Gires Usup

Alexandrium(Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastalwaters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

Abstract: The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species.

EFFECTS OF LIGHT AND TEMPERATURE ON GROWTH, NITRATE UPTAKE, AND TOXIN PRODUCTION OF TWO TROPICAL DINOFLAGELLATES: ALEXANDRIUM TAMIYAVANICHII AND ALEXANDRIUM MINUTUM (DINOPHYCEAE)†

The two tropical estuarine dinoflagellates, Alexandrium tamiyavanichii Balech and A. minutum Halim, were used to determine the ecophysiological adaptations in relation to their temperate counterparts. These species are the two main causative organisms responsible for the incidence of paralytic shellfish poisoning (PSP) in Southeast Asia. The effects of light (10, 40, 60, and 100 μmol photons·m−2·s−1) and temperature (15, 20, and 25°C) on the growth, nitrate assimilation, and PST production of these species were investigated in clonal batch cultures over the growth cycle. The growth rates of A. tamiyavanichii and A. minutum increased with increasing temperature and irradiance. The growth of A. tamiyavanichii was depressed at lower temperature (20°C) and irradiance (40 μmol photons·m−2·s−1). Both species showed no net growth at 10 μmol photons·m−2·s−1 and a temperature of 15°C, although cells remained alive. Cellular toxin quotas (Qt) of A. tamiyavanichii and A. minutum varied in the range of 60–180 and 10–42 fmol PST·cell−1, respectively. Toxin production rate, Rtox, increased with elevated light at both 20 and 25°C, with a pronounced effect observed at exponential phase in both species (A. tamiyavanichii, r2=0.95; A. minutum, r2=0.96). Toxin production rate also increased significantly with elevated temperature (P<0.05) for both species examined. We suggest that the ecotypic variations in growth adaptations and toxin production of these Malaysian strains may reveal a unique physiological adaptation of tropical Alexandrium species.

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.

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.

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.

Marked Differences in Fatty Acid Profiles of Some Planktonic and Benthic Marine Dinoflagellates from Malaysian Waters

G. Usup, S.Z. Hamid, P.K. Chiet, C.K. Wah and A. Ahmad. 2007. Marked differences in fatty acid profiles of some planktonic and benthic marine dinoflagellates from Malaysian waters. Phycologia 47: 105–111. DOI: 10.2216/07-55.1 This study was carried out to characterize the fatty acid profiles of some planktonic and benthic marine dinoflagellates from Malaysian waters. Clonal batch cultures of Alexandrium affine, A. leei, A. minutm, A. tamarense, A. tamiyavanichii, Coolia monotis, Prorocentrum emarginatum, P. mexicanum, Ostreopsis ovata and Amphidinium sp. were harvested at late exponential phase, and total lipid was extracted. Samples were derivatized to produce fatty acid methyl esters (FAMEs). FAMEs were analyzed on a gas chromatograph with flame ionization detection. The total number of fatty acids detected in the clones ranged from 10 in the A. tamarense AtPA04 clone to 22 in the C. monotis CmPL01 clone. Fatty acids found in all clones were myristic acid (14 : 0), palmitic acid (16 : 0), stearic acid (18 : 0), linoleic acid (18 : 2ω6c) and oleic acid (18 : 1ω9c). In all clones only a few fatty acids were dominant. In the Alexandrium clones the dominant fatty acids were 16 : 0, 18 : 0, cis-13,16-docosadienoic acid (22 : 2), 18 : 2ω6c and 18 : 1ω9c. There was almost complete absence of omega-3 polyunsaturated fatty acids (PUFA) in the Alexandrium clones. In the benthic species the major fatty acids were 16 : 0, eicosapentaenoic acid (EPA, 20 : 5ω3), docosahexaenoic acid (22 : 6ω3), 18 : 2ω6c and 18 : 1ω9c. In the Prorocentrum clones the major fatty acids were 14 : 0, 16 : 0, palmitoleic acid (16 : 1) and EPA. Total PUFA content in the benthic species were 37%–56%, while in the planktonic species the content was 19%–44%. The fatty acid profiles could not differentiate between species. However, cluster analysis and principal components analysis were able to clearly discriminate between the Alexandrium group, Prorocentrum group and benthic species group.

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.