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Fulltext Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay

Lau WLS, Teng ST, Lim HC, Hii KS, Leong SCY, Leaw CP, et al.

Trop Life Sci Res, 2023 Mar;34(1):99-120.
PMID: 37065805 DOI: 10.21315/tlsr2023.34.1.7

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.
Fulltext Identification of Fish Species and Toxins Implicated in a Snapper Food Poisoning Event in Sabah, Malaysia, 2017

Dao HV, Uesugi A, Uchida H, Watanabe R, Matsushima R, Lim ZF, et al.

Toxins (Basel), 2021 09 15;13(9).
PMID: 34564661 DOI: 10.3390/toxins13090657

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian countries, few have been confirmed by ciguatoxins identification, resulting in limited information for the correct diagnosis of this food-borne disease. In the present study, ciguatoxin-1B (CTX-1B) in red snapper (Lutjanus bohar) implicated in a CFP case in Sabah, Malaysia, in December 2017 was determined by single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Continuous consumption of the toxic fish likely resulted in CFP, even when the toxin concentration in the fish consumed was low. The identification of the fish species was performed using the molecular characterization of the mitochondrial cytochrome c oxidase subunit I gene marker, with a phylogenetic analysis of the genus Lutjanus. This is the first report identifying the causative toxin in fish-implicated CFP in Malaysia.
Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae)

Lim PT, Ogata T

Toxicon, 2005 May;45(6):699-710.
PMID: 15804519

Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.
Toxic bloom of Pseudo-nitzschia cuspidata (Bacillariophyceae) and domoic acid contamination of bivalve molluscs in Malaysia Borneo

Teng ST, Abdullah N, Hanifah AH, Tan SN, Gao C, Law IK, et al.

Toxicon, 2021 Sep 30;202:132-141.
PMID: 34600910 DOI: 10.1016/j.toxicon.2021.09.018

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.
Corrigendum to "Toxic bloom of Pseudo-nitzschia cuspidata (Bacillariophyceae) and domoic acid contamination of bivalve molluscs in Malaysia Borneo" [Toxicon 202 (2021) 132-141]

Teng ST, Abdullah N, Hanifah AH, Tan SN, Gao C, Law IK, et al.

Toxicon, 2022 Jan 15;205:99.
PMID: 34872755 DOI: 10.1016/j.toxicon.2021.12.002
Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays

Shang L, Xu Y, Leaw CP, Lim PT, Wang J, Chen J, et al.

Sci Total Environ, 2021 Aug 01;780:146484.
PMID: 33774286 DOI: 10.1016/j.scitotenv.2021.146484

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24 h exposed to the live culture (2000-6710 cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48 h when exposed to 500-6000 cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100 cells·mL-1 within 24 h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.
Regional comparison on ciguatoxicity, hemolytic activity, and toxin profile of the dinoflagellate Gambierdiscus from Kiribati and Malaysia

Zhu J, Lee WH, Yip KC, Wu Z, Wu J, Leaw CP, et al.

Sci Total Environ, 2023 May 10;872:162236.
PMID: 36791857 DOI: 10.1016/j.scitotenv.2023.162236

The dinoflagellates Gambierdiscus and Fukuyoa can produce Ciguatoxins (CTXs) and Maitotoxins (MTXs) that lead to ciguatera poisoning (CP). The CP hotspots, however, do not directly relate to the occurrence of the ciguatoxic Gambierdiscus and Fukuyoa. Species-wide investigations often showed no association between CTX level and the molecular identity of the dinoflagellates. In the Pacific region, Kiribati is known as a CP hotspot, while Malaysia has only three CP outbreaks reported thus far. Although ciguatoxic strains of Gambierdiscus were isolated from both Kiribati and Malaysia, no solid evidence on the contribution of ciguatoxic strains to the incidence of CP outbreak was recorded. The present study aims to investigate the regional differences in CP risks through region-specific toxicological assessment of Gambierdiscus and Fukuyoa. A total of 19 strains of Gambierdiscus and a strain of Fukuyoa were analyzed by cytotoxicity assay of the neuro-2a cell line, hemolytic assay of fish erythrocytes, and high-resolution mass spectrometry. Gambierdiscus from both Kiribati and Malaysia showed detectable ciguatoxicity; however, the Kiribati strains were more hemolytic. Putative 44-methylgambierone was identified as part of the contributors to the hemolytic activity, and other unknown hydrophilic toxins produced can be potentially linked to higher CP incidence in Kiribati.
Ciguatera fish poisoning: First reported case in Sabah, Malaysia

Lee HG, Leaw CP, Lim PT, Jipanin SJ

Med J Malaysia, 2019 12;74(6):545-546.
PMID: 31929485

Ciguatera fish poisoning (CFP) is the most common natural marine toxin poisoning worldwide and yet under recognised in Malaysia. We report the first confirmed case of CFP in Sabah with severe neurological, cardiovascular and gastrointestinal manifestations after consumption of emperor snapper. Early recognition of CFP is important because it will result in improved patient care and public health intervention.
Clinical outcomes of children with COVID-19 infection in a low-risk centre in Malaysia

Tan MS, Zulkifli NA, Teng W, Lim PT

Med J Malaysia, 2023 Dec;78(7):853-856.
PMID: 38159917

INTRODUCTION: According to WHO, long-COVID or post- COVID-19 condition is defined as the continuation or development of new symptoms 3 months after the initial SARS-CoV-2 infection, with these symptoms lasting for at least 2 months with no other explanation. A systematic review and meta-analyses published in 2022, which mainly focus on the Western population, revealed that the prevalence of long COVID was 25.24%. Literature regarding long-COVID in children in Asia was scarce. The objectives of our study were to assess the long-term effect of COVID-19 infection in children and its correlation to their acute COVID- 19 infection.
MATERIALS AND METHODS: This study was conducted in Hospital Kepala Batas (HKB), a district hospital in Penang State, Malaysia, which was the designated regional COVID hospital during the pandemic. It was a retrospective observational study, where children who were admitted from November 2020 to March 2021, and attended follow-up clinics from Jan 2021 to May 2021, were recruited.
RESULTS: This study comprised 90 subjects, from 3 months old to 12 years old, mean of 6.5 years old. When comparing asymptomatic and symptomatic children, children with comorbidities were more likely to be symptomatic with a pvalue of 0.045 using the Pearson Chi-square test. All our patients' symptoms resolved upon discharge. During followup at 2-4 months after COVID-19 infection, all children were reported as back to their usual selves. Fifteen patients had recurrent symptoms. Most of their symptoms pointed towards an acute infection. One patient had two episodes of illness, while the rest had one. The most common symptoms were cough, fever and runny nose. The average duration of illness of these 16 episodes was 4.5 days with a standard deviation of 2.48. None of these symptoms lasted more than seven days. None of them required hospital admission. None of them had recurrent COVID-19 infections. Tweleve out of 72 children who had been going to school stopped physical school after COVID-19 infection. Our findings differed from other studies. These could be due to the limitations that we faced.
CONCLUSION: Most children who contracted COVID-19 infection recovered fully after acute infection, and most of them recovered fully without long-term sequelae.
Fulltext Effects of substratum and depth on benthic harmful dinoflagellate assemblages

Lee LK, Lim ZF, Gu H, Chan LL, Litaker RW, Tester PA, et al.

Sci Rep, 2020 07 09;10(1):11251.
PMID: 32647125 DOI: 10.1038/s41598-020-68136-6

Microhabitats influence the distribution and abundance of benthic harmful dinoflagellate (BHAB) species. Currently, much of the information on the relationships between BHABs and microhabitat preferences is based on non-quantitative anecdotal observations, many of which are contradictory. The goal of this study was to better quantify BHAB and microhabitat relationships using a statistically rigorous approach. Between April 2016 to May 2017, a total of 243 artificial substrate samplers were deployed at five locations in the Perhentian Islands, Malaysia while simultaneous photo-quadrat surveys were performed to characterize the benthic substrates present at each sampling site. The screen samplers were retrieved 24 h later and the abundances of five BHAB genera, Gambierdiscus, Ostreopsis, Coolia, Amphidinium, and Prorocentrum were determined. Substrate data were then analyzed using a Bray-Curtis dissimilarity matrix to statistically identify distinct microhabitat types. Although BHABs were associated with a variety of biotic and abiotic substrates, the results of this study demonstrated differing degrees of microhabitat preference. Analysis of the survey results using canonical correspondence analysis explained 70.5% (horizontal first axis) and 21.6% (vertical second axis) of the constrained variation in the distribution of various genera among microhabitat types. Prorocentrum and Coolia appear to have the greatest range being broadly distributed among a wide variety of microhabitats. Amphidinium was always found in low abundances and was widely distributed among microhabitats dominated by hard coral, turf algae, sand and silt, and fleshy algae and reached the highest abundances there. Gambierdiscus and Ostreopsis had more restricted distributions. Gambierdiscus were found preferentially associated with turf algae, hard coral and, to a lesser extent, fleshy macroalgae microhabitats. Ostreopsis, almost always more abundant than Gambierdiscus, preferred the same microhabitats as Gambierdiscus and were found in microbial mats as well. With similar habitat preferences Ostreopsis may serve as an indicator organism for the presence of Gambierdiscus. This study provides insight into how BHAB-specific microhabitat preferences can affect toxicity risks.
Fulltext Sexual reproduction and genetic polymorphism within the cosmopolitan marine diatom Pseudo-nitzschia pungens

Kim JH, Ajani P, Murray SA, Kim JH, Lim HC, Teng ST, et al.

Sci Rep, 2020 06 30;10(1):10653.
PMID: 32606343 DOI: 10.1038/s41598-020-67547-9

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.
Fulltext Wind-driven development and transport of Gymnodinium catenatum blooms along the coast of Fujian, China

Zhang C, Lim PT, Li X, Gu H, Li X, Anderson DM

Reg Stud Mar Sci, 2020 Sep;39.
PMID: 33241099 DOI: 10.1016/j.rsma.2020.101397

Gymnodinium catenatum is a cosmopolitan, bloom-forming dinoflagellate known to produce a suite of potent paralytic shellfish poisoning (PSP) toxins. Here, we revisit two major blooms of G. catenatum along the Fujianese Coast, China, in 2017 and 2018. The impact area of the 2017 bloom was larger than that of the 2018 event. Field sampling and remote satellite sensing revealed that alongshore transport driven by the southwest wind, as well as physical accumulation driven by the northeast wind, played important roles in the development and distribution of the two bloom events. The relationship between wind-induced hydrodynamic conditions and the unprecedented HAB events established in this study adds greatly to our understanding of algal bloom dynamics along the Fujianese coast. These results improve our ability to detect, track, and forecast G. catenatum blooms, thereby potentially minimizing the negative impacts of future HAB events.
Blooms of diatom and dinoflagellate associated with nutrient imbalance driven by cycling of nitrogen and phosphorus in anaerobic sediments in Johor Strait (Malaysia)

Chai X, Li X, Hii KS, Zhang Q, Deng Q, Wan L, et al.

Mar Environ Res, 2021 Jul;169:105398.
PMID: 34171592 DOI: 10.1016/j.marenvres.2021.105398

Coastal eutrophication is one of the pivotal factors driving occurrence of harmful algal blooms (HABs), whose underlying mechanism remained unclear. To better understand the nutrient regime triggering HABs and their formation process, the phytoplankton composition and its response to varying nitrogen (N) and phosphorus (P), physio-chemical parameters in water and sediment in Johor Strait in March 2019 were analyzed. Surface and sub-surface HABs were observed with the main causative species of Skeletonema, Chaetoceros and Karlodinium. The ecophysiological responses of Skeletonema to the low ambient N/P ratio such as secreting alkaline phosphatase, regulating cell morphology (volume; surface area/volume ratio) might play an important role in dominating the community. Anaerobic sediment iron-bound P release and simultaneous N removal by denitrification and anammox, shaped the stoichiometry of N and P in water column. The decrease of N/P ratio might shift the phytoplankton community into the dominance of HABs causative diatoms and dinoflagellates.
Disturbance of mangrove forests causes alterations in estuarine phytoplankton community structure in Malaysian Matang mangrove forests

Hilaluddin F, Yusoff FM, Natrah FMI, Lim PT

Mar Environ Res, 2020 Jun;158:104935.
PMID: 32217292 DOI: 10.1016/j.marenvres.2020.104935

To assess the effects of environmental changes on phytoplankton community structure in a mangrove ecosystem, phytoplankton distribution in Matang mangrove, Malaysia was examined. Phytoplankton and water samples, and in situ environmental parameters from three estuaries with differing levels of disturbance were examined monthly for one year. Two species, Cyclotella choctawhatcheeana and Skeletonema costatum, were dominant in the least disturbed and moderately disturbed areas, respectively. Skeletonema costatum was also the most dominant in the most disturbed area. Significant differences in phytoplankton density and biodiversity between the least and most disturbed areas were also observed. Principle component 1 (salinity, conductivity, total solids/water transparency and nitrogenous compounds) and PC2 (dissolved oxygen, pH and temperature) explained 60.4% of the total variance. This study illustrated that changes in phytoplankton community structure in Matang mangrove estuaries were significantly correlated with environmental parameters which were in turn influenced by ecosystem disturbance levels as well as seasonal changes.
Three novel species in the Pseudo-nitzschia pseudodelicatissima complex: P. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov. (Bacillariophyceae) from the Strait of Malacca, Malaysia

Lim HC, Teng ST, Leaw CP, Lim PT

J Phycol, 2013 Oct;49(5):902-16.
PMID: 27007315 DOI: 10.1111/jpy.12101

A study on the morphology and phylogeny of 18 strains of Pseudo-nitzschia established from the Strait of Malacca, Peninsular Malaysia, was undertaken. Morphological data combined with molecular evidence show that they constitute three new species, for which the names, P. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov., are proposed. The three new species closely resemble species in the P. pseudodelicatissima complex sensu lato. Morphologically, P. batesiana differs from other species in the complex by having a smaller part of cell overlapping in the chain, whereas P. lundholmiae differs by having fewer poroid sectors and P. fukuyoi by having a distinct type of poroid sectors. Nucleotide sequences of the LSU rDNA (D1-D3) of the three new species reveal significant nucleotide sequence divergence (0.1%-9.3%) from each other and from other species in the P. pseudodelicatissima complex s.l. The three species are phylogenetically closely related to species in the P. pseudodelicatissima complex, with P. batesiana appearing as a sister taxon to P. circumpora, P. caciantha, and P. subpacifica; whereas P. lundholmiae and P. fukuyoi are more closely related to P. pseudodelicatissima and P. cuspidata. The three species show 2-3 compensatory base changes (CBCs) in their ITS2 transcripts when compared to the closely related species. The ITS2 with its structural information has proven its robustness in constructing a better resolved phylogenetic framework for Pseudo-nitzschia.
MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) FROM MALAYSIAN BORNEO, INCLUDING THE NEW SPECIES PSEUDO-NITZSCHIA CIRCUMPORA SP. NOV

Lim HC, Leaw CP, Su SN, Teng ST, Usup G, Mohammad-Noor N, et al.

J Phycol, 2012 Oct;48(5):1232-47.
PMID: 27011282 DOI: 10.1111/j.1529-8817.2012.01213.x

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.
A non-toxigenic but morphologically and phylogenetically distinct new species of Pseudo-nitzschia, P. sabit sp. nov. (Bacillariophyceae)

Teng ST, Lim PT, Lim HC, Rivera-Vilarelle M, Quijano-Scheggia S, Takata Y, et al.

J Phycol, 2015 Aug;51(4):706-25.
PMID: 26986792 DOI: 10.1111/jpy.12313

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.
Pseudo-nitzschia nanaoensis sp. nov. (Bacillariophyceae) from the Chinese coast of the South China Sea

Li Y, Dong HC, Teng ST, Bates SS, Lim PT

J Phycol, 2018 12;54(6):918-922.
PMID: 30270437 DOI: 10.1111/jpy.12791

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.
Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum

Luo Z, Hu Z, Tang Y, Mertens KN, Leaw CP, Lim PT, et al.

J Phycol, 2018 10;54(5):744-761.
PMID: 30144373 DOI: 10.1111/jpy.12780

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.
Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution

Lim HC, Tan SN, Teng ST, Lundholm N, Orive E, David H, et al.

J Phycol, 2018 04;54(2):234-248.
PMID: 29377161 DOI: 10.1111/jpy.12620

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.

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