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Pseudo‐nitzschia nanaoensis sp. nov. is described from waters around Nan'ao Island (South China Sea), using morphological data and molecular evidence. This species is morphologically most similar to P. brasiliana, but differs by a denser arrangement of fibulae, interstriae, and poroids, as well as by the structure of the valvocopula and the narrow second band. Pseudo‐nitzschia nanaoensis constitutes a monophyletic lineage and is well differentiated from other species on the LSU and ITS2 sequence‐structure trees. Pseudo‐nitzschia nanaoensis makes up the basal node on the LSU tree, and forms a sister clade with a group of P. pungens and P. multiseries on the ITS2 tree. The ability of cultured strains to produce domoic acid was assessed, including its possible induction by the presence of a copepod and brine shrimp, by liquid chromatography–tandem mass spectrometry. However, no strains showed detectable domoic acid. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Species of the diatom Pseudo-nitzschia are known to produce domoic acid that is responsible for amnesic shellfish poisoning (ASP). To investigate the potential risk of ASP and species occurrence of toxic or potentially toxic Pseudo-nitzschia in Malaysian waters, plankton samples were collected from 17 locations. Samples were examined through transmission electron microscopy. Species of Pseudo-nitzschia were identified on the basis of the frustule morphology and morphometric measurements. Twenty-two well-described species were recorded, of which 14 are new records for Malaysia. A new morphotype, closely resembling species in the pseudodelicatissima complex, was also discovered. The morphotype differs from other species in the complex by its lower densities of fibulae and striae. Nine of the species have previously been associated with ASP events worldwide. Our study recorded for the first time high species richness of Pseudo-nitzschia in the confined coasts of Malaysia. In addition, we performed a morphology-based phylogeny and proposed a key to Pseudo-nitzschia species, with a special emphasis on the poroid structure of the striae, to aid in species identification. [ABSTRACT FROM AUTHOR]

• Toxic blooms increased till early 2000s then stabilized or lessened. • Number of HAB species observed have increased. • Sites affected by Alexandrium and Pyrodinium still expanding. • Increase in reported fish killing species in Southeast Asia. • Enhanced long-term monitoring and establishing HAB species distributions needed. In the Southeast Asian region, the Philippines and Malaysia are two of the most affected by Harmful Algal Blooms (HABs). Using long-term observations of HAB events, we determined if these are increasing in frequency and duration, and expanding across space in each country. Blooms of Paralytic Shellfish Toxin (PST)-producing species in the Philippines did increase in frequency and duration during the early to mid-1990s, but have stabilized since then. However, the number of sites affected by these blooms continue to expand though at a slower rate than in the 1990s. Furthermore, the type of HABs and causative species have diversified for both toxic blooms and fish kill events. In contrast, Malaysia showed no increasing trend in the frequency of toxic blooms over the past three decades since Pyrodinium bahamense was reported in 1976. However, similar to the Philippines, other PST producers such as Alexandrium minutum and Alexandrium tamiyavanichii have become a concern. No amnesic shellfish poisoning (ASP) has been confirmed in either Philippines or Malaysia thus far, while ciguatera fish poisoning cases are known from the Philippines and Malaysia but the causative organisms remain poorly studied. Since the 1990s and early 2000s, recognition of the distribution of other PST-producing species such as species of Alexandrium and Gymnodinium catenatum in Southeast Asia has grown, though there has been no significant expansion in the known distributions within the last decade. A major more recent problem in the two countries and for Southeast Asia in general are the frequent fish-killing algal blooms of various species such as Prorocentrum cordatum, Margalefidinium polykrikoides, Chattonella spp., and unarmored dinoflagellates (e.g., Karlodinium australe and Takayama sp.). These new sites affected and the increase in types of HABs and causative species could be attributed to various factors such as introduction through mariculture and eutrophication, and partly because of increased scientific awareness. These connections still need to be more concretely investigated. The link to the El Niño Southern Oscillation (ENSO) should also be better understood if we want to discern how climate change plays a role in these patterns of HAB occurrences. [ABSTRACT FROM AUTHOR]

• The ITS2 phylogeny and population genetic analysis of 354 N. navis-varingica strains from the Western Pacific (WP) identified eight haplogroups, designated A to H. • Low genetic differentiation indicated high connectivity among N. navis-varingica populations in WP. • Inference of molecular clock suggested historical events influenced species dispersal and widespread distributions in WP. The benthic pennate diatom Nitzschia navis - varingica , known for producing domoic acid (DA) and its isomers, is widely distributed in the Western Pacific (WP) region. To investigate the genetic differentiation and gene flow patterns among the populations in the WP, the genetic diversity of 354 strains of N. navis-varingica was analysed using two nuclear-encoded rDNA loci: the large subunit rDNA (LSU rDNA) and the internal transcribed spacer 2 (ITS2). Frustule morphology of each strain was examined by TEM. The LSU rDNA phylogeny revealed a monophyletic lineage encompassing all strains, with sequence divergences of <0.9 %. Phylogenetic and population genetic analyses of ITS2 identified eight distinct clades (designated as Groups A to H) with moderate to high genetic heterogeneity (0.5–19.7 %). The low genetic differentiations between the geographically separated populations (pairwise F ST of <0.03) suggested high gene flow and lack of spatial genetic structuring. Molecular clock analysis of the ITS2 phylogeny traced the evolutionary history of N. navis-varingica to the Eocene Epoch, and the split between clades likely occurred from the mid-Miocene to Pleistocene Epochs (10.8–1.2 Ma). The population dispersal in the WP were likely influenced by historical events like the Quarternary glacial cycles during the period, contributing to its homogenous distributions in the region. [ABSTRACT FROM AUTHOR]

To determine the species distribution of an amnesic shellfish poisoning (ASP) toxins-producing diatom Nitzschia navis-varingica outside its current restricted geographical distribution range in Asian coastal waters, samples were collected from two sites of Bootless Bay, located on southwest coast of Papua New Guinea near Port Moresby. A total of twenty-one strains of N. navis-varingica were isolated and the clonal cultures established. The species identity was confirmed by molecular characterization based on the ribosomal DNA markers. The LSU rDNA phylogenetic inference revealed a monophyletic clade of all strains, clustered with N. navis-varingica with high bootstrap supports. ASP toxin production in the strains was investigated by HPLC with fluorescence detection and subsequently confirmed for the representative isolates by LC-MS/MS with multiple reaction monitoring (MRM) mode. All eleven strains from site A showed presence of domoic acid (DA) and isodomoic acid (IB); the toxin quota ranged from 0.70 to 4.63 pg cell−1 (average 2.75 ± 1.26 pg cell−1, n = 11), with the composition of DA and IB of 21 DA: 79 IB. While for strains from site B, four out of ten strains showed presence of DA and IB, with the toxin quota ranged from 1.40 to 3.84 (average 2.57 ± 1.17 pg cell−1, n = 4); the composition was 52 DA: 48 IB. The strains examined in this study were divided into toxic and probably non-toxic groups in ITS2 phylogeny. This represents the first record of domoic acid-producing Nitzschia navis-varingica from Papua New Guinea. [Display omitted] • First recorded finding of the toxic diatom Nitzschia navis-varingica in Papua New Guinea. • Verification of domoic acid and isodomoic acid B in the strains by HPLC and LC-MS/MS. • Confirmation of the two toxin composition types, i.e. domoic acid-isodomoic acid B and only isodomoic acid B. • Taxa of N. navis-varingica were divided into toxic and probably non-toxic groups in ITS2 phylogeny. [ABSTRACT FROM AUTHOR]

Highlights: [•] A Pseudo-nitzschia morphotype, P. sp. Port Dickson, found in the Strait of Malacca, Malaysia, was described morphologically and characterized molecularly based on both field and culture specimens. [•] The morphotype was designated as Pseudo-nitzschia kodamae sp. nov. [•] The species produces the amnesic shellfish poisoning toxin, domoic acid. [•] This is the first report of a toxigenic Pseudo-nitzschia species found in the Southeast Asian region. [Copyright &y& Elsevier]

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. [Display omitted] • First record of Pseudo-nitzschia cuspidata bloom in Western Pacific region. • Low level of domoic acid was detected with cellular contents of 257–504 fg cell−1. • Pseudo-nitzschia cuspidata bloom associated with domoic acid contamination in shellfish of up to 8 μg g−1 tissue. • The phylogenetic tree revealed three distinct clades of Pseudo-nitzschia cuspidata. [ABSTRACT FROM AUTHOR]

The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80–83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, r s = 0.67, p < 0.0001; V9, r s = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies. • qPCR and HTS methods for the detection of Alexandrium tamiyavanichii showed high occupancy profiles across samples. • Minimal discordance between qPCR and HTS methods in detecting A. tamiyavanichii cells. • Estimates of A. tamiyavanichii cell abundances by HTS read abundances was comparable to qPCR quantification. [ABSTRACT FROM AUTHOR]

• Fourteen Pseudo-nitzschia taxa were identified in the Taiwan Strait. • Two novel species were described, i.e., P. chiniana sp. nov. and P. qiana sp. nov. • Three taxa were detected to produce domoic acid, i.e. P. multiseries , P. pseudodelicatissima and P. lundholmiae. • It was proofed that brine shrimp can induce toxic Pseudo-nitzschia to produce more DA. 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 < 0.05) in P. lundholmiae (strain MC4218) and P. multiseries (strain MC4177), from 2.5 ± 0.4 to 8.9 ± 0.7 and 1.6 ± 0.3 to 37.2 ± 2.5 fg cell−1 respectively. [ABSTRACT FROM AUTHOR]

• Spatial variability of HAB species in the micro-eukaryotic protist community of the eastern Malaysian Peninsula, South China Sea was investigated by DNA metabarcoding. • The results revealed 29 molecular HAB taxa, dominated by Alexandrium tamiyavanichii. • The diversity and community structure of HABs varied in space with different HAB taxa occupied in different environmental niches across the region. 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. [ABSTRACT FROM AUTHOR]

The distribution of the toxic pennate diatom Nitzschia was investigated at four mangrove areas along the coastal brackish waters of Peninsular Malaysia. Eighty-two strains of N. navis-varingica were isolated and established, and their identity confirmed morphologically and molecularly. Frustule morphological characteristics of the strains examined are identical to previously identified N. navis-varingica , but with a sightly higher density of the number of areolae per 1 μm (4–7 areolae). Both LSU and ITS rDNAs phylogenetic trees clustered all strains in the N. navis-varingica clade, with high sequence homogeneity in the LSU rDNA (0–0.3%), while the intraspecific divergences in the ITS2 data set reached up to 7.4%. Domoic acid (DA) and its geometrical isomers, isodomoic A (IA) and isodomoic B (IB), were detected in cultures of N. navis - varingica by FMOC-LC-FLD, and subsequently confirmed by LC–MS/MS, with selected ion monitoring (SIM) and multiple reaction monitoring (MRM) runs. DA contents ranged between 0.37 and 11.06 pg cell −1 . This study demonstrated that the toxigenic euryhaline diatom N. navis-varingica is widely distributed in Malaysian mangrove swamps, suggesting the risk of amnesic shellfish poisoning and the possibility of DA contamination in the mangrove-related fisheries products. [ABSTRACT FROM AUTHOR]

• Two novel ribotypes (C and D) of A. minutum were identified in Chinese and Malaysian coastal waters. • Alexandrium minutum ribotype c produced predominantly gonyautoxin 1/4. • Alexandrium minutum ribotype d did not produce detectable toxins. • Cells and cysts of ribotype c were detected in the East China Sea through metabarcoding. The bloom-forming dinophyte Alexandrium minutum comprises biogeographic inferred, global and Pacific clades with both toxic and nontoxic strains reported. A. minutum has a wide distribution in the Western Pacific, but to date only a few strains have available DNA sequences. To fully understand its genetic diversity, sampling was undertaken from the Yellow Sea, the East and South China Sea, and five strains of A. minutum and two strains of its sister species, A. tamutum, were established. Their morphology was examined by light and scanning electron microscopy. In addition, sequences were obtained from both large subunit (LSU) ribosomal DNA and/or internal transcribed spacer (ITS) region. Strains of A. minutum are morphologically indistinguishable, characterized by a smaller cell size and a narrow sixth precingular plate. In contrast, A. tamutum has a wider sixth precingular plate. High nucleotide divergences of LSU (D1–D3) rDNA and ITS were revealed amongst strains of A. minutum (10% and 25%, respectively), and A. tamutum (3% and 13%, respectively). Molecular phylogenies based on LSU rDNA and ITS revealed three ribotypes (B–D) of A. minutum, and two ribotypes of A. tamutum in the Western Pacific. Seasonal sampling in the East China Sea to detect A. minutum using the DNA metabarcoding targeting ITS1 region was also performed. Our results showed that the ribotypes B and C of A. minutum co-occurred in the water. Paralytic shellfish toxin (PSTs) of all seven strains was analysed using liquid chromatography with tandem mass spectrometry (LC–MS/MS). PSTs were detected only in A. minutum ribotypes B and C with predominance of gonyautoxins 1/4. Our results suggest high diversity and risk potential of this toxic species in this region. [ABSTRACT FROM AUTHOR]

Abstract: Nitzschia navis-varingica is a diatom that is known to produce significant levels of amnesic shellfish poisoning (ASP) toxins. A total of 33 N. navis-varingica strains were isolated from four brackish water localities in the Philippines and Indonesia, and cultured to characterize the toxins produced. The isolates were analyzed for domoic acid (DA) and isodomoic acids A (IA) and B (IB) by HPLC with fluorescence detection. Two toxin composition types were detected that have not been previously described: strains producing only IB and strains producing DA–IA–IB. These two types were isolated from two different localities. Eighteen strains were isolated from the Philippines (northern Luzon Island). Among them, 10 isolates from Alaminos produced only IB with an average toxin content of 3.05pgcell−1, seven isolates from Bulacan produced DA and IB with average toxin contents of 0.68pgcell−1 and 1.18pgcell−1, respectively. One isolate from Cavite produced DA, IA, and IB with a toxin content of 0.58, 0.20, and 0.92pgcell−1, respectively. Fifteen isolates from Indonesia (Bone, South Sulawesi) produced only DA (four isolates) or DA with trace amounts of IB (eleven isolates), with an average toxin content of 2.38pgcell−1 and 0.06pgcell−1, respectively. Sub-strains were established from strains producing either of the three toxin types: IB, DA–IA–IB, and DA-trace IB. Results showed that the toxin composition type was the same for parent and sub-strains, indicating that the toxin composition is a stable character for a strain. [Copyright &y& Elsevier]

• Alexandrium fragae was reported firstly outside its type locality. • Azadinium spinosum ribotype A was reported firstly in the Asia Pacific. • Vulcanodinium rugosum was reported firstly in the Southeast Asia. • Karenia selliformis and Karlodinium digitatum were detected in the Gulf of Thailand. Information on the diversity and distribution of harmful microalgae in the Gulf of Thailand is very limited and mainly based on microscopic observations. Here, we collected 44 water samples from the Gulf of Thailand and its adjacent water (Perhentian Island, Malaysia) for comparison in 2018. DNA metabarcoding was performed targeting the partial large subunit ribosomal RNA gene (LSU rDNA D1–D3) and the internal transcribed spacers (ITS1 and ITS2). A total of 50 dinoflagellate genera (made up of 72 species) were identified based on the LSU rDNA dataset, while the results of ITS1 and ITS2 datasets revealed 33 and 32 dinoflagellate genera comprising 69 and 64 species, respectively. Five potentially toxic Pseudo-nitzschia (Bacillariophyceae) species were detected, with four as newly recorded species in the water (Pseudo-nitzschia americana / brasilliana, Pseudo-nitzschia simulans / delicatissima, P. galaxiae and P. multistriata). The highest relative abundances of P. galaxiae and P. multistriata were found in Trat Bay and Chumphon (accounting for 0.20% and 0.06% of total ASVs abundance, respectively). Three paralytic shellfish toxin producing dinoflagellate species were detected: Alexandrium tamiyavanichii, Alexandrium fragae , and Gymnodinium catenatum. The highest abundance of A. tamiyavanichii was found in the surface sample of Chumphon (CHO7 station), accounting for 1.95% of total ASVs abundance. Two azaspiracid producing dinoflagellate species, Azadinium poporum ribotype B, Azadinium spinosum ribotype A, and a pinnatoxin producing dinoflagellate species Vulcanodinium rugosum , with two ribotypes B and C, were revealed from the datasets although with very low abundances. Six fish killing dinoflagellate species, including Margalefidinium polykrikoides group IV, Margalefidinium fulvescens, Karenia mikimotoi, Karenia selliformis ribotype B, Karlodinium australe , and Karlodinium digitatum were detected and all representing new records in this area. The findings of numerous harmful microalgal species in the Gulf of Thailand highlight the potential risk of human intoxication and fish killing events. [ABSTRACT FROM AUTHOR]