Chui Pin Leaw

Phylogenetic relationships, morphological variation, and toxin patterns in the Alexandrium ostenfeldii (Dinophyceae) complex: implications for species boundaries and identities

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.

Phylogenetic analysis of Alexandrium species and Pyrodinium bahamense (Dinophyceae) based on theca morphology and nuclear ribosomal gene sequence

C.P. Leaw, P.T. Lim, B.K. Ng, M.Y. Cheah, A. Ahmad and G. Usup. 2005. Phylogenetic analysis of Alexandrium species and Pyrodinium bahamense (Dinophyceae) based on theca morphology and nuclear ribosomal gene sequence. Phycologia 44: 550–565. A phylogenetic analysis of Alexandrium species and Pyrodinium bahamense was carried out. The analysis was based on nucleotide sequences of the large subunit ribosomal RNA gene and 16 morphological characters considered taxonomically informative. Maximum likelihood, maximum parsimony and Bayesian approaches were used. Molecular and morphological data were analysed independently and in combination. The outcomes of all the analyses were the same. Pyrodinium was consistently grouped in the same clade with Alexandrium, specifically with the subgenus Gessnerium, A. pseudogoniaulax and A. taylori. Two monophyletic clades were resolved. The first comprised A. tamarense, A. fundyense, A. catenella, A. tamiyavanichii, A. affine and A. concavum, with the base formed by A. pseudogoniaulax, A. taylori and P. bahamense. The second clade comprised the species A. minutum, A. insuetum, A. tamutum, A. andersoni, A. ostenfeldii and A. leei, with A. margalefi forming the base. Mapping of morphological characters onto the phylogenetic trees indicated that posterior sulcal plate probably has the highest value in the taxonomy of Alexandrium. Some other characters considered taxonomically important, such as the ventral pore and position of the anterior attachment pore, are most probably homoplastic.

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.

MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF A NEW SPECIES OF THECATE BENTHIC DINOFLAGELLATE, COOLIA MALAYENSIS SP. NOV. (DINOPHYCEAE)1

Coolia Meunier is an important component of benthic dinoflagellate assemblages in tropical and subtropical seas. In this study, detailed morphological observation of Coolia species from Malaysian waters was carried out using light and electron microscopy in parallel with molecular characterization of nuclear‐encoded partial LSU rDNA, and internal transcribed spacer (ITS) regions. Live specimens were collected from seaweed samples and established into clonal cultures. There are significant morphological variations between the Malaysian isolates in comparison to the type species, C. monotis Meunier. The feature that differentiates the new species is the third postcingular plate (3′′′), which is the largest hypothecal plate in the Malaysian isolates, whereas in C. monotis, the 3′′′ and 4′′′ plates are almost equal in size. Detailed observations of the thecal pores also revealed the presence of fine perforations within the pores of the Malaysian isolates, but these perforations are absent in C. monotis. Comparisons between Malaysian isolates and C. monotis nucleotide sequence of the ITS region showed high genetic divergence at 28%, in contrast to the 0.3%–3% divergence observed among populations of the same species. Structural comparison of the second internal transcribed spacer (ITS2) rRNA transcript between the two species showed compensatory base changes (CBCs) in the three helices of ITS2 rRNA. Based on morphological and molecular data, the Malaysian isolates are considered to represent a new species, for which the name Coolia malayensis is proposed.

Growth and toxin production of tropical Alexandrium minutum Halim (Dinophyceae) under various nitrogen to phosphorus ratios

Effects of nitrogen to phosphorous (N/P) ratios of two nitrogen sources (nitrate and ammonium) on growth and toxin production of a tropical estuarine dinoflagellate, Alexandrium minutum Halim, were examined using a strain isolated from a bloom at Tumpat Estuary, Malaysia in September 2001. Experiments were carried out in batch cultures, using either nitrate (N-NO3) or ammonium (N-NH4) as the nitrogen source at a constant amount, and with initial N/P ratios ranging from 5 to 500. Cell density, residual N and P in the medium, cellular toxin quota (Qt), and toxin composition were analyzed throughout the growths. Our results showed that cell densities and growth rates of A. minutum were severely suppressed under high N/P ratios (>100) in both N-NO3 and N-NH4 treatments. Cells tended to be larger at lower growth rate and P-limited cultures. Toxin profile was relatively constant throughout the experiments, with GTX4/GTX1 as the dominant toxin congeners. Cellular toxin quota (Qt) increased with elevated N/P ratios in both N-NO3 and N-NH4 treatments. Toxin production rate, Rtox, however was enhanced in N-NH4-grown cultures when P was limited, but showed no difference between N-NO3- and N-NH4-grown cultures when P was replete. Our results clearly showed that N/P ratios as well as the nitrogen compounds not only affected the growth of A. minutum, but also the cellular toxin quota and its toxin production rate.

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.

Genetic diversity of Pseudo-nitzschia brasiliana (Bacillariophyceae) from Malaysia

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.

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.