads (Supplementary Table S1). Biecheleria Moestrup, Lindberg & Daugbjerg (mostly Biecheleria halophile), a thin-walled nanosized dinoflagellate, was widely and abundantly distributed (Supplementary Table S1). High reads of Biecheleria occurred in the northwestern area, with the highest at station B5 (Fig. 6e).
Toxic and harmful algal species other than dinoflagellates mostly occurred in the east, with the highest reads at station B11 (Fig. 6f–h). Many species in Raphidophyceae could produce ichthyotoxins (Basti et al., 2016). Three OTUs of raphidophytes were detected in the present study (Supplementary Table S1), and they were identified as Chattonella marina (OTU658), Fibrocapsa japonica (OTU722), and Heterosigma akashiwo (OTU518). Chattonella marina was distributed at eight stations, with higher reads at station B11 (Fig. 6f). Fibrocapsa japonica and Heterosigma akashiwo occurred in low sequence numbers (Supplementary Table S1).
Thalassiosira was the most abundant diatom genus in this study, and two of its OTUs were detected (Supplementary Table S1). Chaetoceros Ehrenberg is a common genus of planktonic diatoms that form resting spores, and seven OTUs of Chaetoceros were identified in this study; however, all OTUs had low sequence numbers (Supplementary Table S1). Higher sequence numbers of Thalassiosira and Chaetoceros occurred in the western sea area, with the highest at station B11 (Fig. 6g–h). Another algal bloom species, Skeletonema marinoi, occurred at eight stations but in very low sequence numbers (Supplementary Table S1).
Two Pelagophyceae OTUs were detected in this study, including the brown tide species Aureococcus anophagefferens, which only occurred at two stations (B2 and B4) with very low sequence numbers (Supplementary Table S1).4 DISCUSSION 4.1 Community structure of eukaryotic algae
A total of 98 algal OTUs from 17 surface sediment samples were detected from the central Bohai Sea in this study. Xu et al. (2017) derived 409 OTUs of the eukaryotic phytoplankton community from samples of the coastal waters of the Bohai Sea, within which 75 OTUs were identified at the species level. The OTU richness of phytoplankton was far higher than that of eukaryotic algae in the sediments. Most phytoplankton vegetative cells are generally thought to not be able to survive in sediments for a long time, and only resting stages of phytoplankton are well preserved in sediments after long-term cold-dark storage (reviewed by Ellegaard and Ribeiro, 2018). In the present study, the sediment samples were stored in a dark place at 8 ℃ for 6 months before DNA extraction to ensure that the algal DNA extracted from sediments was mostly from resting stages (Piredda et al., 2017). Therefore, the algal OTU richness in sediments is generally lower than that obtained from the water column, because only a small component of phytoplankton could form resting stages (Head, 1996; McQuoid and Hobson, 1996).
Most taxa identified at the species level in this study have been reported to form resting stages (Supplementary Table S1). However, whether the other taxa could form cysts was not known. For example, Azadinium trinitatum occurred abundantly in sediments from the central Bohai Sea (Supplementary Table S1) and the southern Chinese coasts (Liu et al., 2020), suggesting that it is a cyst-forming species. Indeed, cyst formation is an important characteristic in genus Azadinium (Tillmann et al., 2016). The wide and abundant occurrences of some parasitic taxa, such as the parasitic Syndiniales (OTU234), were probably due to the presence of their hosts. In addition, the dark cold treatment before DNA extraction could not exclude the fragmental DNA released from the dead cells. These DNA fragments should generally be represented as rare OTUs according to Shang et al. (2019). The rare OTUs with 1–2 reads (singletons and doubletons) were removed in the present study. Therefore, the algal sequences we detected in this study mostly existed as resting stages.
Dinoflagellate cysts constitute an important component of sedimentary assemblages of microalgal resting stages (Ellegaard and Ribeiro, 2018). In addition, high numbers of DNA copies (up to tens or hundreds of thousands of copies per cell) have been found in dinoflagellates (Lin, 2011) and thus may be over-represented in sequencing (Liu et al., 2017; Chen et al., 2021). Dinoflagellates have been also found to make substantial contributions to DNA reads and OTU richness in the phytoplankton communities in the Bohai Sea (Xu et al., 2017), though diatoms have constituted the predominant phytoplankton group represented in microscopic observations (Guo et al., 2014; Wang et al., 2019). The DNA reads and OTU richness of diatoms only ranked the third among the eukaryotic algae in the present study. Resting spores have been reported only in some centric diatoms, and they are seldom reported in pennate species (McQuoid and Hobson, 1996). Diatom OTUs obtained from sediment metabarcoding are mostly a few resting stages forming species, such as the centric diatoms Chaetoceros, Skeletonema Greville, and Thalassiosira (Montresor et al., 2013; Piredda et al., 2017; Dzhembekova et al., 2018). Therefore, the diversity and abundance of diatoms are usually underestimated in sediment metabarcoding studies. Quantification discrepancies of taxa are a common problem of the current approaches used for metabarcoding, and some experts suggested that presence/absence should be used in metabarcoding instead of relative abundance data (Buchner et al., 2019; Bailet et al., 2020).
The high abundance of chrysophyte sequences was accounted for by the predominance of some particular OTUs at some stations, including OTU757 at stations B4 and B12 and OTU10 at station B1. The results agreed with the findings of previous studies on eukaryotic algae and germinated phytoplankton in sediments from the South China Sea, where chrysophytes occurred predominantly at some particular stations (Liang, 2018; Liu et al., 2020). Cyst formation is a characteristic feature of chrysophytes, and several chrysophyte isolates were observed to form cysts under laboratory conditions (Findenig et al., 2010). Although the dominant chrysophyte OTUs could not be identified at the species level in the present study, the wide and abundant occurrence of these sequences suggested their ubiquitous distribution and cyst-formation characteristics.
Diverse chlorophytes (green algae) were notably detected in this study, some of which occurred widely and abundantly (Fig. 5 and Supplementary Table S1), including Mamiella gilva (Parke & Rayns) Moestrup (Mamiellophyceae) and Pterosperma cristatum Schiller (Pyramimonadophyceae). Xu et al. (2017) found a wide diversity of chlorophytes, including five species in Mamiellophyceae, two in Nephroselmidophyceae, and two in Pycnococcaceae, in the phytoplankton community in the coastal waters of the Bohai Sea based on 18S rDNA sequencing. Mamiella gilva and Pterosperma cristatum were regarded as previously unreported species in the Bohai Sea. Tragin and Vaulot (2018) analyzed the Ocean Sampling Day metabarcoding dataset, which provides sequences from the V4 region of 18S rDNA for 157 samples collected at 143 coastal stations, and they found the chlorophytes to be ubiquitous and dominant in coastal waters, especially those in Mamiellophyceae. In the present study, Mamiellophyceae was also the most diverse and abundant chlorophyte class. Most chlorophyte taxa detected in this study were pico-sized and diffcult to sample and distinguish in traditional phytoplankton surveys. In addition, green algae are ubiquitous in nature, and they are likely to be introduced during DNA extraction and sequencing. However, the dominant green algae in this study were all marine species, and they occurred widely in the sediments. Few reports of resting stages in the prasinophyte green algae Pyramimonas gelidicola McFadden, Moestrup & Wetherbee (Van den Hoff et al., 1989) and Mantoniella Desikachary (Ellegaard et al., 2016) could be found. The wide and abundant distribution of these marine green algae in benthic (present study) and planktonic communities (Xu et al., 2017) suggests their perennial occurrence in the Bohai Sea.4.2 Occurrence of small-sized species and HAB species
The small thin-walled dinoflagellate Biecheleria halophila was the most dominant dinoflagellate OTU in this study, and it was present at all stations (Supplementary Table S1). Biecheleria is a group of nanosized phytoplankton taxa, most of which could form resting cysts (Takahashi et al., 2014). Species of Biecheleria have rarely been reported in routine phytoplankton surveys due to their small sizes and thin walls (Luo et al., 2013; Takahashi et al., 2014). Based on electron microscopy and HTS techniques, diverse Biecheleria species have been reported (Takahashi et al., 2014). The cysts and motile cells of Biecheleria are widely distributed in coastal sea areas worldwide (Takahashi et al., 2014; Dzhembekova et al., 2018; Salonen et al., 2019). Luo et al. (2013) reported the first occurrence of Biecheleria species (Biecheleria cincta (Siano, Montresor & Zingone) Siano) in China after sediment incubation. Biecheleria cincta has also been found to occur abundantly and widely in sediments from the southern Chinese coasts (Liu et al., 2020). However, Biecheleria halophila has not yet been reported in the Chinese sea areas in either planktonic or benthic communities. The wide distribution of this species suggests that it could be a common dinoflagellate in the Bohai Sea; however, it may be ignored during routine phytoplankton investigations.
Another nanosized dinoflagellate, Azadinium trinitatum, occurred at all stations in high abundance (Supplementary Table S1). Azadinium trinitatum is a new Azadinium species recently described in the North Atlantic by Tillmann et al. (2014). Many species of Azadinium are responsible for AZA shellfish poisoning (AZP); however, no known AZAs were found in Azadinium trinitatum (Tillmann et al., 2014). Though Azadinium poporum, which causes AZP, has been previously reported to be widely distributed along the Chinese coasts (Gu et al., 2013; Liu et al., 2020), the present study was the first to report Azadinium trinitatum in the Chinese sea areas. The cells of Azadinium are generally small (ca. 15 μm in length and 10 μm in width) and rather inconspicuous under light microscopy. Determination of the tiny diﬀerences in morphological characteristics requires electron microscopy or tedious high-resolution light microscopy (Tillmann et al., 2014). Identification of Azadinium from fixed field samples is thus problematic and further complicated by other species with similar sizes and shapes (Tillmann et al., 2014). The wide distribution of Azadinium along the Chinese coasts (this study, Gu et al., 2013; Luo et al., 2013; Liu et al., 2020) and worldwide in sea areas (Akselman and Negri, 2012; Tillmann et al., 2014) indicates a global distribution of this genus (Tillmann et al., 2014).
Among all microalgal taxa, 16 potentially algal bloom-forming species were detected in this study (e.g., PSP-causing Alexandrium affne; YTX producers Gonyaulax spinifera and Protoceratium reticulatum; ichthyotoxic Polykrikos hartmannii, Chattonella marina, Fibrocapsa japonica, and Heterosigma akashiwo; and bloom-causing species, such as Noctiluca scintillans, Polykrikos geminatus, Scrippsiella acuminate, Chaetoceros spp., Skeletonema marinoi, and the brown tide species Aureococcus anophagefferens). These taxa may produce toxins harmful to humans or form blooms associated with mortality of fish or birds in marine coastal waters (Grattan et al., 2016). Some of these harmful algal taxa and other OTUs were detected in very few sequences (< 20 reads), including Alexandrium affne, Noctiluca scintillans, and Aureococcus anophagefferens (Supplementary Table S1). These taxa were detected in very small amounts in this study, suggesting that they rarely occur in nature or were the outcome of biases in the analysis (Chariton et al., 2015; Gran-Stadnicze ko et al., 2019; Bailet et al., 2020).
HABs have frequently occurred in the coastal sea areas of the Bohai Sea starting from the late 1990s (Song et al., 2016). Aureococcus anophagefferens, Phaeocystis globosa Scherﬀel, Heterosigma akashiwo, Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup, Margalefidinium polykrikoides (Margalef) Gómez, Richlen & Anderson, Noctiluca scintillans, and Skeletonema costatum (Greville) Cleve have been the most frequently occurring bloom species (Song et al., 2016). Brown tides caused by Aureococcus anophagefferens have recurred frequently in the Northwestern Bohai Sea since 2009 (Huang et al., 2020). Most of these blooms occurred in coastal sea areas of the Bohai Bay due to the luxuriant nutrients discharged from the terrestrial sources (Song et al., 2016). However, resting stages formed after the nearshore blooms may be carried by water masses and settled into the deeper bottom sediments of the central Bohai Sea. These resting stages may germinate into vegetative cells and release them to the upper water column when the environmental conditions are suitable, which has the potential to form algal blooms in nutrient-rich waters. Therefore, the diverse HAB species recorded in this study and the high DNA reads of some particular species, such as Azadinium trinitatum, Scrippsiella acuminata, Chattonella marina, and Polykrikos hartmannii, suggests the potential risk of HABs in the central Bohai Sea.5 CONCLUSION
The obtained environmental metabarcoding data improved the understanding of the composition of the microalgal community in surface sediments from the central Bohai Sea. Eukaryotic algae were dominated by chrysophytes and dinoflagellates. Dinoflagellates were the most diverse microalgal group. The identification of 16 potentially toxic and/or algal bloom species, some of which were found at all stations, highlighted the ecological significance of the benthic resting stages for HAB events. Some small taxa, such as Biecheleria halophila and Azadinium trinitatum, occurred widely and abundantly in sediments, indicating that they are common dominant species in phytoplankton communities; however, they may have been ignored in previous surveys relying on microscopic observations. The capacity of metabarcoding to detect morphologically cryptic species and better detect rare taxa makes this method suffciently sensitive to assess the composition of benthic microalgae in sediments.6 DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/Traces/sra) with the accession number PRJNA722689.
Electronic supplementary material
Supplementary material (Supplementary Table S1) is available in the online version of this article at https://doi.org/10.1007/s00343-021-0481-7.
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