Hong Chang Lim

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

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.

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)

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.

High diversity of Pseudo-nitzschia along the northern coast of Sarawak (Malaysian Borneo), with descriptions of P. bipertita sp. nov. and P. limii sp. nov. (Bacillariophyceae).

Forty‐eight isolates of Pseudo‐nitzschia species were established from the Miri coast of Sarawak (Malaysian Borneo) and underwent TEM observation and molecular characterization. Ten species were found: P. abrensis, P. batesiana, P. fukuyoi, P. kodamae, P. lundholmiae, P. multistriata, P. pungens, P. subfraudulenta, as well as two additional new morphotypes, herein designated as P. bipertita sp. nov. and P. limii sp. nov. This is the first report of P. abrensis, P. batesiana, P. kodamae, P. fukuyoi, and P. lundholmiae in coastal waters of Malaysian Borneo. Pseudo‐nitzschia bipertita differs from its congeners by the number of sectors that divide the poroids, densities of band striae, and its cingular band structure. Pseudo‐nitzschia limii, a pseudo‐cryptic species in the P. pseudodelicatissima complex sensu lato, is distinct by having wider proximal and distal mantles, a higher number of striae, and greater poroid height in the striae of the valvocopula. The species were further supported by the phylogenetic reconstructions of the nuclear‐encoded large subunit ribosomal gene and the second internal transcribed spacer. Phylogenetically, P. bipertita clustered with its sister taxa (P. subpacifica + P. heimii); P. limii appears as a sister taxon to P. kodamae and P. hasleana in the ITS2 tree. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of both hemi‐ and compensatory base changes. Toxicity analysis showed detectable levels of domoic acid in P. abrensis, P. batesiana, P. lundholmiae, and P. subfraudulenta, but both new species tested below the detection limit.

New scenario for speciation in the benthic dinoflagellate genus Coolia (Dinophyceae)

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.

18S rDNA phylogeny of Pseudo-nitzschia (Bacillariophyceae) inferred from sequence-structure information

Abstract: We explored the 18S rDNA sequences of Pseudo-nitzschia and its close relatives, together with their individual secondary structure information to reconstruct phylogenies. Forty-eight taxa classified in the Bacillariaceae and Achnanthaceae were analyzed by character-based (maximum parsimony and maximum likelihood) and distance-based (neighbour-joining) methods. Three raphid diatoms from the Eunotiaceae were used as out-groups. The sequence-structure alignment was generated by 4SALE (i.e. a one-letter encoded alignment that contained the individual secondary structure information) and applied to the character-based phylogeny reconstruction. All analyses highly support Pseudo-nitzschia + Fragilariopsis as monophyletic. The Pseudo-nitzschia/Fragilariopsis clade is further divided into three subclades. One clade comprises members in the P. seriata and P. americana complexes and species of Fragilariopsis; the second and third clades constitute members in P. pseudodelicatissima and P. delicatissima complexes. Among all analyses, the distance-based method yielded a more highly resolved phylogenetic framework for the Pseudo-nitzschia/Fragilariopsis clade than did the character-based analyses. Other than those currently known genetic markers, 18S rDNA was also useful in exploring the intrageneric relationships.

Diatom Nitzschia navis-varingica (Bacillariophyceae) and its domoic acid production from the mangrove environments of Malaysia

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 1μm (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 11.06pgcell-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.

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

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.

Quantitative real-time PCR detection of a harmful unarmoured dinoflagellate, Karlodinium australe (Dinophyceae): qPCR detection of Karlodinium australe

We investigated a harmful algal bloom (HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green‐based real‐time PCR (qPCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species‐specific qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species specificity of the primers designed was evaluated by screening on the non‐target species (Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross‐detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 ± 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R2 = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single‐cell PCR and rDNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose.