DISTRIBUTION AND GENETIC DIVERSITY OF THE LUCIFERASE GENE WITHIN MARINE DINOFLAGELLATES1

Dinoflagellates are the most abundant protists that produce bioluminescence. Currently, there is an incomplete knowledge of the identity of bioluminescent species arising from inter‐ and intraspecific variability in bioluminescence properties. In this study, PCR primers were designed to amplify the dinoflagellate luciferase gene (lcf) from genetically distant bioluminescent species. One of the primer pairs was “universal,” whereas others amplified longer gene sequences from subsets of taxa. The primers were used to study the distribution of lcf and assess bioluminescence potential in dinoflagellate strains representing a wide variety of taxa as well as multiple strains of selected species. Strains of normally bioluminescent species always contained lcf even when they were found not to produce light, thus demonstrating the utility of this methodology as a powerful tool for identifying bioluminescent species. Bioluminescence and lcf were confined to the Gonyaulacales, Noctilucales, and Peridiniales. Considerable variation was observed among genera, or even species within some genera, that contained this gene. Partial sequences of lcf were obtained for the genera Ceratocorys, Ceratium, Fragilidium, and Protoperidinium as well as from previously untested species or gene regions of Alexandrium and Gonyaulax. The sequences revealed high variation among gene copies that obscured the boundaries between species or even genera, some of which could be explained by the presence of two genetic variants within the same species of Alexandrium. Highly divergent sequences within Alexandrium and Ceratium show a more diverse composition of lcf than previously known.     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). I. RFLP ANALYSIS OF SSU rRNA GENES1

Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni. The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.     

DEVELOPMENT OF SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS FROM INTERSIMPLE SEQUENCE REPEAT FINGERPRINTS FOR THE MOLECULAR DETECTION OF TOXIC PHYTOPLANKTON ALEXANDRIUM CATENELLA (DINOPHYCEAE) AND PSEUDO‐NITZSCHIA PSEUDODELICATISSIMA (BACILLARIOPHYCEAE) FROM FRENCH COASTAL WATERS1

Harmful algal blooms are a serious threat to shellfish farming and human health all over the world. The monitoring of harmful algae in coastal waters originally involved morphological identification through microscopic examinations, which was often difficult unless performed by specialists and even then often did not permit identification of toxic species. More recently, specific molecular markers have been used to identify specific phytoplankton species or strains. Here we report on the use of the intersimple sequence repeat (ISSR) technique to develop specific sequence characterized amplified region markers (SCAR) and to identify with these tools two toxic species in French coastal waters, the diatom Pseudo‐nitzschia pseudodelicatissima (Hasle) Hasle and the dinoflagellate Alexandrium catenella (Whedon and Kofoid 1936), Balech 1985. Six polymorphic ISSR regions were selected among amplified fingerprints of a representative sample of phytoplankton species. After cloning and sequencing the selected polymorphic ISSR regions, pairs of internal primers were designed to amplify a unique and specific sequence designed as a SCAR marker. Of the six selected SCAR markers, three were specific to P. pseudodelicatissima and one for A. catenella. The SCAR marker specificity was confirmed by using basic local alignment search tool comparison, by experimental assays on different strains from 11 countries, and by checking that the sequence amplified was the expected one. When tested on water samples collected along the French shores, the four specific SCAR markers proved to be efficient tools for fast and low‐cost detection of toxic phytoplankton species.     

DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR SIMULTANEOUS DETECTION OF SIX ALEXANDRIUM SPECIES (DINOPHYCEAE)1

In Japan, the bloom seasons of two toxic species, namely, Alexandrium catenella (Whedon et Kof.) Balech and Alexandrium tamiyavanichii Balech, sometimes overlap with those of three nontoxic Alexandrium species, namely, Alexandrium affine (H. Inouye et Fukuyo) Balech, Alexandrium fraterculus (Balech) Balech, and Alexandrium pseudogoniaulax (Biecheler) T. Horig. ex Y. Kita et Fukuyo. In this study, a multiplex PCR assay has been developed that enables simultaneous detection of six Alexandrium species on the basis of differences in the lengths of the PCR products. The accuracy of the multiplex PCR system was assessed using 101 DNA templates of the six target Alexandrium species and 27 DNA templates of 11 nontarget species (128 DNA templates in total). All amplicons obtained from the 101 DNA templates of the target species were appropriately identified, whereas all 27 DNA templates of the nontarget species were not amplified. Species‐specific identification by the multiplex PCR assay was certainly possible from single cells of the target species.     

Diversity of the Luciferin Binding Protein Gene in Bioluminescent Dinoflagellates – Insights from a New Gene in Noctiluca scintillans and Sequences from Gonyaulacoid Genera

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re‐organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within‐strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems.     

BIODIVERSITY OF PLANKTONIC DINOFLAGELLATE SPECIES IN MANGROVE PONDS,PELICAN CAYS,BELIZE

Information on the population structure of planktonic dinoflagellates is reported in the coral reef‐mangrove ecosystem at Pelican Cays, Belize. Six sites examined included: Cat Cay, Douglas Cay, Elbow Cay, Fisherman's Cay, Lagoon Cay and Manatee Cay. A spectacular and rich dinoflagellate taxa including oceanic, coastal and offshore species are illustrated. The presence of oceanic species in the studied cays is an unexpected observation since dinolfagellate assemblages are virtually enclosed within ponds bordered by coral ridges that limits water exchange with the open ocean except during storm events. I am also reporting significant differences in the dinoflagellate associations among the studied cays. Dominant taxa included 16 Proroperidinium species, 11 Gonyaulax species, and ten Ceratium species. Only six planktonic species were harmful. Bloom forming species included Ceratium furca and Gonyaulax polygramma. A much more diverse authotrophic and heterotrophic dinoflagellate population characterizes the Pelican Cays than previously suspected. Some species are reported the first time: Protoperidinium belizeanum sp. nov., P. pyrum Balech, P. steidingerae Balech, P. depressum (Bailey) Balech, and P. divergens (Ehrenberg) Balech. These results demonstrate that the Belizean coral reef‐mangrove ecosystem is a delicate and species‐rich environment, and as such, should be protected and preserved.     

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Nei's genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fisher's combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.     

Molecular phylogenetic study of the heterotrophic dinoflagellate genus Protoperidinium (Dinophyceae) inferred from small subunit rRNA gene sequences

In the present study, we investigated the intrageneric and intergeneric phylogenetic relationships of the heterotrophic marine dinoflagellate genus Protoperidinium. Using single‐cell polymerase chain reaction methods, we determined small subunit ribosomal RNA gene sequences for 10 Protoperidinium species belonging to four sections and two subgenera. Phylogenetic trees were constructed using maximum parsimony, neighbor joining and maximum likelihood methods. We found intraspecific variability of small subunit rDNA sequences in Protoperidinium conicum (Gran) Balech, Protoperidinium crassipes (Kofoid) Balech and Protoperidinium denticulatum (Gran et Braarud) Balech, but not in other species. The small subunit rDNA phylogeny revealed that the genus is monophyletic, but its phylogenetic position within the Dinophyceae could not be determined because of ambiguous basal topologies. Within the genus Protoperidinium, species of the subgenus Archaeperidinium with two anterior intercalary plates (2a) were shown to be monophyletic, but species of the subgenus Protoperidinium with three anterior intercalary plates (3a) were resolved as paraphyletic. The sections Avellana, Divergentia and Protoperidinium were shown to be monophyletic, while the section Conica was paraphyletic. Based on the trees obtained in the present study, most of the traditionally defined sections are supported by molecular phylogeny. It was also indicated that the section Avellana evolved from one of the Conica‐type dinoflagellates.     

IDENTIFICATION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING PCR AND rDNA-TARGETED PROBES

A PCR (polymerase chain reaction)-based assay for the detection of Alexandrium species in cultured samples using rDNA-targeted probes was developed. The internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S ribosomal RNA gene (rDNA) from cultured isolates of A. tamarense (Lebour) Taylor, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech and A. lusitanicum Balech were amplified using PCR and sequenced. Sequence comparisons showed that the 5.8S and ITS1-ITS2 regions contain sequences specific for the Alexandrium genus, especially at the 3' end of the 5.8S coding region. PCR primers and a radioactive ³²P-labeled DNA probe were devised for this region. The cross-reactivity of the PCR primers and probe was tested against cultured isolates of Alexandrium and other dinoflagellates and diatoms. All the Alexandrium isolates screened reacted toward the genus-specific probe; in contrast, the other groups of microalgae (dinoflagellates and diatoms) did not react with the probe. Furthermore, the PCR amplification technique combined with the use of the rDNA-target probe allowed us to develop a method for the detection of Alexandrium cells in cultured samples. This PCR method might offer a new approach for the identification and enumeration of the HAB (harmful algal bloom) species present in natural phytoplankton populations.     

DESCRIPTION OF THECADINIUM MUCOSUM SP. NOV. (DINOPHYCEAE), A NEW SAND‐DWELLING MARINE DINOFLAGELLATE,AND AN EMENDED DESCRIPTION OF THECADINIUM INCLINATUM BALECH1

A new thecate, phototrophic, marine, sand‐dwelling dinoflagellate, Thecadinium mucosum Hoppenrath et Taylor sp. nov., is described from a culture isolated from Boundary Bay, British Columbia, Canada. It was illustrated with LM as well as SEM and TEM, and its position in the phylogenetic tree of dinoflagellates was investigated using molecular methods. Cells are asymmetrical, oval, laterally flattened, and strongly pigmented, with the plate formula P 3′ 1a 6′′ 7/8c 5 s 6′′′ 2′′′′. Thecal plates are smooth with scattered pores, and there is a distinctive anterior intercalary plate that could be involved in mucus secretion. Thecadinium inclinatum Balech (=Sabulodinium inclinatum (Balech) Saunders et Dodge), a thecate, marine, sand‐dwelling species that has been previously confused with what we now call T. mucosum, was also examined and illustrated through LM and SEM. New information on T. inclinatum is provided, including its plate formula P 3′ 6′′ 7c ?s 5′′′ 1p 1′′′′; we consider T. inclinatum to be related to most other Thecadinium species and not to Sabulodinium. Molecular phylogenetic analyses based on the small subunit ribosomal gene of T. mucosum, T. kofoidii (the type species of the genus), and T. dragescoi weakly support earlier suspicions based on morphology that T. dragescoi is not a member of Thecadinium. Tabulational patterns of the species suggest a relationship to the genus Amphidiniopsis.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.     

SEASONAL VARIABILITY OF THE ORGANIC‐WALLED DINOFLAGELLATE CYST PRODUCTION IN THE COASTAL UPWELLING REGION OFF CAPE BLANC (MAURITANIA): A FIVE‐YEAR SURVEY1

A 5‐year sediment trap survey in the upwelling area off Cape Blanc (NW Africa) provides information on the seasonal and annual resting cyst production of dinoflagellates, their sinking characteristics and preservation potential. Strong annual variation in cyst production characterizes the region. Cyst production of generally all investigated species, including Alexandrium pseudogonyaulax (Biecheler) T. Horig. ex T. Kita et Fukuyo (cyst genus Impagidinium) and Gonyaulax spinifera (Clap. et J. Lachm.) Diesing (cyst genus Nematosphaeropsis) was enhanced with increasing upper water nutrient and trace‐element concentrations. Cyst production of Lingulodinium polyedrum (F. Stein) J. D. Dodge was the highest at the transition between upwelling and upwelling‐relaxation. Cyst production of Protoperidinium americanum (Gran et Braarud) Balech, Protoperidinium monospinum (Paulsen) K. A. F. Zonn. et B. Dale, and Protoperidinium stellatum (D. Wall) Balech, and heterotrophic dinoflagellates forming Brigantedinium spp. and Echinidinium aculeatum Zonn., increased most pronouncedly during upwelling episodes. Production of Protoperidinium conicum (Gran) Balech and Protoperidinium pentagonum (Gran) Balech cysts and total diatom valves were related, providing evidence of a predator–prey relationship. The export cyst‐flux of E. aculeatum, P. americanum, P. monospinum, and P. stellatum was strongly linked to the flux of total diatom valves and CaCO₃, whereas the export production of Echinidinium granulatum Zonn. and Protoperidinium subinerme (Paulsen) A. R. Loebl. correlated with total organic carbon, suggesting potential consumption of diatoms, prymnesiophytes, and organic matter, respectively. Sinking velocities were at least 274 m · d^?1, which is in range of the diatom‐ and coccolith‐based phytoplankton aggregates and “slower” fecal pellets. Species‐selective degradation did not occur in the water column, but on the ocean floor.     

Characterization of mycosporine‐serine‐glycine methyl ester,a major mycosporine‐like amino acid from dinoflagellates: a mass spectrometry study

Several unknown mycosporine‐like amino acids (MAAs) have been previously isolated from some cultured species of toxic dinoflagellates of the Alexandrium genus (Dinophyceae). One of them, originally called M‐333, was tentatively identified as a shinorine methyl ester, but the precise nature of this compound is still unknown. Using a high‐resolution reversed‐phase liquid chromatography mass spectrometry analyses (HPLC/MS), we found that natural populations of the red tide dinoflagellate Prorocentrum micans Ehrenberg showed a net dominance of M‐333 together with lesser amounts of other MAAs. We also documented the isolation and characterization of this MAA from natural dinoflagellate populations and from Alexandrium tamarense (Lebour) Balech cultures. Using a comparative fragmentation study in electrospray mass spectrometry between deuterated and non‐deuterated M‐333 compounds and synthesized mono and dimethyl esters of shinorine, this novel compound was characterized as mycosporine‐serine‐glycine methyl ester, a structure confirmed by nuclear magnetic resonance. These isobaric compounds can be differentiated by their fragmentation patterns in MS³ experiments because the extension and the specific site of the methylation changed the fragmentation pathway.     

INTER‐ AND INTRASPECIFIC COMMUNITY STRUCTURE WITHIN THE DIATOM GENUS PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE)1

Pseudo‐nitzschia‐specific PCR primers (PnAll F/R) were designed to amplify a polymorphic region of the internal transcribed spacer 1 (ITS1) from at least 11 Pseudo‐nitzschia species. The primers were used to generate environmental clone libraries from Puget Sound, Washington, and Vancouver Island, British Columbia, to confirm that the primers were specific for Pseudo‐nitzschia and to determine the extent of ITS1 sequence diversity within individual species. All environmental ITS1 sequences generated with PnAll primers displayed the greatest similarity to known Pseudo‐nitzschia ITS1 sequences. The length of cloned ITS1 fragments differed among species but was conserved within a species. Intraspecific genotypes exhibited <3% sequence divergence for seven of the 10 species detected in clone libraries. Several ITS1 genotypes unique to the Pacific Northwest were identified in environmental samples, and other genotypes were more broadly distributed. The Pseudo‐nitzschia primers were also used to develop an automated ribosomal intergenic spacer analysis (ARISA) to rapidly identify Pseudo‐nitzschia species in environmental samples based on species‐specific variation in the length of the targeted ITS1 region. The ARISA peaks were then associated with the environmental clone sequences for Pseudo‐nitzschia species. Surveying the genetic composition of communities at both the inter‐ and intraspecific levels will enhance our understanding of Pseudo‐nitzschia bloom dynamics.     

COMPARATIVE STUDY OF LUMINESCENT AND NONLUMINESCENT STRAINS OF GONYAULAX EXCAVATA (PYRRHOPHYTA)1,2

Three of ten cultures of Gonyaulax excavata (Braarud) Balech isolated from the 1972 New England red tide are nonluminescent, Biochemical components of dinoflagellate bioluminescence were not detected in the extracts from these three isolates. Cells of the nonluminescent cultures were identical to those of luminescent cultures as compared by light microscopy, major body plate tabulation, cell size and growth. Both luminescent and nonluminescent cells were toxic as determined by using the mouse bioassay for paralytic shellfish poisoning. All the 122 clones made from one of the luminescent isolates were luminescent suggesting this feature is a stable trait. We conclude that these isolates represent luminescent and luminescent strains of G. excavata. This is the first intraspecific investigation of in vitro bioluminescent components between nonluminescent and luminescent strains of a dinoflagellate.     

Diversity of luciferase sequences and bioluminescence production in Baltic Sea Alexandrium ostenfeldii

The toxic dinoflagellate Alexandrium ostenfeldii is the only bioluminescent bloom-forming phytoplankton in coastal waters of the Baltic Sea. We analysed partial luciferase gene (lcf) sequences and bioluminescence production in Baltic A. ostenfeldii bloom populations to assess the distribution and consistency of the trait in the Baltic Sea, and to evaluate applications for early detection of toxic blooms. Lcf was consistently present in 61 Baltic Sea A. ostenfeldii strains isolated from six separate bloom sites. All Baltic Sea strains except one produced bioluminescence. In contrast, the presence of lcf and the ability to produce bioluminescence did vary among strains from other parts of Europe. In phylogenetic analyses, lcf sequences of Baltic Sea strains clustered separately from North Sea strains, but variation between Baltic Sea strains was not sufficient to distinguish between bloom populations. Clustering of the lcf marker was similar to internal transcribed spacer (ITS) sequences with differences being minor and limited to the lowest hierarchical clusters, indicating a similar rate of evolution of the two genes. In relation to monitoring, the consistent presence of lcf and close coupling of lcf with bioluminescence suggests that bioluminescence can be used to reliably monitor toxic bloom-forming A. ostenfeldii in the Baltic Sea.     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). II. SEQUENCE ANALYSIS OF A FRAGMENT OF THE LSU rRNA GENE1

A fragment of the large-subunit (LSU) ribosomal RNA gene (rDNA) from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech was cloned and sequenced to assess inter- and intraspecific relationships. Cultures examined were from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo vessels and included both toxic and nontoxic isolates. Parsimony analyses revealed eight major classes of sequences, or “ribotypes,” indicative of both species- and strain-specific genetic markers. Five ribotypes subdivided members of the A. tamarense/catenella/ fundyense species cluster (the “tamarensis complex”) but did not correlate with morphospecies designations. The three remaining ribotypes were associated with cultures that clearly differ morphologically from the tamarensis complex. These distinct sequences were typified by 1) A. affine, 2) A. minutum and A. lusitanicum, and 3) A. andersoni. LSU rDNA from A. minutum and A. lusitanicum was indistinguishable. An isolate's ability to produce toxin, or lack thereof, was consistent within phylogenetic terminal taxa. Results of this study are in complete agreement with conclusions from previous work using restriction fragment-length polymorphism analysis of small subunit rRNA genes, but the LSU rDNA sequences provided finer-scale species and population resolution. The five divergent lineages of the tamarensis complex appeared indicative of regional populations; representatives collected from the same geographic region were the most similar, regardless ofmorphotype, whereas those from geographically separated populations were more divergent even when the same morphospecies were compared. Contrary to this general pattern, A. tamarense and A. catenella from Japan were exceptionally heterogeneous, displaying sequences associated with Australian, North American, and western European isolates. This diversity may stem from introductions of A., tamarense to Japan from genetically divergent sources in North America and western Europe. Alexandrium catenella from Japan and Australia appeared identical, suggesting that these two regional populations share a recent, common ancestry. One explanation for this genetic continuity was suggested by A. catenella cysts transported from Japan to Australia via ships' ballast water: the cysts contained LSU rDNA sequences that were indistinguishable from those of known populations of A. catenella in both Japan and Australia. Ships ballasted in South Korea and Japan have also fostered a dispersal of viable A. tamarense cysts to Australia, but their LSU rDNA sequences indicated they are genetically distinct from A. tamarense/catenella previously found in Australia and genetically distinct from each other, as well. Human-assisted dispersal is a plausible mechanism for inoculating a region with diverse representatives of the tamarensis complex from geographically and genetically distinct source populations. The D1-D2 region of Alexandrium LSU rDNA is a valuable taxo-nomic and biogeographic marker and a useful genetic reference for addressing dispersal hypotheses.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.