Red tides and associated fisheries damage caused by the harmful raphidophyte Chattonella were reassessed based on the documented local records for 50 years to understand the distribution and economic impacts of the harmful species in the Western Pacific. Blooms of Chattonella with fisheries damage have been recorded in East Asia since 1969, whereas they have been only recorded in Southeast Asia since the 1980s. Occurrences of Chattonella have been documented from six Southeast Asian countries, Indonesia, Malaysia, Philippines, Singapore, Thailand and Viet Nam, with mass mortalities mainly of farmed shrimp in 1980-1990s, and farmed fish in 2000-2010s. These occurrences have been reported with the names of C. antiqua, C. marina, C. ovata, C. subsalsa and Chattonella sp., owing to the difficulty of microscopic species identification, and many were not supported with molecular data. To determine the distribution of C. marina complex and C. subsalsa in Southeast Asia, molecular phylogeny and microscopic observation were also carried out for cultures obtained from Indonesia, Malaysia, Japan, Philippines, Russia, Singapore and Thailand. The results revealed that only the genotype of C. marina complex has been detected from East Asia (China, Japan, Korea and Russia), whereas both C. marina complex (Indonesia and Malaysia) and C. subsalsa (Philippines, Singapore and Thailand) were found in Southeast Asia. Ejection of mucocysts has been recognized as a diagnostic character of C. subsalsa, but it was also observed in our cultures of C. marina isolated from Indonesia, Malaysia, Japan, and Russia. Meanwhile, the co-occurrences of the two harmful Chattonella species in Southeast Asia, which are difficult to distinguish solely based on their morphology, suggest the importance of molecular identification of Chattonella genotypes for further understanding of their distribution and negative impacts.
In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis.
A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.
Coastal ecosystems are often subjected to anthropogenic disturbances that lead to water quality deterioration and an increase in harmful algal bloom (HAB) events. Using the next-generation molecular tool of 18S rDNA metabarcoding, we examined the community assemblages of HAB species in the Johor Strait, Malaysia between May 2018 and September 2019, covering 19 stations across the strait. The molecular operational taxonomic units (OTUs) of HAB taxa retrieved from the dataset (n = 194) revealed a much higher number of HAB taxa (26 OTUs) than before, with 12 taxa belong to new records in the strait. As revealed in the findings of this study, the diversity and community structure of HAB taxa varied significantly over time and space. The most common and abundant HAB taxa in the strait (frequency of occurrence >70%) comprised Heterosigma akashiwo, Fibrocapsa japonica, Pseudo-nitzschia pungens, Dinophysis spp., Gymnodinium catenatum, Alexandrium leei, and A. tamiyavanichii. Also, our results demonstrated that the HAB community assemblages in the strait were dependent on the interplay of environmental variables that influence by the monsoonal effects. Different HAB taxa, constitute various functional types, occupied and prevailed in different environmental niches across space and time, leading to diverse community assemblages and population density. This study adds to the current understandings of HAB dynamics and provides a robust overview of temporal-spatial changes in HAB community assemblages along the environmental gradients in a tropical eutrophic coastal ecosystem.
Species of the benthic dinoflagellate Gambierdiscus produce polyether neurotoxins that caused ciguatera fish/shellfish poisoning in human. The toxins enter marine food webs by foraging of herbivores on the biotic substrates like macroalgae that host the toxic dinoflagellates. Interaction of Gambierdiscus and their macroalgal substrate hosts is believed to shape the tendency of substrate preferences and habitat specialization. This was supported by studies that manifested epiphytic preferences and behaviors in Gambierdiscus species toward different macroalgal hosts. To further examine the supposition, a laboratory-based experimental study was conducted to examine the growth, epiphytic behaviors and host preferences of three Gambierdiscus species towards four macroalgal hosts over a culture period of 40 days. The dinoflagellates Gambierdiscus balechii, G. caribaeus, and a new ribotype, herein designated as Gambierdiscus type 7 were initially identified based on the thecal morphology and molecular characterization. Our results showed that Gambierdiscus species tested in this study exhibited higher growth rates in the presence of macroalgal hosts. Growth responses and attachment behaviors, however, differed among different species and strains of Gambierdiscus over different macroalgal substrate hosts. Cells of Gambierdiscus mostly attached to substrate hosts at the beginning of the experiments but detached at the later time. Localized Gambierdiscus-host interactions, as demonstrated in this study, could help to better inform efforts of sampling and monitoring of this benthic toxic dinoflagellate.
Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0-1.6 pg cell-1) and P. caipirignum (3.1 pg cell-1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.
Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.
A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.
This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.
Field sampling was undertaken to investigate the occurrence of Pseudo-nitzschia Peragallo species in eight locations along the coast of Malaysian Borneo. A total of 108 strains of Pseudo-nitzschia species were isolated, and their morphology examined with SEM and TEM. Additionally, molecular data from nuclear-encoded partial LSU rDNA, and ITS regions, were characterized. A total of five species were confidently identified based on a combination of distinct morphological characteristics and supporting molecular evidence: P. brasiliana Lundholm, Hasle & Fryxell, P. cuspidata (Hasle) Hasle, P. dolorosa Lundholm & Moestrup, P. micropora Priisholm, Moestrup & Lundholm, and P. pungens (Grunow) Hasle var. pungens. However, one morphotype from Sarawak, while somewhat similar to P. caciantha, showed significant morphological distinction from this and any other of the currently described species. Most notably this morphotype possessed a characteristic pore arrangement in the poroids, with the fine pores in each perforation sector arranged in circles. Pair-wise sequence comparison of the LSU rDNA between this unidentified morphotype and P. caciantha Lundholm, Moestrup & Hasle, revealed 2.7% genetic divergence. Phylogenetic analyses strongly supported the monophyly of the morphotype. Based upon these supporting data it is here described as a new species, Pseudo-nitzschia circumpora sp. nov. A key to the six species of Pseudo-nitzschia from Malaysian Borneo is presented. Molecular signatures for all species were established based on structural comparisons of ITS2 rRNA transcripts.
Different clades belonging to the cosmopolitan marine diatom Pseudo-nitzschia pungens appear to be present in different oceanic environments, however, a 'hybrid zone', where populations of different clades interbreed, has also been reported. Many studies have investigated the sexual reproduction of P. pungens, focused on morphology and life cycle, rather than the role of sexual reproduction in mixing the genomes of their parents. We carried out crossing experiments to determine the sexual compatibility/incompatibility between different clades of P. pungens, and examined the genetic polymorphism in the ITS2 region. Sexual reproduction did not occur only between clades II and III under any of experimental temperature conditions. Four offspring strains were established between clade I and III successfully. Strains established from offspring were found interbreed with other offspring strains as well as viable with their parental strains. We confirmed the hybrid sequence patterns between clades I and III and found novel sequence types including polymorphic single nucleotide polymorphisms (SNPs) in the offspring strains. Our results implicate that gene exchange and mixing between different clades are still possible, and that sexual reproduction is a significant ecological strategy to maintain the genetic diversity within this diatom species.
The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.
In a field survey in the Taiwan Strait during April 2016, the species composition and the domoic acid production of the diatom genus Pseudo-nitzschia were investigated. A total of 80 strains of Pseudo-nitzschia were established, and species identification was determined based on a combination of morphological and molecular data. Fourteen taxa were recognized, i.e., P. americana, P. brasiliana, P. calliantha, P. cuspidata, P. galaxiae, P. lundholmiae, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens var. aveirensis, P. pungenus var. pungens and P. sabit, as well as two novel species P. chiniana C.X. Huang & Yang Li and P. qiana C.X. Huang & Yang Li. Morphologically, P. chiniana is characterized by striae comprising one or two rows of poroids, and valve ends that are normally dominated by two rows of poroids within each stria. Whereas P. qiana is unique by having a narrow valve width (1.3-1.5 μm) and sharply pointed valve ends. Both taxa constitute their own monophyletic lineage in the phylogenetic analyses inferred from LSU and ITS2 rDNA, and are well differentiated from other Pseudo-nitzschia species. Pseudo-nitzschia chiniana forms a group with P. abrensis and P. batesiana in LSU and ITS trees, whereas P. qiana is sister to P. lineola. When comparing ITS2 secondary structure, five CBCs and seven HCBCs are recognized between P. chiniana and P. abrensis, and four CBCs and ten HCBCs between P. chiniana and P. batesiana. Two CBCs and eight HCBCs are found between P. qiana with P. lineola. The ability of the strains to produce domoic acid was assessed, including a potential toxin induction by the presence of brine shrimps. Results revealed production of domoic acid in six strains belonging to three species. Without presence of brine shrimps, cellular DA (pDA) was detected in four P. multiseries strains (1.6 ± 0.3, 26.6 ± 2.7, 68.3 ± 4.2 and 56.9 ± 4.7 fg cell-1, separately), one strain of P. pseudodelicatissima (0.8 ± 0.2 fg cell-1) and one strain of P. lundholmiae (2.5 ± 0.4 fg cell-1). In the presence of brine shrimps, pDA contents increased significantly (p
In March 2018, an algal bloom of Pseudo-nitzschia was detected, for the first time, in a semi-enclosed lagoon in Miri, Sarawak, Malaysia Borneo. The plankton samples were collected for cell enumeration and species identification by electron microscopy and molecular characterization. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to detect and quantify the neurotoxin domoic acid (DA) in both the plankton and shellfish samples. The abundance of Pseudo-nitzschia cells ranged from 5.6 × 105 to 3.5 × 106 cell L-1 during the bloom event. Morphological observation of the cells by transmission electron microscopy showed that the plankton samples comprised a single Pseudo-nitzschia morphotype resembling P. cuspidata. The ITS2 sequence-structure phylogenetic inference further supported the species identity as Pseudo-nitzschia cuspidata. Low levels of DA were detected in the plankton samples, with cellular DA, particulate DA, and dissolved DA of 257-504 fg DA cell-1, 676 ng L-1, and 15 ng L-1, respectively. The amount of DA, 8 μg g-1 tissue, was found present in the shellfish sample (Magallana sp.) which is below the regulatory limit of 20 μg DA g-1 tissue. The study documented, for the first time, DA contamination in shellfish that associated with bloom of P. cuspidata in the Western Pacific region.
Thirty-four strains of Heterocapsa were established from Malaysian waters and their morphologies were examined by light, scanning, and transmission electron microscopy. Three species, H. bohaiensis, H. huensis, and H. rotundata, and three new species, H. borneoensis sp. nov., H. limii sp. nov., and H. iwatakii sp. nov. were described in this study. The three species were differentiated morphologically by unique characteristics of cell size, shape, displacement of the cingulum, shape and position of nucleus, the number and position of pyrenoids, and body scale ultrastructure. The species delimitations were robustly supported by the molecular data. A light-microscopy-based key to species of Heterocapsa is established, with two major groups, i.e., species with a single pyrenoid, and species with multiple pyrenoids. Bioassays were conducted by exposing Artemia nauplii to Heterocapsa densities of 1-5 × 105 cells mL-1, and treatments exposed to H. borneoensis showed naupliar mortality, while no naupliar death was observed in the treatments exposed to cells of H. bohaiensis, H. huensis, H. limii, and H. iwatakii. Naupliar death was observed during the initial 24 h for both tested H. borneoensis strains, and mortality rates increased up to 50% after 72-h exposure. This study documented for the first time the diversity and cytotoxic potency of Heterocapsa species from Malaysian waters.
Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL-1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL-1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.
Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species' identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.
The diatom genus Pseudo-nitzschia, which has been associated with amnesic shellfish poisoning events globally, is also one of the key harmful microalga groups in Guangdong coastal waters, off the north coast of the South China Sea. In order to explore the diversity and toxigenic characteristics, Pseudo-nitzschia isolates were established. Based on a combination of morphological and molecular features, in total 26 different Pseudo-nitzschia taxa were identified, including two new species, P. uniseriata H.C. Dong & Yang Li and P. yuensis H.C. Dong & Yang Li. Morphologically, P. uniseriata is unique by having striae mainly comprising one row of poroids, which are simple without divided hymen internally, and each poroid containing one, seldom two sectors. Pseudo-nitzschia yuensis is characterized by having striae comprising one to two rows of poroids. In biseriate striae, the poroids are polygonal and irregularly distributed, and a discontinuous row of poroids may be present in the middle. In uniseriate striae, the poroids usually contain 1-5 sectors. Both taxa are well differentiated from other Pseudo-nitzschia species in phylogenetic analyses inferred from ITS2 sequence-structure information. Pseudo-nitzschia uniseriata is sister to P. lineola, whereas P. yuensis forms a group together with P. micropora and P. delicatissima. When comparing ITS2 secondary structure, two hemi-compensatory base change (HCBCs) are found between P. uniseriata and P. lineola. One compensatory base change (CBC) and four HCBCs are found between P. yuensis and P. delicatissima, and there is one CBC and five HCBCs between P. yuensis and P. micropora. The ability of cultured strains to produce particulate DA (pDA) revealed production of pDA in twenty-nine strains belonging to seven species: P. bipertita, P. caciantha, P. cuspidata, P. fraudulenta, P. fukuyoi, P. lundholmiae and P. multiseries. This is the first report of P. bipertita being toxic, with pDA content of 15.6 ± 2.1 fg cell-1. The presence of brine shrimps significantly increased pDA content in P. cuspidata, P. fukuyoi, P. lundholmiae and P. multiseries 1.4 to 7 times, and induced pDA production in P. fraudulenta from below detection limit to 17.5 ± 1.6 fg cell-1. The highest pDA concentration, 4830.5 ± 120.3 fg cell-1, was detected in P. multiseries, a level much lower than previous reports on P. multiseries from North America and Europe. Overall, the cellular toxin levels in Pseudo-nitzschia spp. were low in Guangdong coastal isolates.
To explore the species diversity and toxin profile of Pseudo-nitzschia, monoclonal strains were established from Chinese southeast coastal waters. The morphology was examined under light and transmission electron microscopy. The internal transcribed spacer region of ribosomal DNA was sequenced for phylogenetic analyses, and the secondary structure of ITS2 was predicted and compared among allied taxa. A combination of morphological and molecular data showed the presence of two new species, Pseudo-nitzschia hainanensis sp. nov. and Pseudo-nitzschia taiwanensis sp. nov. Pseudo-nitzschia hainanensis was characterized by a dumpy-lanceolate valve with slightly blunt apices and a central nodule, as well as striae comprising two rows of poroids. Pseudo-nitzschia taiwanensis was characterized by a slender-lanceolate valve, and striae comprising one row of split poroids. The poroid structure mainly comprised two sectors. Both taxa constituted their own monophyletic lineage in the phylogenetic analyses inferred from ITS2 rDNA and were well differentiated from other Pseudo-nitzschia species. Morphologically, P. hainanensis and P. taiwanensis could be assigned to the Pseudo-nitzschia delicatissima and the Pseudo-nitzschia pseudodelicatissima complex, respectively. Particulate domoic acid was measured using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), but no detectable pDA was found. With the description of the two new species, the species diversity of genus Pseudo-nitzschia reaches 58 worldwide, among which 31 have been recorded from Chinese coastal waters.